BIOL 308: STUDY QUESTIONS PREP

LECTURE 1
1. Define (or compare and contrast): a) gene expression; transcription;
replication; translation; b) gene; allele

Gene: functional unit of heredity (subunits of DNA); the entire DNA

sequence necessary for synthesis of RNA or functional polypeptide
(protein) which includes coding and regulatory regions (up/downstream)
Allele: alternative forms of a single gene, even as little of a difference as
one base only

Gene expression: process through which genetic instructions are used to

synthesize gene products (i.e. proteins or functional RNA products) that
are essential to cell
Transcription: the process by which DNA is copied to mRNA/RNA (RNA
synthesis)
Replication: the ds DNA molecule is copied to produce two identical DNA
molecules (essential because the daughter cells contain the same
genetic information as parent) (DNA synthesis)
Translation: RNA is used to produce proteins (protein synthesis)

2. Explain by using your own words the meaning/significance of gene

expression.

The goal is to understand the products of gene expression (protein, r-, t- and
small RNA) which serve functionality and performance because everything is
ultimately about proteins
Proteins: structural, enzymes, regulators, hormones, receptors,
neurotransmitters, transporters, antibodies
RNA: directly involved in expression of genes that code for proteins or control it
Proteins synthesis is crucial for survival of cell because proteins AND
regulatory RNA set genetic predisposition for how we look, our health,
shyness, aggressive, substance abuse tendencies, money handling
matters, selection of friends and attraction to someone
Environment is important: nurture vs nature (all environment variables that
impact who we are vs all genes/hereditary that influences who we are)
Epigenetics: studying heritable changes in gene expression (active vs
inactive genes) which doesnt involve changes to underlying DNA
sequence (genetic code)

3. What information(s) could you obtain from a genetic approach of studying

mutants defective in a particular process?

Through the identification/isolation of mutants, you can discover:

 - Potential function of protein of interest and the importance of that protein for
survival
- Essential genes and proteins (absolutely necessary for survival)
- Permissive/non-permissive conditions
- Number of genes involved and the order of these genes (complementation
analysis)
- Interaction between different genes (gene suppression)

4. How would you define permissive conditions in respect to temperature

sensitive mutants?

Permissive conditions for temperature sensitive mutants are conditions under

which the temperature sensitive mutant gene will convert into a product that
results in a normal, functional phenotype. When grown in a permissive condition,
the temperature sensitive mutant grows as it would normally, despite the fact that
it carries a mutated gene/allele. Example: isolating temperature sensitive mutants
in yeast! Grow at 23C, add mutagen (distribute in aliquots). Let grow at constant
temp (23C) for 5hours, replicate. One half grows at 23C and the other at 36C
(which displays no growth because the conditions are not permissive: the mutant
is temperature sensitive hence it will not allow growth (protein formation) at non-
permissive temperatures!

5. What are the roles of model organisms in molecular biology studies?

Choose two model organisms and explain your reasoning.

Model organisms allow performing biochemical and genetic approaches and

collecting data because they are used in experiments. They allow us to study
complex organisms through a simplified molecular level by (carefully)
extrapolating the results obtained from model organisms to complex organisms.
- Simple
- Small
- Cheap (in comparison)
- Easy to maintain
- Have short reproductive cycles
- Lots of data available (informatics)
- Facilities, availability
2 model organisms that are commonly used in studies are E coli and drosophila
(These model organisms are important/studied because they help understand
complex biological processes with the expectation that results/understanding
from model organisms provide insights into the processes of other organisms)

LECTURE 2
6. What are three main functions of DNA? Explain the importance of each of
them.
*Ultimately everything is about proteins!
 Stores information it contains the instructions to build other components
that are essential for the survival of the cell (i.e. proteins and RNA)
(information has genes which have coding and regulatory sequences)
Replicates faithfully by replicating and preserving information, DNA
maintains enough genetic material for creating essential proteins/RNAs
Has the ability to mutate a change in an organisms DNA can cause
changes to the survivability/development of that organism. Mutations are
important for evolution because they provide genetic variation (formation
of new alleles, no product (knockout) and altered regulation of expression
of product (if the mutation is in regulatory sequence)

7. What is meant by saying that a DNA strand has polarity? That two strands
of DNA are antiparallel? That the strands are complementary to one
another?

DNA polarity means that DNA has direction (there is a 5 and 3 end)
The strands are also complementary: means that base pairs complement each
other (A T) and (C-G) One strand predicts the sequence of the other.
And antiparallel: each strand is different at both ends (5 and 3) and each strand
in complementation, run in opposite directions. At each end there is a 5 and 3 (a
phosphate group and an oxygen group): the numbers come from the carbons
that are participating in the phosphodiester bonds

8. Describe Meselson-Stahl experiment and explain how it showed that DNA

replication is semiconservative?

They had three models for how replication could happen set up:
- Semiconservative (each daughter DNA has one old and one new strand)
- Conservative (old duplex is replicated completely so that the daughter cells
have one old DNA and one new DNA)
- Dispersive (sections of the old DNA are dispersed randomly into the two
new daughter DNA)

The E coli was grown for several generations in 15 nitrogen (so that the DNA
contained 15N), the cells were transferred to a medium that had 14N and was
allowed to replicate. The dna was extracted and dissolved in cesium chloride
solution and centrifuged (which allowed for a concentration gradient to form: so
the dna molecules moved to a place where their density matches the CsCl)
They noticed that first generation had 15N: second generation had somewhere
between 14N and 15N, third generation had 14N and somewhere in between)
The inbetween indicates that only one strand becomes part of the DNA
when replication happens
Parent strands are separated, complementary base pairing allows new
nucleotides to join and form the daughter strand and completing replication

[Link]
 9. Learn to recognize nitrogenous bases (A,T,G,C,U) and respective
nucleotides.

Nitrogenous bases: adenine and guanine (purines) and thymine, cytosine and
uracil (pyrmidines)
Nucleotides: adenine monophosphate, guanine monophosphate, thymine
monophosphate, cytosine monophosphate, uracil monophosphate
*Could be di and tri depending on how many phosphate groups are attached

10. What is the difference between nucleoside and nucleotide? What does
dNTP stand for?

Nucleoside: has a base and sugar, and a nucleotide has a phosphate-sugar

group with a base (nucleotides are building blocks of DNA)
dNTP : deoxyribonucleoside triphosphate (Ribose has OH group, not deoxyrib.)

11. What is (are) the role(s) of phospho-diester bonds in DNA structure? What
is (are) the role(s) of hydrogen bonds in DNA structure? What is (are) the
role(s) of hydrophobic interactions in DNA structure?

Phosphodiester bonds: linkage of the 3 carbon of one sugar molecule to

the 5 carbon of another sugar molecule (which leads to sugar-phosphate
backbone) using phosphate groups that link the two sugar molecules
together
Hydrogen bonds: form the steps of the DNA structure by attaching the
complementary bases together through H- attraction (keep chain together)
Hydrophobic interactions: the highly negative backbone repels polar
forces

12. If a C content of a preparation of double-stranded DNA is 20%, what is the T

content?

C has to be the same as G, so C and G make up (40%) the other 60% is divided
among A-T so T is 30%. Chargaffs Rule :# purines = # pyrimidines (A=T, C=G)

13. Who received a Nobel Prize for 3D DNA structure?

In 1962 Watson, Crick and Wilkins got the Nobel Prize

14. What noncovalent interactions are involved in maintaining the double-

helical conformation of DNA?

Ionic: when salts interact with the backbone (+ve salts) stabilize the
negative charge of the backbone (which protects the DNA)
 Van der Waals: interactions between bases (weak but theres lots of them
so its strong in a DNA molecule)
Hydrophobic interactions: there is a highly negative backbone is on the
outside which repels positive charges of water (outside) and non-polar
hydrophobic bases (inside)
Hydrogen bonding: responsible for complementary base pairing

15. Describe the conformational characteristics of B DNA (or A DNA, Z DNA,

triple-helical DNA). When does this (any of the above) form of DNA occur?

B-DNA: right-handed DNA (10 nucleotides, or 3.4 nm in length every turn,

2nm in diameter) Mostly of the in vivo DNA!

A-DNA: right-handed DNA (11 nucleotides or 2.8 in length every turn,

2.6nm in diameter)
o Found in low-water content so you dont find it as often in cells
(Mostly in vitro RNA/DNA helixes are A-DNA)

Z-DNA: left-handed DNA (12 nucleoties, 4.5 nm every turn, 1.8 in

diameter)
o IN VITRO with very high salt concentrations
o Z-DNA are involved in regulation of gene expression so found in
regulatory sequences or promoter regions of genes
o Not as permanent as B-DNA because the negative charge
repulsion of the backbone along grooves is very high

H-DNA (hinge-DNA): formed when purines make one strand and

pyrimidines make the other (found in introns of genes coding for cell
membrane proteins)

LECTURE 3
16. What are action or agents that denature DNA vs forces that keep the
strands together?

Actions/agents that denature:

- heat (breaks bonds) Factors that keep strands together:
- low ionic strength - ionic (stabilizes backbone)
- agents that enhance solubility - van der waals (interaction
of hydrophobic regions between bases)
(i.e. organic solvents) - hydrophobic (bases steps)
- agents that influence H-bonds - hydrogen (steps)
(competition/covalent modification)
- high pH
High salt concentration masks the negative charge/repulsive force of the sugar-
phosphate backbone so it decreases denaturation because it stabilizes DNA

17. How does high salt concentration influence denaturation kinetics of DNA? Explain
your reasoning.

Low salt concentration low ionic strength, there are not as many salts around to
stabilize the phosphate backbone thus causing repulsion and separating the
strands (Quicker kinetics)

High salt concentration higher ionic strength, there is a stabilized phosphate

backbone thus LESS REPULSION, strands want to stay together (Slower kinetics)
- The higher the salt concentration, the higher the melting point needs to be to break
the intramolecular hydrogen bonds

18. How can DNA denaturation be monitored?

The progress of DNA denaturation can be monitored by examining the properties that
would change when the strands separate
- Absorbance (optical density: common lab technique)
- Viscosity (rarely used because difficult)
Absorbance: measurement of absorbed light (the measurement changes depending on
whether the purines/pyrimidines are stacked)
- In dsDNA bases are stacked: less absorbance because hydrogen bonding limits
resonance so the light absorbed (a form of energy) is limited too
(Hypochromic: decrease in absorbance (stacked bases)
Hyperchromic: increase in absorbance because of strand separation)

19. What factors affect the Tm (melting temp) of DNA fragment?

The GC content affects the Tm (melting temp) because the more GC you have (which
share 3 hydrogen bonds) the greater melting temperature is needed to break those
hydrogen bonds
Tm INCREASES BY 0.4C WITH EVERY 1% INCREASE IN GC CONTENT
(UNDER NORMAL CONDITIONS)

Higher salt also increases Tm because DNA is stabilized so more effort to break

20. What can be learned from studying kinetics of renaturation?

Renaturation is the recombination of two complementary single stranded DNA (it tells us
that when strands denature, they reneal with strands that they are complementary with
(which could be from the same or different DNA molecule resulting in hybridization)
Renaturation kinetics: speed at which single strand sequence is able to find a
complementary sequence and base pair with it (the rate of renaturation = measures the
complexity of the DNA/genome)
*So basically: tells us how complex the DNA/genome that we are dealing with is
 21. List and briefly explain factors that influence DNA renaturation kinetics.

DNA concentration (complementary single strands must find each other) (the
greater the DNA concentration, the higher the chance of finding)
Salt concentration (ionic conditions (+ive ions) mask the repulsion forces (-ve) of
phosphate backbone (the greater the salt concentrations, the less the repulsive
forces between the two strands so the quicker the rate)
Temperature (20-25 below Tm: the lower it is from Tm, the quicker)
Time (reaction time: faster the time, the quicker the rate)
Size of DNA fragment (inversely proportional: the bigger, the slower)
Level of sequence complexity (basic properties of DNA sequence (i.e. some
simple sequences re-nature faster than others, even if all of the conditions above
are the same for both)

22. What is Cot analysis?

Cot analysis explores genome structure in individual species, looks at similarities among
organisms and evaluates diversity in ecological samples
- tells us whether the size of the genome indicates its complexity and what simple
sequences renature faster than complex ones
- Its a method used to measure the complexity of DNA/genome
Cot (Co = starting conc. , t = time usually t1/2 (time needed for half of the DNA to
renature)
- The measurement of complexity is done in terms of nucleotides
a. If no repeptitive nucleotide sequences then : complexity = # of nucleo.
b. If some unique & some repetive sequences: complx. = # of unique nucleotides +
total # of nucleo. from one copy of each repetitive sequence

23. What are the classes of DNA sequences in genomic DNA (based on renaturation
kinetics)?

Unique (no rep. sequences, single copy), moderately repetitive & highly repetitive

24. You have found a new species of insects. To evaluate the complexity of the genome
of this species, you isolate genomic DNA, fragment the DNA to uniform 500 base
pair pieces, denature the DNA and measure the rate of re-association. Your data is
represented in the curve below (sorry for the bad drawing):
(a) How many classes of DNA (in respect to sequence complexity) are found in this
organism? Three classes: highly rep., moderately rep, & unique

(b) What can you say about the relative complexity of each class? What fraction of the
genome falls into each class? Unique class is the most complex because it takes the
greatest amount of time to re-nature (sequences have difficulty finding each other) highly
rep. is the least complex (re-association rate is the quickest!)
- Unique: 50%, moderately rep.: 25% (from 50 75) , highly rep.: 25% (75 100)

25. If you had two solutions of DNA, one single-stranded and one double-stranded, with
equivalent absorbance at 260 nm, how would the concentrations of DNA compare in
these two solutions? (You can use a diagram if it makes it easier for you to explain.)

In single-stranded: the bases are unstacked (hyperchromic because absorbance

increases), in ds: bases are stacked so the hydrogen bonding limits resonance structures
(so limited light absorbance (hypochromic)
In order to have equivalent concentration, ds dna must be higher in concentration
(represent more of the solution) and ss dna must be lower in concentration

26. You preformed Cot analysis using genomic DNA samples obtained from a 2 year-old
child and a 76 year-old individual. Results of this analysis show that one of the most
rapidly reassociating classes of DNA is substantially reduced in the older individual
with respect to the 2 year-old. How can you explain this finding? (hint: think about
termination of linear DNA replication.)

Highly repetitive sequences are lower in elders than children due to the termination of
linear DNA replication. Highly repetitive sequences are mostly located around
centromeres and telomeres, which decrease the more the cell divides (so as the
person ages)
Telomeres: specific sites at the ends of DNA to prevent loss of genetic information
due to the shortening of the molecule because of the 3 overhang after each
replication cycle. Telomeres shorten the more the cell divides (so the repetitive
sequences are being lost)
Centromeres: the site where the sister chromatids are held together by cohesions
and the complex that forms kinetochores to assist in pulling the strands apart. During
 anaphase, when the strands are pulled apart, the centromeres are split in half so the
repetitive sequences are reduced from the parent to the daughter cell each time

27. How does complexity of bacterial genome differ from that of eukaryotic (calf)
genome?
E coli (model organism for bacteria) has no repetitive sequence: the whole genome is one
unique sequence so it is highly complex: re-naturation rate is very slow because the
strands have difficulty finding each other

Calf genome has lots of repetitive sequences: so its not as complex because the
renaturation rate is pretty quick (almost instantly), it only has moderately repetitive and
unique sequences
- If there are no unique sequences then the complexity is (theoretically) equal to the
genome size
- If there are unique sequences and repetitive sequences then the complexity is equal
to # of nucleotides in unique + # of different nucletides in each repetitive sequence
(ATATAT 2)

28. Explain C value paradox.

There is no correlation between the amount of DNA (size of genome) and the apparent
complexity of the organism

29. What are the differences between primary (or secondary, or tertiary) structures of
RNA and DNA?
DNA:
Primary phosphate backbone, sec - h bonding, tertiary supercoiling

RNA:
Primary: similar but different conformations because the 2 OH group doesnt allow a
formation of B-helix so A-helix forms
Secondary: stem-loop or hairpin because of self-complementarity base pairing
Tertiary: high degree of rotation in the backbone around the non-base-paired regions so it
folds into tertiary structures
Base triple: U:A:U OR A:G:C
Pseudoknots: when the base pairing happens in not-adjacent-sequences

30. Explain the importance of DNA supercoiling for the cell survival?

DNA supercoiling is important for cell survival because

a) It packages DNA (condenses it more)
b) Dictates DNA replication and transcription (based on topographical state: the dna
can either be topographically linearized (locally uncoiled) to allow the strands to
separate and replication and transcription processes begin
c) Reduce the stress induced by the twisting of the double helix (i.e. in DNA
denaturation, the double helix is twisted so that the strands separate) (because
 when you untwist one side of a side, the other side instead of twisting bends it into
supercoils)
d) It is the most energetically favorable for the cell to bend long DNA molecules rather
than to untwist it (supercoils adds kind of like that extra layer of protein, first you
have to uncoil and then denature DNA)
Positive (right handed for b-dna): form infront of the protein (overwound)
Negative (left handed for b-dna): form behind the protein (underwound)

31. What are topological isomers of DNA?

Topological isomers are DNA differing in only their states of supercoiling (The shape of
the molecule or structure changes based on how it supercoiled)

LECTURE 4
32. You have a small 4800 bp long circular DNA. It has a linking number of 450 (L=450).
What are the twist (T) and the writhe (W) of this DNA? What assumptions about the
structure of the DNA have you made in your answer? (Hint: what is the definition of
the twist #?)

The assumption made is that the DNA is in the conformation of B-DNA (not specified)
L=T+W
= 450
T = # bp total (length) / # of bp every W = # of supercoils
turn W=LT
( in B-DNA there are 10 every turn) = 450 480
= 4800 / 10 = -30 (So it has 30 negative
= 480 (480 twists in the DNA) supercoils)
32. List three (3) differences between prokaryotic Topoisomerase I and Gyrase

Topoisomerase 1: Gyrase (opposite of topo 2)

Relaxes negative supercoils Induces negative supercoils
Single stranded cut (relaxes +ve supercoils!)
No ATP Double stranded cut
Requires ATP

33. Does the degree of chromosomal condensation play a role in controlling

replication and transcription? How (explain briefly; use your own words)?

The degree of chromosomal condensation DOES control replication and

transcription because in interphase, the chromatin in the chromosome structures
are euchromatin so they are less tightly packed which allows replication and
transcription mechanisms to access the DNA. In metaphase, metaphase
chromosome is the tightest packaged form of the DNA structure which does not
allow transcription and replication to occur (this is when the 30nm fibres:
heterochromatin structures are condensed into an even tighter packaging to
protect the DNA from the stress of dividing and separating)

34. What are some of the distinctive features of eukaryotic chromosomes? (note:
I expect you to first define chromosomes and after that you have to briefly
explain nucleosomes/histone proteins/octet +H1/wrapped DNA, different
levels of chromosome condensation, centromere and telomere regions)

Chromosomes are the most condensed form of DNA (tightly packed chromatin
fibers which arise from chromatin structure)
Nucleosomes: the fundamental unit of organization which carries histone
octet and wrapped core DNA complex
Histones are small +ve charged proteins rich in lysine/arginine which
interact with DNA through electrostatic interactions. There are 5 main types:
which are H3, H4, H2A, H2B, and H1. H3, H4, H2A, H2B form a histone
octet (based on H3-H4 tetramer and 2 H2A-H2B dimers)
H1 is a histone protein that influences the bonding between the wrapped
DNA and the octet (linker region: binding of two different dsDNA regions)
Wrapped DNA is 147 bp wrapping around the octets approx. 1.65 times
Different levels of chromosome condensation:
o Nucleoproteins (complex between nucleic acid and specific protein
complexes) are like beads on a string and they form chromatin
o Chromatin condenses to form chromatin fibres (10nm or 30nm fibres;
through nucleosomes) which condenses into chromosomes
Centromeres: specific nucleotide sequences that allow the binding of
microtubules (kinetochores and spindle fibers) to separate sister chromatids
Telomeres: nucleotide sequences at the ends of DNA (loop 3 OH overhang
after replication) to protect the DNA molecule from degradation after
successive replications!
35. What are heterochromatin and euchromatin? What is their importance in DNA
replication and transcription?

Euchromatin and heterochromatin are variations of chromatin structure

Euchromatin:
- Transcriptionally active DNA
- Susceptible to DNase

Heterochromatin:
- Transcriptionally inactive
- Less susceptible to DNase
a. Constitutive: highly condensed, very few genes (because repetitive
sequences), inactive
b. Facultative: not active in some tissues! (forms rarely: only under special
conditions like:
X-chromsome inactivation
Imprinting

The chromatin structure (euchromatin or heterochromatin) leads the replication

fork because euchromatin allows the replication/transcription processes to attach
to it. Both participate in gene expression regulation, transcription and replication.
The importance is that indicates when the DNA is available for replication /
transcription and when it is not available it gets tightly condensed to protect the
DNA from damage! It allows some genes to be available and easily transcribed
while protecting others until transcription is not happening (keeping majority of
the structure condensed hence saving space!)

36. Would you expect there to be more histones per kilobase in euchromatin or
heterochromatin? Explain your reasoning.

There would be more histones in heterochromatin because they are more tightly
packed so they need more octets to wrap the DNA around tightly!

37. Name few nonhistone proteins which are a part of chromatin structure and
explain why you would expect them to be found there.

Some non-histone proteins that are part of the chromatin structure are:
Structural maintenance of chromosome proteins (SMC): responsible for
scaffolding (when you are building a bridge, the rods you stick in place as
the blueprint which are the most important in providing stability is scaffold)
DNA replication proteins and transcriptional factors (attached to the
chromatin structure in euchromatin (which is actively transcribing and during
interphase when the structure is looser so allows binding for replication)
Chaperone proteins are proteins that find and carry the histone proteins to
form the histone complex (octet)
38. What is unusual about the amino acid composition of histones? How is the
function of histones related to their amino acid composition?

Amino acid sequence is very conserved! This is important because the fewer the
differences in amino acid sequences across species for the histone proteins, the
less genetic variation, which gives less change of mutations to occur which would
be lethal to organisms! Chromosome structure would not form without histones.

LECTURE 5
39. The human gametes have about 3 billion bp of DNA in their chromosomes.
a. If the entire DNA was in relaxed B-DNA form, what would be the average
length of a chromosome in the cell?
(3 billion bp / 23 chromosomes) x (0.34 x 10-9) = 0.044 m (44.35 mm or 44
347 826 nm)

b. On average, how many complete turns would be in each chromosome?

T = # of total bp / # of bp every turn
= (3 billion bp/23 chromosomes) / 10 bp
= 13 043 478.26 complete turns

c. If there are around 30-40,000 genes in a human gamete, how many

genes are there in an average chromosome?
Average number of genes: (30000 + 40000)/2 = 35 0000
If there is on average, 35 000 genes then each chromosome would have
35 000 genes / 23 chromosomes = 1521. 74 genes

40. What is the purpose of cell division in Prokaryotes? In Eukaryotes?

Prokaryotes:
Reproduction (binary fission)

Eukaryotes:
- Reproduction (germ cells (diploids) undergo specialized cell division
(meiosis) to create gametes (haploids))
- Tissue growth/development (larger bones/muscles)
- Repair of damaged tissues (broken bones, muscle tears, renewal of skin
cells, broken blood vessels) (MITOSIS)

41. Distinguish between DNA replication and cell division.

DNA replication: passing down of genetic info. from parent to daughter cells
(synthesize DNA)

Cell division: division of a parent cell into two new daughter cells (create more
cells)

42. What is a cell cycle? What are the stages of cell cycle?
Cell cycle: the set of events that take place from the formation of a new cell to the
division of that parental cells into 2 new daughter cells
There are two main stages (which have divisions of their own) in the cell cycle:
a. Interphase (normal cell functions and preparation for mitosis (division; i.e.
replication)
G1: growth occurs as organelles double
S: DNA replication
G2: growth occurs to prepare the cell to divide
G0: cell that just finished dividing (temporary or permanent phase)

b. Mitosis/cytokinesis (nuclear division & cytoplasmic division)

PROMETA ANATELA (prophase, metaphase, anaphase, telophase)

43. List and briefly describe the checkpoints in cell cycle. What is their purpose?
A checkpoint in the cell cycle is when specific proteins (i.e. cyclins, cyclin-
dependent kinases, etc.) stop the cell cycle to make programmed
assessments & controls (Apoptosis: programmed cell death)
Start (G1 checkpoint) (cell growth (size) and environment (favorability))
S phase (S chckpnt) (DNA replication machinery -> is rep. complete?
Enter M (G2 chck) (cell growth and environment)
Exit M (metaphase checkpoint) (are all chromosomes aligned?)
Purpose:
Energy/resources are saved (no damaged cells)
Internal control (through signals i.e. late attachment of kinetochores delays
ana)
External control (growth factors, density dependent inhibition, etc.)

44. What can trigger arrest during the cell cycle?

When checkpoints are triggered, it causes arrest in the cell cycle. This happens
if:
- DNA tries to divide before replication is complete (MITOSIS)
- Mass of the cell is not doubled (daughter cells wont get enough material)
(START)
(Lack of coordination between these two events: leads to cell cycle arrest)
The DNA must not replicate more than once or try to divide before the replication
is finished or before the mass has doubled!

45. Distinguish between reason/purpose for/of mitosis and reason/purpose for/of

meiosis?
The purpose of mitosis is growth and renewal and repair
The purpose of meiosis is reproduction (germ cells -> gametes)

46. Define homologous chromosomes.

Same genes, at the same loci (same gene order) but different alleles (mom/dad)

47. Define non-homologous chromosomes.

Contain alleles for different types of genes

48. How many homologous chromosomes are there in a germ cell of a woman?
Germ cell: diploid. In a human diploid, there are 23 pairs of homologous
chromosomes (MALE: 22 pairs because X AND Y arent homologous)

49. Distinguish between homologous chromosomes and sister chromatids.

Sister chromatids: same genes with the same alleles, replicated copies of each
other
Homologous chromosomes: same gene, same loci, but different allele (one
derived from mother and one from father)

50. How many chromosomes are there in a somatic cell of a person with Down
syndrome (trisomy of chromosome 21) A normal somatic cell has 46
chromosomes: so a trisomy has 47 chromosomes

a. How many autosomes does this person have in a somatic cell?

22 pairs (one with trisomy) so 45 autosomal chromosomes
b. How many sex chromosomes does person have in a somatic cell? 2
In a germ cell? 2 In a gamete? 1 In a spermatozoid? 1 In an ovum? 1 In
a zygote? 2
c. How many DNA molecules does this person have in mitotic
metaphase? In G1 phase? 94 DNA molecules (47 chromosomes: 47
DNA molecules: each replicates so 94 DNA molecules). In G1 phase
there are 47 DNA molecules (prior to replication)
d. How many telomeres are there in a persons somatic cell during G2
phase? (188 telomeres: 2 for each DNA molecule)
e. There are 4 alleles for a certain gene carried by chromosome 21 in
human population. How many alleles does a person with Down
syndrome have in a somatic cell in G1 phase? 3 alleles in the G1 phase
How many different alleles for this gene could the same person have?
This person can have 2 different alleles for this gene (one mom/dad)
f. There are 13 alleles for a certain gene carried by chromosome 21 in
human population. How many alleles does a person with Down
syndrome have in a somatic cell in G1 phase? 3 alleles in G1 phase
How many different alleles for this gene could the same person have?
This person can have 2 different alleles (one from each parent)
g. There are 13 alleles for a certain gene carried by chromosome 21 in
human population. How many alleles does a person with Down
syndrome have in a cell which is in meiosis I anaphase? 6 copies of
alleles (3 in DNA molecule, but after replication it doubles) How many
different alleles for this gene could the same person have in a cell
which is in meiosis I anaphase? You can only have 2 different alleles

51. How is variability of genetic information attained by meiosis and fertilization?

Variability increases in meiosis:
 1. Independent assort of homologous chromosomes
(The material and parental set could be combined in metaphase because of
the way that the chromosomes separate)

2. Crossing over (homologous chromosomes cross over each other and

exchange genetic information

LECTURE 6
52. When does chromosome segregation happen in mitosis? In meiosis? When
does chromatid segregation happen in mitosis? In meiosis
Chromosome segregation:
Mitosis: doesnt occur
Meiosis: anaphase I
Chromatid separation:
Mitosis: anaphase
Meiosis: anaphase II

53. What is meant by replication being bidirectional? Semiconservative?

Continuous and discontinuous?
Bidirectional: it starts from a specific site (origin of replication) and
replicates in both directions from that site because of the antiparallel
nature of DNA (that is why a replication bubble expands both ways)
Semiconservative: dsDNA unwinds into ssDNA, strands separate, each
strand serves as a template to synthesize a daughter strand. So every
new DNA molecule is 50% old, 50% new
o Continuous: in the leading strand (there is only one initial primer and DNA is
continually synthesized in the 5 to 3 direction using that primer and
complementary base pairing)
o Discontinuous: lagging strand (primers are continually added because the
direction is opposite to the replication fork, replication still continues in 5 to 3
direction but only for a limited number of bases and then primer is added)

54. List proteins (enzymes and other factors) involved in the process of DNA
replication in E. coli. Explain the role of each of these proteins in replication.

DnaA: initiates replication at OriC by recognizing four DnaA boxes (9bps)

only if DNA is negatively supercoiled
DnaB/helicase: moves along dsDNA, unzips & separates strands (w/ ATP)
DnaC: clamp loader req. to be used with DnaB which escorts it to DnaA
where it forms a pre-priming complex
Single-stranded DNA binding proteins (ssBP): small, bind to ssDNA and
prevent the double helix from re-annealing
DnaG/primase/RNAP: synthesizes RNA primers, works with DnaB to
attach to a priming site (primosome: the site where the primer forms)
DNA pol2: repair
DNA pol3: KEY ENZYME! Catalyzes synthesis of DNA using RNA primer
 DNA pol1: works on lagging strand, removes RNA primers, esp. the ones
that are linked directly to DNA
Rnase H: removes most of the RNA primers expect the last one that is
directly linked to DNA (only the one in between okazaki fragments)
Ligase: links okazaki fragments by forming phosphodiester bonds
between 3OH and 5phosphate group
Topoisomerase: recognize and regulate supercoiling (topo 1 and topo 2)

55. How can discontinuous synthesis of the lagging strand keep up with
continuous synthesis of the leading strand?
Help from enzymes: DNA poly 1, ligase, DnaG (primase) which maintain speed.
Also it works very well with the core polymerase enzyme (especially the tau
subunits) which continually synthesizes DNA from a RNA primer (until approx.
135 bp) and then jumps onto the next RNA primer to continue synthesis there
In eukaryotes, the leading strand rate of synthesis is slowed down to match
the rate of synthesis of the lagging strand (by the C-M-G complex)

56. What are cis- elements? What are trans-factors?

Cis elements: specific nucleotide sequences (cant move around), the
sequence contains a precise distribution of acceptors and donors that
regulate gene expression of the strand it is located on
o Ex: DnaA box, OriC (e. coli), TATA box
Trans factors: diffuse through the cell/nucleus because their function
involves moving around
o Ex: PROTEINS: they recognize cis-elements and bind to them
o DnaA, DnaB, DnaC

57. Describe the events that occur at an origin of replication during initiation of
replication in E. coli?
a. OriC is recognized by specific proteins
b. The A in GATC (11 times in ORIC) is methylated by Dam methylase
c. DnaA binds (by recognizing cis- elements) and initiates replication by
breaking the hydrogen bonds between complementary bases
d. DnaB (helicase) unzips/separates the strands
e. ssBP attach to each individual strand to prevent re-annealing
f. DnaC loads the clamps (the beta-subunit of DNA POLY 3
holoenzyme), unloading the clamps allows binding to replication fork
g. DnaG (primase) lays down RNA primers for elongation
h. Topo 2 creates ve supercoils (as the replication fork is extended)
i. DNA poly 3 binds: INITIATION OVER!!!

58. Contrast the role of DNA polymerase I and III in E. coli DNA replication.
DNA poly 1: removes RNA primers and inserts DNA only to lagging strand (using
the complementary base pairing system), 5 to 3 exonuclease activity, 5 to 3
addition of DNA (polymerization) and 3 to 5 exonuclease activity for mismatch
bases
DNA poly 3: the most key enzyme for replication, forms a holoenzyme complex
compiled of tau protein, gamma complex, and beta subunit. 3 to 5 exonuclease
activity, adds DNA to both the leading and the lagging strand (requires a primer
to elongate, BUT it does not remove these primers)

59. Which subunit of DNA polymerase III provides processivity? Which protein
complex loads this subunit onto the DNA?
The beta subunit provides the highest processivity (it is part of the core
polymerase), the gamma complex loads this subunit onto the RNA primer of DNA

60. What would be the components necessary to make DNA in vitro by using
DNA polymerase I.
dNTP (deoxynucleoside tri phosphate), RNA primers, DNA of interests that
contains a target sequence you wish to copy, DNA polymerase 1

61. What properties would you expect an E. coli cell to have if it had a
temperature sensitive mutation in the gene for DNA ligase?
The role of DNA ligase is to attach the lagging strand together (through
phosphodiester bonds: 3 OH and 5 phosphate). At high temperatures (non-
permissive conditions) temperature-sensitive mutated DNA ligase would fail to
ligate the strands, which would result in fragmented DNA, which is non-functional
hence will result in apoptosis.

62. What properties would you expect an E. coli cell to have if it had a
temperature sensitive mutation in the gene for DNA polymerase I?
The function of DNA polymerase I is to remove RNA primers, add DNA (through
polymerization) and proof-read for any mismatched bases. At high temperatures,
the lagging strand, which is what DNA poly 1 works with, will be non-functional
because it would not be able to lose the primer.

LECTURE 7
63. Why is decantation required after replication of circular DNAs?
After replication, the daughter helices are linked to the together, catenated, but
some parts still have to be replicated. Thus, at the site of linking between the
parental duplex and the daughter duplex: denaturation occurs, followed by
supercoiling, replication is completed, and then decatenation occurs, which is
when topo 4 makes a dsDNA cut, relaxes the supercoils, segregate the two
duplex (by passing the parental through the gate) and then re-anneals

64. Is the following statement true or false: Regardless of whether a gene is

expressed in a given cell type, it will replicate at the same, characteristic time
during S phase. Explain your reasoning.
False. In S phase, DNA undergoes replication only once, which is when the
whole genome is replicated regardless of whether the gene is expressed.
However, the genome does not replicate at the same time, genes that are
expressed in the euchromatin structure will replicate first because the lesser
condensed structure provides greater accessibility to the DNA for replication.
Since euchromatin replicates in the beginning of S phase, heterochromatin
replicates slower/later on in the S phase because it has greater condensation.

65. What is the major difference between bacterial and eukaryotic replication that
allows a eukaryotic cell to replicate its DNA in a reasonable amount of time?
Eukaryotic cells have multiple replicons/origin sites whereas bacterial replication
machinery only has one in the circular DNA. This allows for multiple replisomes
to be activated at once, speeding up the replication process.

66. Compare and contrast major eukaryotic and prokaryotic DNA polymerases.
Prokaryotic:
DNA POLY 3: Key enzyme in replication (tau protein, gamma complex, beta
subunit, 5 to 3 polymerization, 3 to 5 exonuclease proofreading)
DNA POLY 2: repair mechanism
DNA POLY 1: 5 to 3 polymerization, 5 to 3 exonuclease of RNA primers, 3 to 5
exonuclease of mismatched base pairs

Eukaryotic:
POLY ALPHA: priming DNA synthesis
POLY DELTA: lagging strand replication and repair
POLY EPSILON: leading strand replication and repair

67. Why do eukaryotes need telomeres but prokaryotes do not?

Prokaryotes have circular DNA, so there is always a 3 OH group available for
DNA polymerase to finish synthesis of the lagging strand. Eukaryotes dont have
fixed ends, so when the very last of the lagging strand is reached and the primer
is removed, there is no 3 OH group for the poly Alpha to add to, so there is a 3
overhang. Telomeres are specific nucleotide sequences, G-rich, at the 3
overhang that form loops and are capped by shelterin to physical protect the
ends of the chromosomes.

68. What is telomerase and why is it important?

Telomerase is a nucleoprotein that adds repetitive sequences to the 3 overhang
of the lagging strand, extending the length and reducing the chance of important
original genetic information being lost (solution to the end-replication problem).
It carries its own RNA primer with it, attaches to the tip of the telomeric region,
and adds to the parental strand by repeat units.
IMPORTANCE: Keeps the ends of the chromosomes long, preventing loss
of important information in every replication cycle

LECTURE 8
69. Distinguish between the terms mutation, DNA repair and recombination
Mutation: transmissible permanent change in nucleotide sequence of
chromosome often in a gene that changes or alters the function of product
DNA repair: mechanisms used by the body to repair faults in DNA sequence,
preventing mutations (even if it doesnt repair it back to the original form)
Recombination: exchange of genetic material between two DNA molecules,
causing rearrangement of genes (inc. variability). Could be used as repair
mechanism, i.e. when a nick in DNA is encountered during replication
70. List and briefly explain three major causes for mutation in DNA.
Replication errors: mistakes made during replication that werent detected
and corrected by DNAP 1 / 3 proofreading mechanisms
Spontaneous changes: depurination (hydrolysis of N-glycosol linkage -> a
basic site in DNA -> loss of nucleotide. C deamination (C->U), A
hypoxanthine (A pairs with C, instead of T)
External factors: radiation, temperature changes, mutagens, etc.
o Alkylating chemicals (add alkyl group to DNA: decreased stability)
o Reactive O pairs with bases A / C(Mutagenic)
o Non-ionizing radiation (absorbed highly by bases: thymine dimers)
o Ionizing radiation (double stranded breaks)
o Base analogues (5 bromouracil = T, replaces it)
o Intercalating agents: added between stacked DNA
(insertion/deletion mutations: frameshift)

71. Explain how errors in DNA replication can lead to mutations.

Failure to repair DNA before next round leads to permanent change in sequence
(mutation). The two strands will separate and the mutated strand will become the
parental strand, which will allow complementary base pairing to the mutated
sequence, hence it will be carried on the daughter strands (non-repairable/perm.)

72. Distinguish effects of mutations on somatic / germ cells of multicell. organism.

A mutation in a somatic cell is non-hereditary (i.e. cancer) because it cant be
passed vertically (no meiosis on somatic cells so no contribution to gametes). But
it is still detrimental for the organism carrying the mutation

Germ cell mutation/precursors lead to hereditary disease because the mutated

DNA becomes the original template to make DNA in other cells (i.e. in meiosis,
the mutated DNA is replicated and passed on)

73. List the various types of DNA repair mechanism (we have mentioned seven).
I. Mismatch repair
II. Direct reversal of damage
III. Base excision pair
IV. Nucleotide excision repair
V. Recombination repair
VI. Non-homologous end joining
VII. SOS trans-lesion repair
74. Describe the mismatch repair process of bacteria (pay attention at the ways in
which the daughter and parent strand are recognized by repair system).
Mismatch repair: errors missed by DNA polymerase, during replication, create a
distortion in the backbone (alerting this repair system!).
MutS scans DNA, binds to mismatch, recruits MutL, scans for and binds to GATC
sequence (binding at two locations creates a loop in the DNA). MutSL brings in
MutH (endonuclease): nicks the daughter (UNMETHYLATED) strand and
degrades from 5 to 3 (UvrD helicase helps) starting at GATC to the mutation,
and DNAP III polymerizes, DNA ligase seals.
Dam methylase: methylates the A in GATC on the parental strand, so the
unmethylated strand is the new strand which is where the mutation will be found

75. Describe briefly the mechanism of direct reversal of damage in bacteria.

Direct reversal is repair of thymine dimers. Sometimes UV light (radiation)
causes adjacent T to form dimers:
Photoreactivation (light dependent activity that breaks the covalent bonds
between T dimers). Photolyase: enzyme responsible for carrying out direct
reversal, flips the dimer to get it into its active center, breaks bond and flips back.

76. Give detailed description of the base & nucleotide excision repair in bacteria.
Base excision: LESION-SPECIFIC (i.e. deaminated C (-> U) bound to G)
Glycosylase: recognize/remove damaged base (hydrolysis of glysodic bond)
AP endonuclease/phosphodiesterase: removes abasic sugar (the sugar
connected to the damaged base that was just removed)
DNAP beta: adds new nucleotides, DNA ligase: seals the nick

Nucleotide exicison: NOT LESION SPECIFIC (cuts around the damage too)
UvrAB complex scans the DNA for a distortion in the backbone, UVRA finds
the distortion (UvrB at this moment brings UvrC in: UvrBC complex forms,
UvrA leaves). The UvrBC complex makes two incisions in the DNA molecule:
7 nucleotides up towards the 5 end, 3-4 nucleotides down the 3 end. UvrD
(helicase) binds to the complex, unwinds the damaged region. DNAP 1 adds
new nucleotides, and DNA ligase seals the nick.

LECTURE 9
77. Distinguish between two DSB repair mechanisms we talked about in class.
Homologous recombination repair: activated by nicks in the DNA encountered
during replication (both parent and daughter strand are damaged: replication fork
ends. Loss of nucleotides because the 5 end gets degraded, the 3 end
overhangs and participates in strand invasion. The other original strand duplex
is fine so the gap is filled with stealing the sequence from that other duplex.

Non-homologous end joining (NHEJ): double stranded damage, which is

detected, an incision is made (the ends are degraded) and then the two ends are
ligated together (HIGHLY ERROR PRONE: FAIL SAFE SYSTEM)
 (Homologous recombination repairs the damage accurately by taking
correct information from the undamaged homologous chromosome.

78. Describe the term non-homologous end joining, explain how this process
results in repair of ds break in DNA
NHEJ is an error prone DSB repair: where the broken ends are degraded first,
and then directly ligated without the need for a homologous chromosome
template. Ku70/80 recognizes damage, DNA-PK (DNA dependent protein kinase
brings the Artemis in). The overhangs are trimmed by protein: Artemis, ends are
filled by DNAP and DNA ligase, XRCC4 joins the ds ends.

79. What is SOS repair mechanism, when is it used and why is it important?
SOS repair system: HIGHLY ERROR PRONE, DNAP adds nucleotide randomly
without caring about complementary base pairing because of heavy heat shock
conditions. Important: cell has very low survival rate; this is the cells last resort to
maintain DNA (genetic info) and finish replication/division. If SOS fails: apoptosis.

80. What are biological roles of DNA recombination?

DNA recombination: exchange of genetic material, causing rearrangement
DNA repair
Creation of new gene/allele combination (crossing over during meiosis)
Formulation of new genes (Immuno-globulin rearrangement)
Integration of a specific DNA element

81. List and briefly describe the ways genomic DNA can be rearranged (there are
three of them).
General homologous recombination: exchange of genetic material between
pair of homologous chromosome
Site-specific recombination: between sequences with limited stretch of
similarities, involved with recombination sites
Transposition: mobile DNA element moves from one site to another, usually
little similarity involved

82. What are the key steps in single stranded model of homologous DNA
recombination?
Homologous recombination: genetic exchange between homologous sequences
Alignment of two homologous molecules
Introduction of break IN ONE STRAND of EACH molecule
Strand invasion: ss region from one parental strand pairs with the
complementary strand from the homologous molecule

(WHEN THE STRAND INVADE: THEY CROSS ONE ANOTHER, THIS

CROSSING STRUCTURE IS KNOWN AS HOLIDAY JUNCTION)

Movement of holiday junction (branch migration)

Cleavage of holiday junction (resolution)
83. Explain the relationship between hybrid duplex and heteroduplex. (You can
use diagram.)
Heteroduplex is two strands of different chromosomes of origins that have been
recombined through ss recombination. Hybrid duplex is the pair of
chromosomes of this hetero duplex. (Hetero: different strands, hybrid: a dsDNA
that contain different strands)

84. What is the major role of homologous recombination in prokaryotes?

Major role of homologous recombination in bacteria is to repair DSB; it can only
happen during replication while the daughter strands are still in proximity to one
another (important for strand invasion).

LECTURE 10
85. Describe the function of proteins involved in homologous recombination of E.
coli.
RecBCD: nuclease/helicase (enters the DSB, unwinds and degrades until chi
sequence is reached)
RecA: makes a 3 overhang at the chi sequence (for strand invasion)
RuvAB: recognizes holiday junction and catalyses branch migration
RuvC: resolution of holiday junction

86. Describe the role of chi sequences in homologous recombination of E. coli.

Chi sequences: increase with frequency of recombination, it is a cross over
hotspot. Also, tells RecBCD where to stop the degradation to have a 3 overhang.

87. What are the roles of homologous recombination in eukaryotes?

Repair of DSB
Proper chromosome pairing during meiosis
Gene reshuffling variability

88. List and briefly describe three mechanisms by which genetic elements are
able to move from one site in the genome to another.
DNA-only transposons: encode only for genes for mobilization and insertion (host
replication machinery used for replication)

Retrotransposons: carry LTRs (long terminal repeat sequences) with two

important enzymes: RT and integrase. Cant move from cell to cell. Move only to
new DNA within the cell.

Non-retroviral retrotransposons: poly A at 3 end of RNA transcript. Moves as an

RNA intermediate (the RNA produced from neighboring promoter)
LTRS (long terminal repeat sequences) and SINE

89. When does the programmed creation of DSBs occur in eukaryotes? In which
type of cells? Briefly describe the process.
 Programmed generation of DSBs occurs in meiosis, formed at the
recombination hotspots. The process is of homologous recombination for the
purpose of crossing over (increasing genetic variability), which follows:
- DSB formation, end processing, strand invasion, intermediate processing,
resolution, gene conversion

90. Explain the role of site-specific recombination in infection of E. coli genome by

lambda phage.
Site specific recombination occurs between sequences with a limited stretch
of similarity (that have specific recombination sites). The role of site-specific
recombination is that it participates in both the insertion and deletion of the
lambda phage DNA into E coli (catalyzed by lambda integrase enzyme)

91. What is the role of mismatch repair mechanism in gene conversion?

The mismatch repair system recognizes mispaired bases in heteroduplex
(B/b), it excises and replaces one of the strands to restore
complementarity, converts one allele into another, and transfers genetic
information in non-reciprocal manner (non-Mendelian inheritance patterns)

92. Who was Barbara McClintock and what was her major scientific contribution
[no more then two (2) sentences].
Barbara McClintock was a Nobel prize winner for her discovery of mobile genetic
elements. Through her experimentation with corn, she discovered transposable
elements and their role in mutating genes.

93. What are potential effects of transposons on the genome?

Genomes have lots of transposon-leftovers which potentially effect the
genome because:
- Transposons can cause genetic changes (horizontal transfer
contributions to the evolution of genomes) by inserting into genes/coding
sequences, inserting into regulatory sequences (which induce change in
gene expression)
- Transposons can also form chromosomal rearrangements and relocate
genes

LECTURE 11

LECTURE 12
94. Define: repressor, co-repressor, and aporepressor
Repressor: regulatory proteins that block RNA poly
Aporepressors: a regulatory protein, that when combined with
corepressor, undergo allosteric transformation, combines with operator and
inhibit transcript
Co-repressors: activate aporepressors (stop transcription)

95. Define effector and inducer.

 Effector: small molecules that cause changes in repressors conformation by
binding to it. Two types;
Inducer: inactivate repressor -> allow transcription (induce it) (lac)
Corepressor: activate aporepressor, which binds to the operator and stops
transcription (trp)

96. Discuss why are lac Oc mutants cis-acting.

Oc mutations only affect expression of the genes to which they are physically
attached i.e. the genes at the same DNA (chromosome)
Since O is a specific DNA sequence (cis elements), Oc mutations are cis
acting too as mutations only occur at the operator site/sequence

97. Discuss why are lac I- mutants trans-acting

Lac I mutation is constitutive meaning that the repressor cannot bind to
the operator (but) normal B gal is always synthesized. The mutants are
trans-acting because the repair of this constitutive mechanism occurs by
introducing a plasmid that is carrying a normal Lac I gene (which must carry a
mutated Z gene in order to work). The plasmids lacI gene codes for a
diffusible element that acts in trans by binding to any operator, regardless of
chromosomal locations.
I mutations affect the expression of genes at both chromosome copies in
diploid bacteria, because I codes for regulatory factor (repressor protein)
which can diffuse (therefore trans) throughout the cell and binds to operators
regardless of their chromosomal locations

98. What is catabolite repression? What is the role of Catabolite Activator

Protein? Explain its action.
Catabolite repression is the act of the E coli adapting to the provided, best
energy source available first (i.e. instead of catabolizing glucose into lactose
and then allolactose) the cell just uses gluocuse (or whatever other energy
source is provided) to regulate transcription (no need for the catabolic
operons (ones that catabolize into allolactose) to be on)
In general: in lac operon, when glucose is present in high amounts, its
energetically favorable for the cell to just use glucose as the regulator
CAP (catabolite activator protein) is an inactive activator protein that is
activated by cAMP so the CAP-cAMP complex binds to the CAP binding site
(enhancer element).
a. Promoter is readily bound by RNA polymerase because of the
complex, transcription occurs!

99. Give an example of cis-elements and trans-factors from the Trp operon (or
form the Ara-operon).
TRP OPERON ARA OPERON
Cis: structural genes, operator Cis: araO2, ara O1, ara I1 and 2
Trans: repressor protein (apo) and Trans: araC regulator CAP-cAMP
trp (corepressor)
100. Regarding the regulation of Trp operon, what do we call the amino acid
tryptophan? Why?
Trypophan is effector and also called a corepressor because it can inhibit
transcription indirectly through influencing the main (aprorepressor) - trp is
needed in order to activate the repressor proteins (the aporepressor).
i. When trp is low, there is no repression (because repressor protein is
inactive, so transcription is uninterrupted (trp is needed so it has to be
synthesized)
ii. When trp is high, repression occurs (because the corepressor (trp) is
present and activates the aporepressor) so transcription is halted (trp
is not needed anymore because there is enough of it so trp synthesis
(transcription) has to stop)

101. Discuss positive and negative regulation of L-ara operon

L-ara operon can have positive and negative regulation because AraC is a
Activator: when it binds to ara, it changes conformation and along with
CAP-cAMP (also bounded to initiator) binds to ara I 1 and araI2 which is the
initiator region containing both the main operator and promoter site. The
binding of the two complexes to araI leaves the promoter open for RNA poly
Repressor: when ara is absent, it has a different conformation, through
which, it binds to araO2 and araI1 which forms a loop (bent DNA), the
conformational change hides the promoter so the RNA poly cant bind to it
and transcription is prevented

LECTURE 13
102. What is the role of auxiliary operators?
The presence of auxiliary operators (O2 and O3) near the functional
operator (O1) increases the local concentration of the repressor so that it
can occupy the functional O1 (level of repression is greatest when all
three operators are present, which means that transcription is most
suppressed when auxiliary operators are there)
- Repressor binds as a tetramer (which means it binds to two Os
simultaneously to repress the operon by causing DNA loop and
preventing the RNAP to find the promoter)

103. Discuss the type of regulation of gene expression by two-component

regulatory systems in bacteria?
There are two protein in the two component regulatory systems
- sensor-transmitter protein: monitors specific changes in the
environment (i.e. nutrients level, pH, solvent concentration-
osmolarity)
- response-regulator protein: stimulates or represses regulation of
specific gene (changes gene expression necessarily for E coli to
adapt to environmental change)
104. What are activators? What are enhancers?
Activators: increase binding of RNAP to promoter (positive control of
initiation of transcription) by direct contact, which causes conformational
changes in RNAP to promote formation of open complex
Enhancers are binding sites for activators!

105. List different ways of control of prokaryotic transcription initiation and give
one example for each of them (this question could be separated in few
smaller questions; make sure you understand review slides and that you have
at least one example for each of the control ways).
a. Direct contact of RNAP and activator protein
b. Regulator proteins:
- execute positive/negative regulation depending on
presence/absence of effector (difference in conformation = affinity
for different cis elements)
- bound to promoter, but promote DNA bending (promote RNAP
binding) depending on presence of effector
c. DNA bending: because of binding of trans factor, negative or positive
regulation
- Regulatory protein can directly contact RNAP
- Preventing/helping RNAP to interact with RNAP at start site
(promoter)

106. Glutamine and arginine in DNA-binding proteins tend to make what kind of
bonds with DNA?
DNA-binding proteins recognize base pairs as hydrogen bond acceptors
(A) because they have O or N ions or as hydrogen bond donors (D)
because the hydrogen is bounded to the acceptor
So glutamine and arginine make hydrogen bonds with DNA (in
major and minor grooves) because they are acceptors of H bonds
(Theres usually only about 10-20 contact points because only specific H
bond donors and acceptors on protein and DNA complement each other)

107. Describe the most common structural motif found in a DNA binding
domain of prokaryotic regulatory proteins.
The most common motif in DNA binding domain is helix-turn-helix motif,
which is about 20 amino acids long, it has 2 short alpha helices (7-9 amino
acids long) connected with a short turn
The DNA recognition helix makes most of the contact with DNA and
the other stabilizes the interaction, so the recognition and the stabilizing
helix form almost a 90 angle)
The recognition helix interacts with one face of DNA, and the N-
terminal of the other helix wrap around the other face to promote stability

108. Apart from the DNA binding domain, we have mentioned other two
domains found in prokaryotic binding proteins. What are they?
 i. Effector binding domain: binding of repressor is affected by
small effector molecules that can be inducers or co-repressors,
this results in allosteric conformational change
ii. Oligomerization (dimerization) domain

109. What are the major differences between prokaryotic and eukaryotic
transcription?
- Transcription and translation occur in separate compartments:
eukaryotes have a nucleus to the mRNA has to be transported out of
the nucleus before translation can occur
- Eukaryotic pre-mRNA have to undergo extensive co- and post-
transcriptional modifications (processing)
- Transcription is more tightly regulated in eukaryotes because the
chromatin structure limits accessibility
- Eukaryotic RNAP needs general transcription factors to help
recognize the binding site (promoter)
- Eukaryotes have different cells and tissues (tissue/cell specificity)
- Eukaryotes have three RNA polymerases: different roles

110. Which hypothesis regarding eukaryotic RNAPs was proven with -

amanitin and actinomycin D (be specific)?
Hypothesis: there are 3 distinct RNAPs that have different roles and transcribe 3
different set of genes (the experiments provided definite proof that there are 3
different RNAP with different roles in transcription)
i. Amanitin proved that RNAP II is the most sensitive to amanitin
ii. Actinomycin D proved that RNAP I is the most sensitive to
actinomycin
What were the experiments?
- Amanitin is the product of a specific mushroom, a mouse cell nuclei
was incubated with increasing concentrations of amanitin and the
transcripts were read under electrophoresis
- Actinomycin is a highly toxic antibiotic, which was incubated
similiarly in nuclei, and this intercalated into GC rich regions and
stopped transcription (RNAP I transcribe rRNA, and genetic
information for rRNA is GC rich)

111. Which genes are transcribed by RNAP I? RNAP II? RNAP III?
RNAP I: RNAP II: RNAP III:
rRNA genes (28S Genes for proteins 5S rRNA
& 18S) into mRNA tRNA
NOT 5S! Some snRNA, snRNA
miRNA, snoRNA

LECTURE 14
112. What does CTD stand for? Explain the role of CTD tail in eukaryotic gene
expression?
 CTD: carboxyl terminal domain
It is a stretch of 7 amino acids that are repeated multiple times (in a
consensus repeat)
5 out of the 7 amino acids have OH so this domain is hydrophilic
(water loving) and a phosphorylatable site
Importance: It carries enzymes and factors necessary for each pre-mRNA
processing step (i.e. capping, splicing, and polyadenylation)
The CTD has to be unphosphorylated for RNAP II to bind, but it has
to be phosphorylated for transcription to actually start and proceed

113. What is the role of the TATA box? What happens when TATA box is
removed from the RNAP II promoter?
(IN VITRO: core promoter has to have minimum set of elements for
accurate transcription initiation by RNAP II; core promoter)
The TATA box is a highly conserved sequence, which is 25-35 bp
upstream from start site and it serves similar action as promoter
- The TATA box locates the start of transcription (~30 bp downstream)
- Sometimes important for efficiency of transcription
- TBP (TATA-binding proteins) bind to TATA box and initiate assembly of
other transcription factors and RNA poly)
When TATA box is removed, those genes have to have either initiation (core
element) or GC boxes (upstream elements) to start transcription

114. How would you define enhancers? What are their characteristics? What is
the difference(s) between enhancer and upstream control element?
Enhancers are binding sites for activator proteins (which increase the
binding of RNAP to the start site)

Upstream control elements are regulatory elements that bind regulatory proteins
to influence transcription rate (only sometimes necessary for initiation of
transcription)
The main difference between enhancer and upstream control element is
the distance from the core promoter and activity (i.e. positive or negative effect
on transcription rate)
- Upstream elements are relatively closer to the promoter, some of them
serve as core promoters
- Enhancers are everywhere, their orientation and distance is irrelevant
to function (A negative enhancer is known as silencer)

115. Explain the use of reporter genes for estimation of promoter strength.
Reporter genes usually code for easily assayed gene products (i.e. enzymes) so
the idea is to clone the reporter gene after the promoter of interest (expression of
reporter gene is controlled by the promoter of interest)
Level of expression of measuble gene products is proportional to the
strength of the promoter (the greater the expression the stronger the promoter)
116. Explain briefly 5 deletion series. What kind of information do they reveal?
Starting from the 5 end, you can delete/mutate a part of the promoter and
repeat the experiment of investigating promoter strength
This will give you information about how important the deleted part is to
the strength of the promoter (if gene expression is significantly lower after the
deletion of that part, then that part was very important!
Slight reduction ~ not that important)

117. Explain the tissue (cell type) specificity of eukaryotic cis elements.
The DNA is the same in every cell/tissue and the very same cis elements
(including enhancers) are present in each cell/tissue
However they seem to make different protein products
Tissue specificity is due to presence/absence of particular regulatory
binding proteins (i.e. transcription factors TFS) that are very important at
the level of initiation of transcription.
- TISSUE SPECIFIC REGULATORY PROTEINS DICTATE TISSUE
SPECIFICITY OF CIS ELEMENTS

118. Explain the modular nature of RNAP II promoters.

RNAP II promoters are organized to be initiated on the principle of mix and
match meaning that RNAP II promoters have a modular nature
Variety of elements contribute to promoter functions, but none is essential
or common to all promoters (i.e. individual promoters have different elements,
with different numbers, location, orientation)
- Multiple conditions
- Multiple cis elements
- Different cis elements
- No consistent location of cis elements
i. TATA box: core elements, those that dont have this have initiator sequence
or GC islands
ii. Proximal elements: important for developmental, environmental, and tissue-
specific expression
iii. Same conditions (but control expression of different genes) have the same
cis elements (even though the location differs relative to the start site)
iv. Enhancer: sometimes multiple binding sites for different regulatory proteins
(aka activators) to increase transcription initiation rate
OUTCOME: gene expression induced, increased, decreased, shut-off based
on different conditions because of the modularity of genes promoter
LECTURE 15
119. Distinguish between the function of general transcription factors and
transcription activators in transcriptional regulation.
- General transcription factors (GTFs) bind to core promoter or RNAPII
and position RNAP II at transcription initiation sites, bend DNA
(promote tight binding), prevent nucleosome re-formation
- Transcription activators (TAs) bind to DNA at proximal elements (i.e.
enhancers and silencers) in order to activate/repress (repressor)
transcription (modular proteins: diff domains, diff functions)

120. Distinguish between the function of promoters and enhancers in

transcriptional regulation.
Promoters interact with transcription factors, forming a complex, to recruit
RNAP II at the binding site, they have consensus sequences (TATA box,
initiator elements, etc.) that aid in locating, binding and processing RNAPII
- Must be located in vicinity of start point (+1)
- Contain regulatory elements (one of them is enhancers)
Enhancers are proximal elements (can be anywhere on the DNA) and interact
with transcription activators, that can then interact with co- activators and
regulate transcription (i.e. increase/decrease the rate) by blocking/promoting the
promoter region for RNAPII binding site

121. What are the roles of TFIID in transcription initiation by RNAP II (be as
specific as possible; have to talk about TBP and TAFs)?
TF II D is the largest because it consists of TATA box binding protein (TBP)
and TBP-associated factors (TAFs: different # in diff species).
TWO BASIC ROLES:
- Foundation for the transcriptional complex (TFIID TBP has to be
bound for transcription to start) TBP is the first protein to bind to TATA
box
- Prevention of nucleosome stabilization (= nucleosome destabilization)
in the promoter region (antagonist to H1)

122. What are the roles of TFIIB in transcription initiation by RNAP II? (TFIIB is
pretty important; make sure you know why.)
Once the TBP-TFIID complex binds to TATA box, TFIIB binds to BRE
(TFIIB recognition element; upstream of TATA box)
- Shape: monomeric protein, one end binds to DNA (BRE) and TBP,
other end extends towards startpoint (+1) causes further bending of
DNA downstream from TBP
- Role: the binding of TFIIB is a key rate-limiting step:
o Depends on proximity of certain enhancers & their bound TFs
o Activation domain binds directly to TFIIB
o Important for developmental regulation
- MOST IMPORTANT FUNCTION: since one end of TFIIB is bound at
start site, downstream of TATA box, it is asymmetrically oriented with
 regards to the two DNA strands: so TFIIB orients RNAP on DNA (helps
to position RNAP II on the promoter)

123. What are the roles of TFIIH in transcription initiation by RNAP II?
TFIIH: 9 subunits (promoted/activated by RNAPII bound TFIIF and TFIIE)
- Helicase activity: unwinds DNA downstream from initiator site in
presence of ATP (open complex)
- IMPORTANCE: necessary for promoter clearance, it has protein kinase
activity (phosphorylation of CTD tail of RNAPII)
- Once the CTD is phosphorylated, it detaches RNAPII from TFIID,
which begins transcription

124. When did we mention TFIIH before and how is the previous topic related
to regulation of transcription initiation?
We mentioned TFIIH before during DNA replication: as one component of
TFIIH is the cyclin-activating kinase subcomplex (CDK) which provides
helicase and kinase activity.
The two topic are related because both DNA replication and
transcription need unwound DNA so the same enzyme serves the
same function for the different operations
(Recall: transcription and replication are two different processes that occur
at almost separate times: some level of transcription can be always
expected in the cell, but during S phase (DNA replication) or mitosis
(where the chromosome) is highly condensed, there are lower chances of
transcription occurring)

125. What are TATA-less promoters? How could transcription be initiated at

TATA-less promoters?
TATA less promoters are those that dont have TATA box (~60-70%).
TBP has to be placed at the right spot in respect to the existing core
(initiator) element and transcription start site
i. Initiator element: in order to bind TBP to the promoter containing initiator
element, the whole TF II D can bind through TAFII-250 and TAFII-150 or
interactions between TAFII-250 and TAFII-150 tether TBP to intiator,
waiting for the rest of the TF II D to bind
ii. CG boxes: whole TF II D binds through TAFII-110 and Sp1 or Sp1 proteins
bind to GC and interact with TAFII-110 and (TAFII-250 and TAFII-150: that
anchor TBP), waiting for the rest of the TF II D to bind
Once TF II D binding is complete, the events are the same as in TATA promoters

126. List and briefly explain four major domains in eukaryotic transcription
factors
Transcription factors are modular proteins: diff domains & diff functions
(Domains are tertiary structure of large proteins in distinct regions which have
different functions)
 (Motifs are specific combinations of secondary structures which are organized
into specific 3D structures inside the domains: responsible for the actual
functioning of the protein)
a. DNA-binding domains: interact with specific DNA seqeuences
b. Transcription activation domain: interacts with other proteins to
stimulate transcription from a nearby promoter
c. Dimerization domains (homo or hetero dimers)
d. Ligand-binding domains: binding of accessory small molecule to
regulate transcription factor activity

127. List most frequent structural motifs in eukaryotic DNA binding domains.
i. Zinc finger: proteins with regions around a zinc ion, 3 types:
C2H2 zinc finger: zinc is held between a pair of B (beta) sheets,
bounded by 2 cys and 2 his residues (alpha helix inserts into major
groove of DNA)
C4 zinc fingers: two groups of 4 cys residues that bind to two zinc ions
(two fingers) one for DNA binding and one for dimerization
C6 zinc-finger yeast: 6 cys residues bind to 2 Zn ions into globular
domain + small recognition helix that gets into major groove (a finger
isnt formed) but forms dimers - alpha helix
ii. (one alpha helix in:) Homeodomain proteins: homeotic genes, when
altered, transform one body part for another (i.e. legs in place of antenna)
so they are important in controlling development, very similar to helix-turn-
helix
iii. (basic alpha helix) Leucine zipper proteins: contain hydrophobic AA leucine
(every 7th position of C-terminal region, i.e. every 2nd turn) this region forms
alpha helix, the N-terminal helix grip DNA interacting with 2 major grooves
iv. (basic alpha helix) helix-loop-helix: similar to zipper, hydrophobic residues
on C-terminal which make alpha helix (only diff: non-helical loop forms that
separates two alpha helices, second helix that is responsible for DNA
binding: not together anymore)

128. Describe how steroid hormones regulate transcription (one example is

enough; remember: nuclear receptor could be in cytoplasm or in nucleus,
already bound to DNA)
Steroid hormones regulate transcription
Transcription factors have DNA-binding domains, which have different motifs,
one of which is Zinc fingers which have C4 factors
- Steroid receptors (or just nuclear receptors in general) have these C4
factors (so they can also be transcription factors: in that case, they
regulate transcription just as transcription factors do
Homodimers: two same receptors/hormones, heterodimers: two different
homones are influencing transcription
There are 2 main classes of nuclear receptors:
1. The hormones are activated outside of the nucleus and then they get
transported in to bind to the DNA
 2. Hormones are attached to the DNA (in the nucleus) and then the ligand
comes in (with the receptor) to activate them as transcription factors

129. List three classes of transcription activation domains in eukaryotic

transcription factors.
Acidic, glutamine rich, and proline rich

130. What is achieved by the ability of some transcription factors to form

heterodimers (keep in mind: there are two things/players in this type
of regulation)?
Heterodimers are formed through a coiled structure maintained by
hydrophobic forces: can alter DNA binding specificity of TF: it provides
COMBINATORIAL CONTROL: control by a combination of diff proteins

131. What is meant by the independence of the DNA-binding and transcription-

activating domain of a transcription factor?
Experiments show that activating domain and DNA binding domains are
separate independent molecules:
Lex A (repressor: binds to lexA repression of downstream genes)
Gal4 (activator: binds to upstream activating sequences)
DNA binding domain from repressor, and activation domain from activator

Activation domain is important only for activation, DNA binding domain is

only for binding: they are independent!

132. If you know the binding site for certain transcription factor
(TF), outline experiments you would use to purify this TF and to assay its
activity
The first step is to isolate nuclear proteins (including TFs)
i. Ion exchange chromatography: increasing salt-concentration proteins which
are bound with different affinity (the highest salt concentration will result in
the highly purified TF)
ii. DNA affinity chromatography: beads with attached DNA sequences from
promoter regions (collect fractions)
iii. Specific DNA-affinity chromatography: only specific TF purified

LECTURE 16
133. What is combinatorial control of transcription?
Combinatorial control: gene activity occurs only when the correct
combination of proteins are present (these can happen in
heterodimerization, formation of enhancesomes, etc.)
- Transcription is controlled by a combination of diff proteins interacting

134. Describe an influence of activators and repressors on assembly of

initiation complexes.
 Activators: multiple activators can bind to the multiple binding sites on
enhancers, and interact because cooperative binding creates an
enhanceosome (large nucleoprotein complex: DNA bending/loop)
- Regulates transcription: binding of certain proteins enables assembly
of other proteins (combinatorial control: gene activity occurs only when
the correct combination of these proteins is present)
EG: cAMP response element binding protein (CREB) (the binding site for
this is cAMP response element (CRE)). CREB binds to CRE and then
binds to CBP (CREB-binding protein) which interacts with basal complex:
in this way, the activators influence the promoter regardless of how far
they are because the enhanceosomes are large enough to have DNA loop
or bending!
Repressors: inhibit gene activity by binding to site that overlaps activator
binding site (i.e. silencers: adjacent to activator site),
- It binds upstream to activator sites and interact through mediator with
GTFs to inhibit initiation

135. Describe the role of histone acetylation/deacetylation in regulation of

transcription.
Histones N-terminal tail contain +ve lysine groups that make a tight bond
with DNA so acetylation and deacetylation affects chromatin condensation
- Acetylated +ve charge is neutralized, eliminates interactions with DNA
(remember that DNA is a negatively charged sugar-phosphate
backbone) so chromatin is less condensed, forms a euchromatin
structure (promoter regions are more available (accessible))
- Deacetylation: repression of gene activity (lowered level of
transcription), deacetylases present in co-repressor complexes
- Acetylation: activation of gene activity (increased level of transcription),
catalyzed by histone acetyltransferases (HAT), co-activators have HAT

136. Describe the role of chromatin remodelling complexes in regulation of

transcription.
Histone acetylation (loosening of chromatin structure) is important but not
sufficient for activation because nucleosomes are still in place: so they
have to be repositioned to expose promoter elements: aka chromatin
remodeling protein complexes!
- Swi/Snf, RSC remodeling complexes: nucleosomes move through
DNA translocation (ATP required) and enhancers/promoters get shifted
in nucleosome free regions (these complexes cant bind to specific
DNA sequences so they are recruited by activators/repressors

137. Explain the role of enhancesomes and architectural proteins in regulation

of transcription initiation?
Enhanceosomes: large nucleoproteins
 IMPORTANCE: combinatorial control of gene activity (for specific gene
expression you have to have combination of tissue specific TFs, non-
specific TFs (& co-activators) with GTFs)
- EG: what am I hungry for?
Who did I take with me? (If with BF, youll rethink onion rings)
How much money do I have?
^ In a cell this is combinatory control of gene expression (the correct
combination of different activator proteins (or repressor) form an enhanceosome
which permits or inhibit transcription

Architectural proteins: the purpose of TFs responsible for architectural

changes is:
- Bind to specific binding sites to change DNA shape in control region
(so that other proteins can interact with each other and/or GTFs to
stimulate transcription)
- THE BENDING/LOOPING of DNA is important so that the distance
between core promoter and enhancer are reduced (but if the distance
is already lowered to begin with, then bending/looping can require
more than one architectural TF because its hard)

138. Explain the role of mediators and insulators in regulation of transcription

initiation.
Mediators: proteins that are necessary for assembly of pre-initiation
complex (PIC) because it communicates directly with gene-specific
TFs/GTFs
- Form complexes to control transcription by mediating signals between
DNA-binding TFs and core transcriptional machinery (the conformation
of the mediator complex influences the pace of RNAP re-entry (for the
next round of initiation)
Steps: TFs bind to mediator, mediator forms stable complex with
GTF/RNAP II, mediator forms scaffold around which PIC forms
(molecular bridge: allows communication between TFs and RNAP II)
Insulators: set up boundaries between DNA domains to prevent
activation/repression of genes by close but unrelated activators/repressors
(because enhancers can be anywhere so how do you stop an enhancer from
interacting with a close-by promoter or a promoter on a diff chromosome?)
- Also: prevent gene silencing control boundaries between euchromatin
and heterochromatin (by controlling the chromatin modifications)

139. What is (are) the role(s) of transcription factors during development?

Cell differentiation is a critical point in development: transcriptional control
of genes occurs during development (but these genes are transcription
factors themselves) so you are controlling transcription of transcription
factors (time and space)
- Diff concentrations of transcription factors cause diff gene expression
 ROLE: - respond to signals from other cells (transcription of many TF is
under regulation by extracellular signals: i.e. hormones)
- Other extracellular signals (i.e. growth factors)
- Phosphorylation of serine or threonine regulates activity of some TF
(can enhance/decrease DNA binding ability)

140. What is the role of DNA methylation in regulation of transcription initiation?

Recall: some promoter regions are CG rich (and methylation occurs to C): its
when DNA sequence is not changed but gene expression is changed
because methylated CG sequence will have diff properties which influence diff
regulatory protein binding to DNA (methylated => inactive chromatin
(heterochromatin)
(THE ABSENCE OF METHYLATION IS NECESSARY (BUT NOT
SUFFICIENT) FOR TRANSCRIPTION TO OCCUR)
- Methylation can bring in more histone enzymes (like deacetylase) and
the chromosome gets condensed (prevent transcription)
Recall: deaceylation is when the +ve histone charge is strong enough to
bind strongly to DNA (condensing DNA structure)
- In development, methylation can turn genes on/off
- Cancer: hypomethylation (transcription activated like crazy),
hypermethylation (transcription repressed)
PROMOTERS ARE PROTECTED FROM BEING METHYLATED (thats
why you have CG islands)

LECTURE 17
141. What is meant by polycistronic mRNA? Give an example.
It is a mRNA that encodes several proteins (characteristics of
bacterial/chloroplast mRNAs)
- Consists of leader sequence which precedes first gene, then another
gene, and another
Leader, region 1, region 2, region 3, region 4, structural genes
TRP OPERON SEQUENCE!

142. What is unusual about type 1 and 2 promoters for RNAPIII polymerase?
These promoters are internal promoters:
Type 1: transcribes 5S rRNA and type 2 transcribe tRNA genes
- Efficiency of transcription is altered by changes in region upstream
from the start point
- TFs dont bind to the promoter but rather to cis-elements (box A and
box B) located downstream of the promoter, which then recruits the
only main initiator factor needed: TFIIIB (position at internal promoter)

143. What is the role of Sp1 protein? What is the role of SL1 protein? What do
they have in common?
Sp1 protein: bind to GC (in TATA-less promoters) and interact with TAFs to
recruit TBP/TFIID and initiate transcription
 Sl1 protein: a protein complex mainly responsible for ensuring RNAP is
properly positioned at start point
Common: they both interact with TBP and TAFs (TBP-associated factors: but
different TAFs 110,250,110 vs TAFs 110,41,48,63)

144. Describe the role of TBP during transcription (think about promoters for all
three eukaryotic RNAPs and TATA-less RNAPII promoters how do RNAPs
bind to them; also, think about coordination of activities of all three
polymerases)
TBP is the component of the positioning factor (no matter which RNAP is
transcribing -> it allows each type of polymerase to bind to its promoter)
RNAP I (bind to TBP) using SL1 protein complex
RNAP II (bind to TBP) using TFIID
RNAP III (bind to TBP) using TFIIB
TATA-less promoters (bind to TBP) anchored by TAFs (Sp1 proteins, etc.)
- Coordinate activities of all 3 polymerases: through binding to other
polymerase specific factors;
o Eg: Dr1 protein is activated when rRNA has to be synthesized
(TBP associated with TFII/TFIII is pulled off by Dr1 and
engaged in RNAP I activities)
o Dr1 binds to the TBP that was previously with RNAP II/III and
makes it interact with RNAP I

145. Describe rho-dependant transcription termination

Rho = hexameric RNA helicase (has RNA-binding domain (N-term) and
ATPase (C-term) activities)
- Binds to rut site (rho utilization site) on mRNA (no affinity for RNAP)
- Tracks along RNA until it catches up to RNAP (RNAP is usually paused
at termination site: forming a mini hairpin structure at the site to slow
itself down)
- Rho catches up to RNAP, unwinds DNA-RNA hybrid (ATPase) and
mRNA, rho, RNAP are all released

146. Describe rho-independent transcription termination

Rho-independent termination sites are different because they have:
- GC rich self-complementary region (this region forms a stem loop
structure: recall stem loops are much stronger than hairpins)
- Series of U residues
(BOTH: stem loop formation sequences and U stretch are important)
RNAP transcribes inverted repeat sequences (the stem loop GC rich formation
sequence) and continues along the DNA, until it transcribe polyU region (the U
tail forms very weak bonds with the template)
- the inverted repeats form a hair pin loop which interacts with the
surface of RNAP causing it to pause: (complementary) A regions (on
DNA: recall RNA is single stranded)
 - The poly U tail and A region association is the only thing holding the
RNA transcript and DNA together, so when those very weak bonds
break, the RNA is released
- RNA chain is released, DNA strands re-anneal, RNAP dissociates
Conclusion: inverted repeats stem loop followed by string of U

147. Describe the mechanism by which the leader-attenuator region fine

tunes the extent of transcription of the structural genes in the trp operon
when Trp is available (but not to the point to activate the repressor).
When trp is high only leader sequence (which out of the 14 amino acid
polypeptide only has two trps) is transcribed
When trp is low; whole RNA gets transcribed, attenuator sequence (DNA
sequence where RNAP terminates/continues transcription) overlooked
Fune tuning: balance between leader and total mRNA synthesis is
in accordance with the need for trp

148. Describe the mechanism of attenuation of the Trp operon. Explain the
importance of this mechanism for a bacterium?
Attenuation depends on secondary structure (i.e. stem loop of
polycistronic mRNA), the formation of this stem loop depends on rate of
translation, the rate of translation of leader sequence depends on supply
of trp (tRNA with trp) available, the supply of trp available depends on the
amount of trp made/present

RNAP transcribes genes to produce nascent mRNA very quickly

- Attenuator sequence: the sequence at which RNAP prematurely
terminates transcription when there is enough trp
- Ribosome initiate translation at start codon near 5 end (shine dalgarno
sequence)

149. Could you imagine a mechanism similar to the mechanism of attenuation

of the Trp operon in Eukaryotes? Explain your reasoning.
Attenuation cant happen in eukaryotes because transcription and
translation arent coupled so its not like trp is being made as trp genes are
being translated at the same compartment

150. Describe two distinctly different ways in which the trp operon is controlled
by the overall availability of tryptophan.
WHEN TRP IS HIGH:
- A stem loop forms at regions 3 and 4 (closest to RNAP) which causes
RNAP to be pulled back and terminate
WHEN TRP IS LOW:
- Stem loop forms at regions 2 and 3
- Ribosome gets stuck at the end of region 1 (which has trp codons) so it
is demanding tRNA with trp codons to come in
- The ribosome has to wait because its stuck (the stem loop)
 - RNAP continues transcribing which increases trp and then 3 and 4
base pair when trp is high enough to termination transcription
Attenuation is importance in bacteria because it is a control mechanism
(controlling the amount of trp present in the cell by delaying or activating
transcription of trp genes, to allow for translation to occur, more production of
trp, etc.)

151. Describe the mechanism responsible for shutdown of the trp operon when
a plentiful supply of free tryptophan is available.
RNAP transcribes genes, produces mRNA, translation at start codon (near
5 end) begins
- Regions 1 and 2 are translated
- Regions 3 and 4 form a stem loop (at the attenuator sequence)
- RNAP terminates transcription before reaching trp structural genes

LECTURE 18
152. What are the general steps in processing of a pre-mRNA into a mRNA?
5 capping (CTD tail), RNA splicing (CTD tail) and polyadenylation (CTD tail)

153. What is the relationship between hnRNA and mRNA?

hnRNA collection of prods. made by RNAPII (heterogeneous nuclear RNA)
mRNA final mature RNA (transcripts that are going to be processed into
mRNA are called primary transcripts (pre mRNA)
snRNAs small nuclear RNAs transcribed by RNAP II as well
hnRNA = pre-mRNA + snRNAs

154. How is 5 cap added to the nascent RNA?

Binding through 5 5 triphosphate bridge
- Phosphoryated CTD recruits capping enzymes (enzymes bind to
phosphorylated CTD direcly) (remember phosphorylated CTD means that
transcription is occurring)
i. RNA 5 triphosphatase (RTP) removes 5 phosphate
ii. Guanylyl transferase (GT) attaches GMP
iii. 7-methyltransfase (MT) modifies guanosine terminal (7-met-
guanosine coupled to 5 end)

155. List the roles of 5 methyl cap. List the roles of polyA tail. List the roles of
CTD tail (yes, again).
- 5 methyl cap: protection from degradation, also important in splicing,
nuclear transport and translation (for recognition of mRNA transcript by
ribosome small subunit)
- polyA tail: helps stabilize binding site for cytoplasmic polyA binding protein
I (PAB, or PABP I) to resist 3 exonuclease action and increases
translation efficiency by rapidly recycling ribosomal subunits (function of
PAB 1) and circulating eukaryotic mRNAs
156. What are the roles of PABI? What is the role of PABII?
PABI is cytoplasmic poly(A) binding protein I
- increases translation efficiency by rapid recycling of ribosomal subunits
PABII is nuclear poly(A) binding protein II
- binds to short poly(A) tail, increases affinity of polyadenylate
polymerase for RNA (speeds up polyadenylation)

157. Describe the process of mRNA cleavage and polyadenylation. (Dont

forget the role of CTD tail)
- mRNA cleavage: involved in polyadenylation, GT rich region in DNA is
transcribed into GU rich region in pre-mRNA which occurs after the
AAUAA (polyadenylation consensus sequence) and the CA (the CA is the
site for cleavage and polyadenylation addition site)
o GU rich sequence is cleaved off (important role in splicing)
- polyadenylation: consensus sequence (AAUAAA or AUUAAA) followed by
YA (y = pyrimidine, mostly C) nucleotides, GU rich sequence after CA is
cleaved off (cleavage site) and poly A tail is added (addition site)

GENERAL PROCESS FOR CLEAVAGE AND ADDITION:

- CPSF (cleavage & polyadenylation specificity factor) binds to consensus
- CStF (cleavage stimulatory factor) binds to GU rich region, bends mRNA
- CFI & II (cleavage factor 1 & 2) cleave off mRNA
- PAP (polyadenylation polymerase) binds to mRNA

ROLE OF CTD TAIL: recruit enzymes necessary for polymerization

158. What is the difference between splicing of group I and group II introns?
Between splicing of group II introns and spliceosomal splicing?
Group 1 and 2 introns: RNA is splicing itself (self-splicing introns) no
proteins are involved because RNA is riboenzyme
- group 2 intons are removed as lariat structure (splicing catalzyed by intron
riboenyzme complex to one intron-encoded protein (IEP, e.g maturases)
which increase rate of self-splicing by stabilizing 3D lariat conformation
Spliceosomal splicing; pre-mRNA requires snRNAs and associated
proteins (snRNPS)

General mechanism: 5 PO4 (intron) phosphate-ester bonds with 3OH (extron)...

25 branch formed with first nucleotide of intron (LARIAT) (lec 18, side 25)

159. What is the role of snRNAs in the spliseosome?

Small nuclear RNAs (snRNAs) (5 of them) assist in splicing:
- they are rich in U
- they interact with proteins, mRNA, and with each other because of their
complementarities between their sequences
160. What is the role of Sm proteins in the spliceosome? What other proteins
(apart from Sm) are found to be associated with splicing?
Sm proteins bind to Sm RNA motif in snRNAs (they interact through beta
strands (form circle around RNA)) (U1, U2, U4, U5, U6)
- Sm and other snRNA associated (specific) proteins and splicing factors
form snRNPs (which is a complex involved in splicing)
- Other proteins are tissue/condition specific factors required for splicing
(associated with snRNA and Sm protein) to form a snRNP complex
Role of CTD tali: proteins related to phosphorylated tail aid in:
- splice site recognition and binding to exons (of snRNP)
- branch site recognition
- spliceosomal protein-protein interactions
- RNA/RNA interaction or RNA/protein
- conformational changes during spliceosome assembly/catalysis
- preventing of faulty pre-mRNA folding (sec/tert structure)

161. Describe the current model of spliceosomal splicing.

- U1 attaches to 5 splice site of intron (complementarity between
sequences)
- U2 binds to brand point A (bc complementarity: ATP reqd)
- U4/U6 (base pair with each other) and bind to U5 (tri-complex) which
binds to 3 intron splice site (complementarity)
- U4/U6 change conformation because of the binding, assembly of
spliceosome (B1 complex completed)
- U6 dissocates, shifts U1 from 5 splice site and takes its place (U1 & U4
released)
- spliceosome activated (U6/U2 (active site), U5) (B2 complex)
- U5 (positions and holds) U2/U6 (catayltic activity (mediates splicing at
both sites very fast: formation of C1/C2 complexes)
- second splicing step (ATP reqd)
- Lariet degraded (splicing is irreversible)
- snRNPS most likely released as individual particles and recycled

LECTURE 19
162. What are the two types of transcriptional units in Eukaryotes? Use
diagrams.
Simple (constitutive splicing) Complex (alternative splicing)
transcription unit
163. List three means of control of gene expression that could happen during
pre-mRNA processing
Regulation of splicing, and cleavage/polyadenylation (through using
alternative polyadenylation elements alternative processing)

164. Describe one case of control of gene expression at the level of mRNA
processing by means of splicing

165. What is trans-splicing? Give an example.

mRNAs are made by splicing separate RNA molecules (spliceosome) joins
exon of one gene with exon of another gene
- enables existance of monocistronic mRNA from polycistronic pre-mRNA

EG: in trypanosoma:
- many large leader sequences, each one is transcribed and spliced (into a
mini leader sequence: mini exon)
- these mini exons splice with 5 end exons of all protein coding transcripts
in the polycistronic mRNA, this act trigs polyadenylation of 3 end

166. Explain the connection between pre m-RNA splicing and transport of
mRNA from the nucleus.
Cells have a system that prevents the exit of unspliced (pre mRNA)
- FIRST PROOF: viral expression (first phase in HIV infection) was low, bc
only a few viral mRNAs were fully spliced (host machinery used) and were
exported to the cytoplasm and translated into viral proteins
- Rev mRNA protein is made (outside cytoplasm), rev protein goes back
into the nucleus, binds to specific sequences in viral mRNA, help
unspliced viral mRNAs to get to the cytoplasm through interactions with
nuclear export protein (i.e. Rev binds to intron sites, so that once rev
releases the RNA in the cytoplasm, it takes the introns away)
- transported unspliced viral mRNAs are packed into new viral particles

167. List and briefly explain different mechanisms of post-transcriptional control

of gene expression (think about examples)?
Localization of mRNA
RNA editing
post-transcriptional silencing by siRNA or miRNA
translational control switch
RNA stability (degradation and stabilization)

LECTURE 20
168. What are the roles of three major RNAs in protein synthesis?
mRNA: carries genetic information from DNA in the form of codons
tRNA: translates mRNA code by correlating the carried AA to the mRNA by
codon-anticodon binding specificity
 rRNA: associates proteins to form ribosomes, which catalyzes the
assembly of protein chains (site of protein synthesis)

169. Explain the role of mRNA stability (or editing, or iRNA, or translational
control switch or mRNA localization five different questions possible) in
control of gene expression.
mRNA: The balance between mRNA degradation and synthesis
determines the level of individual mRNA in cells
Instability elements: in the non-translated 3 region there are
elements that call for rapid degradation (although it can happen from both
3 or 5 based on whether these elements are)
Instability elements: AUUUA (AU rich element: ARE) in the 3 UTR;
therefore degradation rate is influenced by interacting with these specific
ARE regions through binding of ARE-binding proteins (stability and
degradation require different ARE-BP)

RNA editing: any process (not splicing) that changes the sequence of
mRNA from the original DNA template
Extreme: many nucleotides, mitochondria (U residues)
- Guide RNAs (U residues: small RNAs that complement the sequence to
be edited those cell who know know who) they pair with the unedited
mRNA (the sequences that are not paired/mismatched are the ones to be
edited (easy to recognize this way)
- It doesnt always have to be that the pairs will stick out or mismatch in
the original mRNA or the gRNA (guide RNA): changes the amount of
nucleotides inserted/deleted
(If it sticks out in the gRNA, then the sequence is added to the
RNA, if it sticks out on mRNA, then the sequence is deleted on
mRNA)

Specific: single nucleotide, mammals (A to I) (C to U)

- A to U is more common (happens to unspliced RNA), I is recognized as G
so it changes protein sequence. C to U: stop codon mutation (nonsense)

Role depends on where it happens:

i. 5UTR: structural change (translation efficiency), introduce or remove
(extend or shorten N terminal) AUG
j. intron: new splice site (i.e. some of the intron becomes exon, intron
structure (which influences splice site), remove splice sites
k. coding region: point mutation, premature stop codon
l. 3UTR: mRNA stability (ARE), mRNA trafficking by mutating ZIP-code

mRNA localization: positioning (transporting) mRNA close to sites where the

protein that they synthesis is required, efficiency increased in protein
diffusion/distribution
 - Important for: synaptic transmission (learning/memory), asymmetric cell
division, cell motility, axis formation during development
- TRANSPORT: elements recognize different mRNAs as transportation
targets to be localized (elements mapped within 3UTR), specific RBP

iRNA: small ds/ss RNA induce sequence specific gene silencing

- specific protein complexes (DICER) will process (cut) short/long dsRNA
hairpins/intron RNA, miRNA/siRNA incorporated in nuclease (RNA-
induced silencing complex: RISC), which recognizes complementary
mRNA-> stop translation, cleave mRNA, induce degradation
Role: regulatory factor (miRNA)

Translational control switch: through 2nd/3rd structure of 5/3 UTR riboswitch

Coordinate/opposite translational regulation, example:
- Fe (protein: elements in 5 UTR to control translation), transferrin receptor
(responsible for transporting Fe into the cell: has elements in 3 UTR to
control degradation (prevent translation))
Low: receptor synthesized (Fe gets into the cell), high: receptor degraded (Fe
cant get more into the cell)

170. What is the name of the region of tRNA molecule which attaches to an
amino acid? Each tRNA is recognized by the enzyme that corresponds to a
specific amino acid, AA is attached to acceptor stem (3 end) to charge
tRNA, so that it can now recognize codon in mRNA and bring AA to the
growing polypeptide chain
(the part that interacts with the codon in mRNA is called anticodon)

171. How many different tRNAs are there in an eukaryotic cell? How many
different aminoacyl tRNA synthetasis are there in an eukaryotic cell?
# of tRNA 62 (Ecoli) 600 (Drosophila)
molecules: 300 (Yeast) 8000 (xenopus)

172. What are the roles of tRNA in translation?

translates mRNA code, each amino acid has its own tRNA which binds to
mRNA due to codon-anticodon complementarity (function depends on 3D
structure)
- to be linked to a particular amino acid (carries AA)
- to recognize codon in mRNA which corresponds to the AA which it carries

LETURE 21
173. What does S in 16S stand for? What is the numerical value of this
constant?
S measures sedimentation of suspended particles when centrifuged
under constant conditions, dependent on shape and size of particle
1S = 10-13s (16S = 16x10-13)
 - S measures relative size if comparing same type of molecules (the
larger the numerical value, the faster the sedimentation velocity)

174. What is the wobble position for an anticodon? For a codon?

Wobble position: a single tRNA is able to recognize more than one codon
corresponding to an amino acid (non-standard pairing)
Codon (5 to 3) and anticodon (3 to 5)
so 1st nucleotide from codon is recognized by 3 rd nucleotide from
anticodon (vice versa)
- Wobble position: 3rd wobble nucleotide in mRNA by 1st wobble
position in tRNA (Codon: 3rd position, anticodon: 1st position)
U A/G/I, C G/I, A U/I, G C/U (E coli)
U G/I, C G/I, A U (deamination), G C

175. Explain what does it mean when we say that the code is degenerate?
Codons are synonymous?

176. What is the role of Shine-Dalgarno sequence?

Ribosomal binding site (RBS) in E coli is the Shine-Dalgarno sequence
(5 AGGAGGU 3)
- It base pairs with complementary sequence at 3 end of 16S rRNA in
the small 30S subunit (positions ribosome correctly in respect to the
initiation codon)

177. What is the role of Kozak sequence?

40S ribosomal subunit binds initiator tRNA (BYOB), this complex binds to
mRNA and scans until it reaches an appropriate AUG and positions tRNA
there, but the first AUG has to be in the correct sequence context
KOZAK SEQUENCE: provides optimal sequence context (so that
the first/appropriate AUG can be recognized) 5 CCRCCAUGG 3

178. What are the major differences in initiation of translation between

eukaryotes and prokaryotes (remember: first AA and the way mRNA and
tRNA bind to ribosomes)?
Prokaryotic; mRNA binds to 30S subunit through interaction of Shine-
Dalgarno sequence with 16S rRNA, then initiator tRNA binds (anti-codon
base pairing) to the P site (30S initiation complex is formed)

Eukaryotic: 40S ribosomal subunit binds to initiator tRNA, then 40S

subunit-initiator tRNA complex attaches to mRNA by recognizing the 57G
capping, scans for AUG in the Kozak Sequence and binds to the AUG

179. Describe in detail events during initiation of the Eukaryotic (or Prokaryotic)
translation.
Prokaryotic:
 - IF3 binds to free 30S subunit (prevents binding to 50S to happen
before mRNA gets a chance to bind)
- IF1 binds (prevents potential binding of tRNA to A site)
- IF2 (GTPase) complexes with GTP and binds
- mRNA binds to 16S rRNA in 30S subunit because of the Shine-
Dalgarno sequence interaction
- initiator tRNA binds to P site (removes IF1)
- 1F3 is removed, 50S subunit binds, GTP is hydrolyzed because the
bringing and attaching of the large subunit is energy consuming)
- IF2 released, so first 30S initiation complex, then 70S complex forms

Eukaryotic:
- Free 40S subunit complexes with EIF3 (large protien) and EIF1A (to
keep the 60S (large) subunit from binding)
- ternary complex forms separately, initiator tRNA, EIF2/GTP (this
complex can be regulated by E1F2B for rate of recyling) bind to 40
(43S pre-initiation complex)
- E1F4 is busy trying to find mRNA, which binds to 43S complex through
5 methyl cap (Role: prevent secondary structure of mRNA and
prevents degradation/damage)
- 43S complex scans mRNA to find right AUG (inside Kozak sequence),
the anticodon binds to the Met codon when it falls in the A site
- E1F5: binds/removes all other factors when AUG is found/bounded to
- 40S initiation complex; 60S subunit binds (80S initiation complex)

180. How is gene expression controlled at the level of translation (mRNA

is now in contact with ribosomes or their subunits/translation factors; three
things mentioned in the class)?
- Phosphorylation of translation initiation factors
- Upstream AUG codons
- IRES

LECTURE 22

LECTURE 23