How to Study DNA

1. Genetic material 2. Expression product

What is gene expression?

The activation of a gene that results in a protein. Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product.
A gene is expressed when its biological product is present and active. Gene expression is regulated at multiple levels.

Expression of Genetic Information

Production of proteins requires two steps:
Transcription involves an enzyme (RNA polymerase) making an RNA copy of part of one DNA strand. There are four main classes of RNA:
i. Messenger RNAs (mRNA), which specify the amino acid

sequence of a protein by using codons of the genetic code. ii. Transfer RNAs (tRNA). iii. Ribosomal RNAs (rRNA). iv. Small nuclear RNAs (snRNA), found only in eukaryotes.

Translation converts the information in mRNA into the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins.

Expression of Genetic Information

Only some of the genes in a cell are active at any given time, and activity also varies by tissue type and developmental stage. Regulation of gene expression is not completely understood, but it has been shown to involve an array of controlling signals.
a. Jacob and Monod (1961) proposed the operon model to explain prokaryotic gene regulation, showing that a genetic switch is used to control production of the enzymes needed to metabolize lactose. Similar systems control many genes in bacteria and their viruses. b. Genetic switches used in eukaryotes are different and more complex, with much remaining to be learned about their function.

Steps of gene expression

Transcription
DNA is read to make a mRNA in the nucleus of our cells

Translation

Reading the mRNA to make a protein in the cytoplasm

Three (3) regulatory elements of transcription

Structural genes: DNA that code for a specific polypeptide (protein) Promoter : DNA segment that recognizes RNA polymerase Operator : Element that serves as a binding site for an inhibitor protein (modulator) that controls transcription

Promoter Region on DNA

Upstream from transcription start site Initial binding site of RNA polymerase and initiation factors (IFs) Promoter recognition: a prerequisite for initiation

Prokaryotic promoter regions

-35 site = TTGACA

-10 site: TATA box

7

Promoter Region on DNA

(TATA box)

Pol II Eukaryotic Promoter Elements

Exon Intron Exon

GC box ~200 bp

CCAAT box ~100 bp

TATA box ~30 bp

Gene

Transcription start site (TSS)

Pol II Eukaryotic Promoter Elements

Cap Region/Signal
n C A G T n G

TATA box (~ 25 bp upstream)

T A T A A A n G C C C

CCAAT box (~100 bp upstream)

T A G C C A A T G

GC box (~200 bp upstream)

A T A G G C G nGA

General modulators of transcription

Modulators:
(1) specificity factors, (2) repressors, (3) activators

1. Specificity factors:
Alter the specificity of RNA polymerase

s70

s32

Standard promoter

Housekeeping gene

Heat shock promoter

Heat shock gene

Modulators of transcription
2. Repressors:
mediate negative gene regulation may impede access of RNA polymerase to the promoter actively block transcription bind to specific operator sequences (repressor binding sites) Repressor binding is modulated by specific effectors

Effector (e.g. endproduct) Operator Promoter

Repressor Coding sequence

Negative regulation
Repressor

Effector Example: lac operon RESULT: Transcription occurs when the gene is derepressed

Negative regulation
Repressor

Effector (= co-repressor) Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism

Modulators of transcription
3. Activators:
mediate positive gene regulation bind to specific regulatory DNA sequences (e.g. enhancers) enhance the RNA polymerase -promoter interaction and actively stimulate transcription common in eukaryotes

Activator

RNA pol.
promoter Coding sequence

Positive regulation
Activator
RNA polymerase

Positive regulation
Activator Effector RNA polymerase

Prokaryotic gene organization

Prokaryotic transcriptional regulatory regions (promoters and operators) lie close to the transcription start site Functionally related genes are frequently located near each other These operons are transcribed into a single mRNA with internal translation initiation sites

Prokaryotic Gene Expression

Expression mainly by controlling transcription

Promoter

Cistron1

Cistron2 CistronN Terminator

RNA Polymerase

Transcription

mRNA 5
1 2

3 Translation
C
N N

N Ribosome, tRNAs, Protein Factors

C N C

2 Polypeptides

Operons
Genes that work together are located together A promoter plus a set of adjacent genes whose gene products function together. They are controlled as a unit They usually contain 2 6 genes (up to 20 genes) These genes are transcribed as a polycistronic transcript. It is relatively common in prokaryotes It is rare in eukaryotes

Operon System

The lactose (lac) operon

Pi I Q3 P Q1 Z Q2 Y A

Contains several elements

lacZ gene = -galactosidase lacY gene = galactosidase permease lacA gene = thiogalactoside transacetylase lacI gene = lac repressor Pi = promoter for the lacI gene P = promoter for lac-operon Q1 = main operator Q2 and Q3 = secondary operator sites (pseudo-operators)

Regulation of the lac operon

Pi

Q3

Q1

Q2

LacZ

LacY

LacA

lacI repressor

Inducer molecules Allolactose:

- natural inducer, degradable IPTG (Isopropylthiogalactoside) - synthetic inducer, not metabolized

The lac operon: model for gene expression

Includes three protein synthesis coding region-sometimes called "genes" as well as region of chromosome that controls transcription of genes Genes for proteins involved in the catabolism or breakdown of lactose When lactose is absent, no transcription of gene since no need for these proteins When lactose is present, transcription of genes takes place so proteins are available to catalyze breakdown of lactose

Eukaryotic gene

Eukaryotic gene Expression

1.Transcripts begin and end beyond the coding region

2.The primary transcript is processed by: 5 capping 3 formation / polyA splicing

3.Mature transcripts are transported to the cytoplasm for translation

Regulation of gene expression

Promoter Gene (red) with an intron (green) Plasmid

1. DNA replication

single copy vs. multicopy plasmids Primary transcript

Mature mRNA

2. Transcription
mRNA degradation 3. Posttranscriptional processing 4. Translation

5. Posttranslational processing

inactive protein active protein

Protein degradation

Regulation of gene expression

Gene expression is regulatednot all genes are constantly active and having their protein produced The regulation or feedback on gene expression is how the cells metabolism is controlled. This regulation can happen in different ways:
1. Transcriptional control (in nucleus):
e.g. chromatin density and transcription factors

2. Posttranscriptional control (nucleus)

e.g. mRNA processing

3. Translational control (cytoplasm)

e.g. Differential ability of mRNA to bind ribosomes

4. Posttranslational control (cytoplasm)

e.g. changes to the protein to make it functional

When regulation of gene expression goes wrongcancer!

Transcription

Eukaryotic gene expression

Gene regulation of the transcription

Condition 1 2
turned turned turned off off on on 4 5 6 7 8

Chr. I

Chr. II
Chr. III
19

10

11 20 21

12 22

13 14 15 16 23 24 25

17 26

18

constitutively expressed gene

induced gene

repressed gene

inducible/ repressible genes

Gene regulation
upregulated gene expression
1 2 3

down regulated gene expression

4 5 6 7 8

Condition 4 3

10 19

11 20 21

12 22

13 14 15 16 23 24 25

17 26

18

constitutively expressed gene

Definitions
Constitutively expressed genes
Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells.

Inducible genes
Genes that are transcribed and translated at higher levels in response to an inducing factor

Repressible genes
Genes whose transcription and translation decreases in response to a repressing signal

Housekeeping genes
genes for enzymes of central metabolic pathways (e.g. TCA cycle) these genes are constitutively expressed the level of gene expression may vary

Post-Transcriptional Modification in Eukaryotes

Primary transcript formed first Then processed (3 steps) to form mature mRNA Then transported to cytoplasm Step 1: 7- methyl-guanosine 5-cap added to 5 end Step 2: introns spliced out; exons link up Step 3: Poly-A tail added to 3 end

mature mRNA 5-cap- exons -3 PolyA tail

Intron Splicing in Eukaryotes

Exons : coding regions Introns : noncoding regions Introns are removed by splicing

GU at 5 end of intron

AG at 3 end of intron

36

Splicesomes Roles in Splicing out Intron

RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes

37

Splicesomes Roles in Splicing out Intron

5 exon then moves to the 3 splice acceptor site where a second cut is made by the spliceosome Exon termini are joined and sealed
1 2

38

Translation
Three parts: 1. Initiation: start codon (AUG) 2. Elongation: 3. Termination: stop codon (UAG)

Translation
Large subunit

P Site

A Site
mRNA

A U G
Small subunit

C U A C U U C G

Initiation
aa1 aa2

2-tRNA 1-tRNA

anticodon
hydrogen bonds

U A C A U G codon

G A U C U A C U U C G A
mRNA

peptide bond
aa3 aa1 aa2

3-tRNA 1-tRNA 2-tRNA

G A A

anticodon
hydrogen bonds

U A C A U G codon

G A U C U A C U U C G A
mRNA

aa1

peptide bond

aa3
aa2

1-tRNA

U A C
(leaves) 2-tRNA

3-tRNA

G A A

A U G

G A U C U A C U U C G A
mRNA

Ribosomes move over one codon

aa1

peptide bonds

aa4
aa2 aa3

4-tRNA
2-tRNA 3-tRNA

G C U

A U G

G A U G A A C U A C U U C G A A C U
mRNA

aa1

peptide bonds aa2 aa3

aa4

2-tRNA

G A U
(leaves) 3-tRNA

4-tRNA

G C U

A U G

G A A C U A C U U C G A A C U
mRNA

Ribosomes move over one codon

aa1

peptide bonds aa2 aa3 aa4

aa5

5-tRNA

U G A
3-tRNA 4-tRNA

G A A G C U G C U A C U U C G A A C U
mRNA

aa1

peptide bonds

aa5

aa2
aa3 aa4

5-tRNA

3-tRNA

U G A
4-tRNA

G A A

G C U G C U A C U U C G A A C U
mRNA

Ribosomes move over one codon

aa4

aa5
aa199
aa200

Termination

aa3 primary structure of a protein aa2 aa1

200-tRNA

terminator or stop codon

A C U
mRNA

C A U G U U U A G

Translation
Ribosome Amino Acids forming Peptide chain

P Site
Met His E Site tRNA anti-codon 5 codon
AUG
UAC

A Site
Val Pro 3

Tyr

CAU
GUA

GGA
CCU

mRNA strand

Translation
The difference
Eukaryotic and prokaryotic translation can react differently to certain antibiotics Puromycin an analog tRNA and a general inhibitor of protein synthesis Cycloheximide only inhibits protein synthesis by eukaryotic ribosomes Chloramphenicol, Tetracycline, Streptomycin inhibit protein synthesis by prokaryotic ribosome

End Product
The end products of protein synthesis is a primary structure of a protein. A sequence of amino acid bonded together by peptide bonds. aa3 aa4 aa5
aa199
aa200

aa2
aa1

Polyribosome
Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides).

incoming large subunit

mRNA

incoming small subunit

polypeptide

Prokaryotes vs eukaryotes: key points

Prokaryotes
Operons (functional grouping) Polycistronic mRNAs (single mRNA, multiple ORFs) No splicing Regulatory sequences lie near (~100 bp) the start site Translation is concurrent with transcription

Eukaryotes
Monocistronic RNAs (One mRNA, one protein) Ribosome scanning Often spliced Regulatory sequences can be far (>1 kb) from the start site RNA processing is concurrent with transcription; translation occurs in a separate compartment

TYPES OF PROTEINS
Enzymes (Helicase) Carrier (Haemoglobine) Immunoglobulin (Antibodies) Hormones (Steroids) Structural (Muscle) Ionic (K+,Na+)

Coupled transcription and translation in bacteria

original base triplet in a DNA strand As DNA is replicated, proofreading enzymes detect the mistake and make a substitution for it:

a base substitution within the triplet (red)

POSSIBLE OUTCOMES:
OR

One DNA molecule carries the original, unmutated sequence

The other DNA molecule carries a gene mutation

VALINE LEUCINE HISTIDINE

PROLINE

THREONINE

VALINE

GLUTAMATE

A summary of transcription and translation in a eukaryotic cell