Lec 6

GENE EXPRESSION:
Proteins and Enzymes
Gene function: proteins and
enzyme synthesis
◼ Function of the gene is to control/influence the
phenotype
◼ Between the gene and the eventual phenotype
are many complex events that make it difficult to
determine exactly how gene control is exercised
◼ Archibald Garrod (1902) – first suggested
specific connection bet genes and enzymes
when he studied the disease alcaptonurea
◼ Alcaptonurea – inborn error of metabolism
◼ Causes arthritis and production of urine that
turns black upon exposure to air
◼ Alcaptonurics- excrete homogentisic acid or
alkapton (intermediate product in a metabolic
pathway)
-unable to metabolize homogentisic acid
oxidase
❑ Garrod showed the relationship bet gene and
enzyme
Metabolites are closest to phenotypes

Genes

mRNAs

Proteins

Metabolites

Phenotypes
Biosynthetic pathway of four major classes of anthocyanins

Biosynthesis of the 4 major classes of anthocyanins (Grotewold, 2006). Names of compounds are indicated and enzymes, in black boxes, are
as follows: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavanone 3’-hydroxylase; F3’,5’H,
flavanone 3’,5’-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX/ANS, leucoanthocyanidin dioxygenase/anthocyanidin synthase.
Grotewold E. 2006, Annu. Rev. Plant-Biol. 57:761-80.
Metabolites provide the petal color phenotypes

Flower displaying the three major types of

pigments and the corresponding structures.

(a) Portulaca (Portulaca grandiflora) flowers

accumulating primarily the betalain pigment,
betanin (R1 = R2 = H).

(b) Marigold (Tagetes patula) flowers

accumulating the carotenoid pigment, lutein.

(c) Petunia (Petunia hybrida) flower

accumulating an anthocyanidin, cyanidin.

Grotewold E. 2006, Annu. Rev. Plant-Biol. 57:761-780

The Central Dogma
◼ Proposed by Francis Crick in 1958
◼ DNA holds the coded hereditary information in
the nucleus
◼ This code is expressed at the ribosome during
protein synthesis (translation) in the cytoplasm
◼ The protein produced by the genetic information
is what is influenced by natural selection
◼ If a protein is modified, it cannot influence the
gene that codes for it
◼ Therefore, there is a one-way flow of
information:
DNA→RNA→Protein
The central dogma of molecular biology is an explanation of
the flow of genetic information within a biological system

Francis Crick (1958, 1970)

“The central dogma of molecular
biology deals with the detailed
residue-by-residue transfer of
sequential information. It states that
such information cannot be
transferred from protein to either
protein or nucleic acid.”

Francis Crick (1970)

One gene –one enzyme
hypothesis
◼ George W. Beadle and Edward L. Tatum –
formulated the one gene-one enzyme
hypothesis (1941)
◼ States that “every gene controls a
particular enzyme and that the ultimate
product of a metabolic process was
affected by a stepwise succession of
enzymes, each produced by a particular
gene”
Ex.Galactosemia

◼ Lacks gal. I-P uridyl transferase ( enzyme

which catalyzes removal of iodine from
iodotyrosine )
◼ Affected individuals have liver
enlargement, galactose in urine and blood
Ex.Galactosemia

Lactase

Lactose Glucose+Galactose
Galactokinase

Galactose -1_phosphate
GA-1-P U tranferase

Glucose-1-Phosphate
Two steps are required in
gene expression
1. Transcription
- the synthesis of mRNA uses the gene on the DNA molecule as a template
-the process of copying a segment of DNA into RNA. The segments
of DNA transcribed into RNA molecules that can encode proteins are
said to produce messenger RNA. Other segments of DNA are copied
into RNA molecules called non-coding RNAs

- happens in the nucleus of eukaryotes

2. Translation
- The synthesis of a polypeptide chain using the genetic code on the
mRNA molecule as its guide

- occurs in the ribosomes

Requirements for transcription
ribonucleic acid (RNA)
1. ribonucleic acid (RNA)

➢ Found all over the cell

(nucleus, mitochondria, chloroplasts, ribosomes and the soluble
part of the cytoplasm)

➢ Types:
Messenger RNA (mRNA) - <5%
Ribosomal RNA (rRNA) - up to 80%
Transfer RNA (tRNA) - about 15%
Structural characteristics of RNA
molecules
◼ Single polynucleotide strand which may be
looped or coiled
◼ composed of ribose sugar
◼ Bases : Adenine, Guanine, Cytosine and
Uracil (instead of Thymine)
 messenger RNA (mRNA)
◼ A long molecule about 1 million Daltons
◼ provides the plan for the polypeptide chain
ribosomal RNA (rRNA)
◼ Coiled
◼ Two subunits:
a long molecule 1 million Daltons
a short molecule 42 000 Daltons
◼ Fairly stable
◼ Found in the ribosomes
◼ Made as subunits in the nucleolus
◼ provides the platform for protein synthesis
Transfer RNA (tRNA)
◼ Short molecule about 25,000
Daltons
◼ Soluble
◼ At least 61 different forms each has
a specific anticodon as part of its
structure
◼ tRNA “translates” the message
on the mRNA into a polypeptide
chain
Transcription: The synthesis of a strand
of mRNA from DNA
◼ Uses an enzyme RNA polymerase
◼ Proceeds in the same direction as replication (5’
to 3’)
◼ Forms a complementary strand of mRNA
◼ It begins at a promotor site which signals the
beginning of the gene and is not much further
down the molecule (about 20 to 30 nucleotides)
◼ After the end of the gene is reached there is a
terminator sequence that tells RNA
polymerase to stop transcribing
Transcription plan
Nucleus

Gene

DNA

Transcription
messenger
RNA
Editing the mRNA
◼ In prokaryotes, the transcribed mRNA goes
straight to the ribosomes in the cytoplasm
◼ In eukaryotes the freshly transcribed mRNA in
the nucleus is about 5000 nucleotides long
◼ When the same mRNA is used for translation at
the ribosome it is only 1000 nucleotides long
◼ The mRNA has been edited
◼ The parts which are kept for gene expression
are called EXONS (exons = expressed)
◼ The parts which are edited out are called
INTRONS.
Translation
◼ Occurs in the ribosomes
◼ The genetic code is brought by the mRNA
molecule
◼ Product is a polypeptide
translation
Translation
What is the genetic code?
◼ The genetic code consists of the sequence of bases
found along the mRNA molecule
◼ There are only four letters to this code (A, G, C and U)
◼ The code needs to be complex enough to represent 20
different amino acids used to build proteins
◼ The genetic code is made of triplets of bases called
codons
Codon and Reading Frame
◼ 4 AA letters → 43 = 64 triplet possibilities
◼ 20 (< 64) known amino acids
◼ Wobbling 3rd base
◼ Redundant → Resistant to mutation
◼ Reading frame: linear sequence of codons in a
gene
◼ Open Reading Frame (ORF), definition varies:
a reading frame that begins with a start codon and
end at a stop codon
 a series of codons in a DNA sequence uninterrupted
by the presence of a stop codon
➔ a potential protein-coding region of DNA sequence
27
Genetic code
Features of the Genetic Code
1. Degenerate - Code contains many synonyms
in that almost all amino acids are represented
by more than one codon
2. The code has a definite structure; synonyms
are not randomly dispersed over the table.
✓ tRNA for an amino acid have more than one
variety
✓ Some tRNAs with a specific anticodon have the
ability to pair with two or more synonymous
codons of a particular amino acid
Features of the Genetic Code
✓ There are three stop or termination
codons, UAA, UAG, and UGA.
Universality of the Genetic Code
➢ Does not mean that DNA base ratios
must be similar in different species for
genes specifying similar proteins.
➢ The fact that the code is degenerate
enables many bases to be changed
by mutation in a sequence of mRNA
➢ This mRNA could still produce the
same amino acid sequence
Universality of the Genetic Code
➢ Exceptions?
✓ Only in mitochondrial protein-
synthesizing system
✓ UGA has the same meaning as UGG
which codes for tryptophan instead of
termination
✓ In mammalian mitochondria, AUA
code for methionine instead of
isoleucine
1.D. Translation (RNA→Protein)

➢ After transcription, the different RNAs enter

the cytoplasm
D.1. Amino acid activation
D.2. Initiation
D.3. Elongation
D.4. Termination
1.D.1. Amino Acid Activation

Amino-acyl synthetase attaches amino acids to their respective

tRNAs.
1.D.2. Initiation

IF1 stimulates dissociation of small and large subunits of

ribosomes
IF3 binds to the 30S subunit to prevent reassociation of
30S and 50S subunits.
1.D.2. Initiation
IF2 with GTP attaches first
aa-tRNA complex to the
30S subunit.
IF1 then attaches to the
30S subunit to complete
initiation complex.
50S subunit then joins the
complex dissociating IF1
and IF3.
Hydrolysis of GTP to
GDP and Pi leads to
dissociation of IF2.
 1.D.3. Elongation
Once fMet is set at the P-
site with EF-Tu and GTP,
a new aa-tRNA complex
enters the A site.
Peptidyl transferase
catalyzes formation of a
peptide bond between
fmet and aa carried by
the tRNA in the A-site.
tRNA in the P-site is
released to cytoplasm to
be recycled.
1.D.3. Elongation
Movement of the
ribosome along the
mRNA in a 5’ to 3’
direction through EF-G
and GTP.
Displacement of aa-
tRNA from A-site to P-
site.
Continues until A site
encounters a
termination/stop codon.
 1.D.4. Termination
➢ When A site encounters any of
the termination or stop codons,
one of the release factors (RFs)
enters the A site and effect the
termination of translation
➢ RF1 – UAA and UAG
➢ RF2 – UAA and UGA
➢ RF3 – assists either RF1 or RF2
Regulation of Gene
Expression
Regulation of gene expression in
eukaryotes:

✓ Levels of control of gene expression

• Short term control

(to meet the daily needs of the
organism)

• Long term control

(gene regulation in
development/differentiation)
Differences in gene expression between
prokaryotes and eukaryotes:

• Prokaryote gene expression typically is

regulated by an operon, the collection of
controlling sites adjacent to polycistronic
protein-coding sequences

• Eukaryotic genes also are regulated in units

of protein-coding sequences and adjacent
controlling sites, but operons are not known
to occur

• Eukaryotic gene regulation is more complex

because eukaryotes possess a nucleus
Differences between prokaryotes and
eukaryotes:

• Two “categories” of eukaryotic gene

regulation exist:

Short-term - genes are quickly turned on

or off in response to the environment and
demands of the cell

Long-term - genes for development and

differentiation
➢ An operon typically includes:
➢ Regulator gene- this codes for a DNA-
binding protein that acts as a repressor
➢ Promoter – DNA sequence that binds RNA
polymerase
➢ Operator- portion of DNA where an active
repressor binds
➢ Structural Genes- codes for enzymes and
proteins needed for the operons
metabolic pathway
Two operons of E. coli:

➢ trp Operon
➢ Regulates the expression of tryptophan
➢ Normally “on” so it expresses the structural
genes needed
➢ If tryptophan is already present, it binds to the
repressor causing it to change shapes so it can
bind to the promoter
lac Operon

➢ Regulation for lactose metabolism

➢ The repressor is normally bound to the

operator to turn “off” gene expression

➢ In the presence of lactose, lactose binds to

the repressor causing it to change shape so
DNA polymerase can easily bind to the
promotor

➢
Eukaryote gene expression is
regulated at six levels:

1. Transcription

2. RNA processing

3. mRNA transport

4. mRNA translation

5. mRNA degradation

6. Protein degradation
1. Transcription control of gene regulation is
controlled by:

1. Promoters

• Occur upstream of the transcription start site.

• Some determine where transcription begins

(e.g., TATA), whereas others determine if
transcription begins.

• Promoters are activated by specialized

transcription factor (TF) proteins (specific TFs
bind specific promoters).

• One or many promoters (each with specific TF

proteins) may occur for any given gene.

• Promoters may be positively or negatively

regulated.
1. Transcription control of gene regulation is controlled by:

2. Enhancers

• Occur upstream or downstream of the transcription

start site.

• Regulatory proteins bind specific enhancer sequences;

binding is determined by the DNA sequence.

• Loops may form in DNA bound to TFs and make contact

with upstream enhancer elements.

• Interactions of regulatory proteins determine if

transcription is activated or repressed (positively or
negatively regulated).
More about promoters and enhancers:

• Some regulatory proteins are common in all cell types,

others are specific

• Each promoter and enhancer possesses a specific set of

proteins (coactivators) that determines expression

• Rate of gene expression is controlled by interaction between

positive and negative regulatory proteins

• Combinatorial gene regulation; enhancers and promoters

bind many of the same regulatory proteins, implying lots of
interaction with fine and coarse levels of control
Short-term - transcriptional control of galactose-utilizing genes in
yeast:

• 3 genes (GAL1, GAL7, & GAL 10) code enzymes that function in the
galactose metabolic pathway.

• GAL1 galactokinase

• GAL7 galactose transferase

• GAL10 galactose epimerase

• Pathway produces d-glucose 6-phosphate, which enters the

glycolytic pathway and is metabolized by genes that are
continuously transcribed.

• In absence of galactose, GAL genes are not transcribed

• GAL genes rapidly induced by galactose and absence of glucose

• Analagous to E. coli lac operon repression by glucose

Galactose metabolizing pathway of yeast
Short-term - transcriptional control of galactose-
utilizing genes in yeast:

• GAL genes are near each other but do not

constitute an operon.

• Additional unlinked gene, GAL4, codes a repressor

protein that binds a promoter element called an
upstream activator sequence (UASG).

• UASG is located between GAL1 and GAL10.

• Transcription occurs in both directions from UASG.

• When galactose is absent, the GAL4 product

(GAL4p) and another product (GAL80p) bind the
UASG sequence; transcription does not occur.

• When galactose is added, a galactose metabolite

binds GAL80p and GAL4p amino acids are
phosphorylated.
Activation model of GAL genes in yeast
2. RNA processing control:

• RNA processing regulates mRNA production from precursor

RNAs.

• Two independent regulatory mechanisms occur:

• Alternative polyadenylation = where the polyA tail is

added

• Alternative splicing = which exons are spliced

• Alternative polyadenylation and splicing can occur together.

• Examples:

• Human calcitonin (CALC) gene in thyroid and neuronal

cells

• Sex determination in Drosophila

Alternative polyadenylation and splicing of the
human CACL gene in thyroid and neuronal cells.

Calcitonin gene-
related peptide
3. mRNA transport control:

• Eukaryote mRNA transport is regulated.

• Some experiments show ~1/2 of primary

transcripts never leave the nucleus and are
degraded.

• Mature mRNAs exit through the nuclear

pores.
4. mRNA translation control:

• Unfertilized eggs are an example, in which

mRNAs (stored in the egg/no new mRNA
synthesis) show increased translation after
fertilization).

• Presence or absence of the 5’ cap and the

length of the poly-A tail at the 3’ end can
determine whether translation takes place
and how long the mRNA is active.

• Conditions that affect the length of the poly-

A tail or leads to the removal of the cap may
trigger the destruction of an mRNA.
5. mRNA degradation control:

• All RNAs in the cytoplasm are subject

to degradation.

• tRNAs and rRNAs usually are very

stable; mRNAs vary considerably
(minutes to months).

• Stability may change in response to

regulatory signals and is thought to
be a major regulatory control point.
6. Post-translational control - protein degradation:

• Proteins can be short-lived (e.g., steroid receptors)

or long-lived (e.g., lens proteins in your eyes).

• This is a cells last change to affect gene expression.

• Protease, enzymes that break down proteins, are

confined to lysosomes and proteasomes.

• When a protein is tagged by a signaling protein, it

enters a proteasome to be degraded.
Summary and contrasts:

Prokaryotes control expression by:

Transcription

Eukaryotes control expression by:

Transcription

RNA processing

mRNA transport

mRNA translation

mRNA degradation
Fig. 18.1
Protein degradation