Gene expression= transcription +

translation

1
 DNA Vs RNA synthesis
• Similarities; In both
1) Synthesis occurs in the 5’ to 3’ direction
2) Synthesis occurs by complementary base pairing
Differences
1) RNA synthesis uses ribonucleotides
2) Only one strand of DNA acts as a template in RNA
synthesis
3) No primers in RNA synthesis
4) Uracil replaces thymine in RNA synthesis
5) No proof reading in RNA synthesis
6) Only a short segment of DNA is copied into RNA
2
rather than the entire genome
 RNA synthesis
• Only one strand of DNA acts as a template in RNA
synthesis (called the non-coding strand)

• The non-template strand is often called the coding

strand because is corresponds to the sequence of the
primary transcript that encodes the protein.

3
 Transcription (DNA to mRNA)
• Occurs in the nucleus and involves 3 main steps
1) Initiation; identification and binding of RNA polymerase to
the promoter and opening of dsDNA.

2) Elongation; assembly of bases along the template strand

by complementary base pairing done by RNA polymerase

3) Termination; occurs when a termination signal appears in

the template strand. In eukaryotes, the appearance of a
poly A tail is the signal. In prokaryotes the rho factor
(protein) detects the termination signal and catalyses
unwinding and separation of the RNA-DNA hybrid. 4
 Transcription
• The starting point of transcription corresponds to the 5’
nucleotide on the mRNA and is designated as +1;
numbers increase as the sequence proceeds down stream

• The nucleotide in the promoter adjacent to the

transcription start site is given the number -1. The
negative numbers increase as the sequence proceeds
upstream

• The designation allows for location of introns and exons

downstream, and regulatory elements that are present
upstream within the promoter region
 The RNA polymerase complex &
promoter recognition
• Mammalian cells have 3 distinct forms of nuclear
dependent RNA polymerases (comprising of 2 large subunits and
over 14 small subunits)
- RNA polymerase I- rRNA
- RNA polymerase II-mRNA
- RNA polymerase III- tRNA, snRNA

 Promoter recognition is very crucial in ensuring efficient

initiation of transcription ( a feature that is given by the
RNA polymerase complex)

 Consensus sequences within the promoter direct binding

6
of the RNA polymerase complex
 Bacterial RNA polymerase
complex
• Has a molecular weight of 400kDa, comprising of 5
subunits; α2ββ’ δ

• The catalytic subunit is β

• The protein factor (sigma factor, δ) allows for the enzyme

to recognize and bind to specific DNA sequences with in
the promoter.

• Additionally, the sigma factor enhances the enzyme’s

affinity for promoter DNA, while decreasing its affinity for
non-promoter DNA 7
Control of transcription (in Bacteria)
• The recognition and association of the RNA polymerase
complex to the promoter ensures accurate initiation of
transcription

• Bacterial promoters have an approximate size of 40

nucleotides

• About 35bp upstream is an 8-nucleotide conserved

sequence (5’TGTTGACA3’ to which RNA polymerase
binds to form a closed complex

• About 10 nucleotides upstream is a 6-nucleotide AT rich

sequence (5’TATAAT3’) called the TATA box 8
Control of transcription (in Bacteria)
• The presence of the TATA box allows for easy
denaturation of the promoter DNA following binding of
RNA polymerase complex on to the promoter

• Once the open complex is formed, transcription proceeds

through the AT region, and continues until when a
termination signal is reached

9
 In Eukaryotes
• Most mammalian genes have a TATA box located 25-
30bp upstream from the transcription start site with a
sequence of 5’TATAAA3’

• For genes that lack a TATA box, an initiator sequence and

down stream promoter elements direct binding of RNA
polymerase to the promoter

Initiator sequence- TCATCAG/TTT/C

Down stream promoter element- A/GGA/TCGTG located 25
bp down stream of the transcription start site.

10
 In Eukaryotes
• Further upstream from the transcription start site are GC
and CAAT boxes

• These regulate the frequency with which the transcription

event occurs by binding to regulatory proteins

• The other sequence elements include;

- Enhancers – Increase the rate of transcription

- Repressors/silencers- decrease the rate of transcription

11
 RNA processing
• Is done minimally in prokaryotes; the processes of
transcription and translation are coupled.

• Eukaryotic RNA undergoes extensive processing between

the time of synthesis and time of translation

• The immediate product generated after transcription is

called the primary transcript

12
 Numerous procedures are involved
1) Splicing (over 50-75%) of the nuclear RNA is lost during
splicing and does not contribute to cytoplasmic mRNA

• The process is accomplished a complex of small

nuclear RNA (U1,U2,U4,U5,& U6) and about 60
ribonucleoproteins called a spiceosome

2) mRNA capping
3) polyA tailing
4) Additional methylations at the 2’-OH and N6 of adenyl
residues. This occurs in the cytoplasm

13
 tRNA molecules are extensively
modified
Involving change of the standard bases A,U,G and C by;

1) Methylations

2) Reduction

3) Deamination

4) Rearrangement of glycosidic bonds

5) Nucleotide alkylations

6) Attachment of the CpCpAOH terminal at the 3’ end

14
 Translation
1) Initiation –Involves ribosomal dissociation and
attachment of a complex of met-tRNA, GTP and mRNA
to the ribosome

• Ends with assembly of the first amino acid (methionine)

with it’s amino terminal at the 5’ end.

2) Elongation
• Involves assembly of subsequent amino acids by
peptide bond formation.

3) Termination - occurs when the termination signal

appears in mRNA reading frame

• Releasing factors (RFs) catalyze hydrolysis of the last

peptide bond and the completed polypeptide 15 then
dissociates from the ribosome
Translation
 Initiation
• Translation begins with formation of the corresponding
amino terminal of the protein near its 5’ terminal end.

• Once mRNA binds to the ribosome, the correct reading

frame is identified and the process of translation begins.

• The entire process involves mRNAs, tRNAs, rRNAs and

various eukaryotic initiation factors (eIFs) and includes the
following stages
i. Ribosomal dissociation
ii. Formation of the 43s preinitiation complex
iii. Formation of the 48s initiation complex
17
iv. Formation of the 80s initiation complex
 Step (i) and (ii)
i. Ribosomal dissociation
The ribosomal unis dissociates into component 40s and
60s subunits. eIF-3 and eIF-1A then bind to the newly
formed 40s subunit preventing its re-association with the 60s
subunit

ii. Formation of the 43s pre-initiation complex

This involves the binding of GTP to eIF-2 forming a binary
complex that binds to met-tRNA.

• The combination of GTP, eIF-2 and met-tRNA forms the

43s pre-initiation complex
18
 Step (iii)
NB; There are 2 tRNAs for methionine; one for binding the
initiator codon AUG (met-tRNA), and another that specifies
other internal methionine molecules

iii. Formation of the 48s initiation complex

The binding of mRNA to the 43s preinitiation complex
forms the 48s initiation complex

• This binding is facilitated by the mRNA 7-methylguanosine

triphosphate cap structure located at the 5’terminal of
mRNA
• Cap binding proteins (eIF-4E, eIF-4G and eIF-4A)
recognize the cap and aid binding of mRNA to the 43s 19
preinitiation complex
 Step (iv)
• Following association of the 43s preinitiation complex with
the mRNA cap, the complex scans the mRNA for a
suitable initiating codon (the 5’ nearest AUG; the code for
methionine)

iv. Formation of the 80s initiation complex

Involves binding of the 60s subunit to the 48s initiation
complex.

• The process involves hydrolysis of GTP bound to the eIF-2

• At this point met-tRNA is at the p-site of the ribosome

20
ready for the elongation cycle to begin.
 Elongation
• Involves assembly of amino acids in the order of codons
as organized on mRNA. Involves the following steps

i. Binding of the aminoacyl tRNA to the acceptor (A) site

on the 80s ribosomal unit.

• This process is aided by the elongation factor (EF1A) that

forms a complex with GTP and the aminoacyl tRNA to be
attached.

• GTP is hydrolyzed in the process to provide energy for

peptide bond formation
21
 Step (ii)
ii. Peptide bond formation

• The formation of peptide bonds is catalyzed by a peptidyl

transferase which is a component of the 28s RNA of the
60s ribosome.

• This reaction results in attachment of the growing peptide

chain to the tRNA in the A site

22
 Step (iii)
ii. Translocation
• After amino acid assembly, the deacylated tRNA is
attached by its anticodon to the P-site

• With the aid of EF2 (elongation factor 2), the peptidyl tRNA
is displaced from the A to the P site and subsequently
released from the ribosome.

• The EF2-GTP complex is hydrolyzed and mRNA moves

forward by one codon

• This leaves the A site open for attachment by another

amino acyl tRNA to continue with another cycle of 23

elongation
 Step (iii)
• With the aid of elongation factors, the process occurs with
great speed and accuracy until a stop/termination codon is
reached.

• A eukaryotic ribosome assembles 6 amino acids /sec

• A prokaryotic ribosome on the other hand can assemble

about 18 amino acids/ sec.

24
 Termination
• Occurs when the stop codon appears in the A site of the
ribosome.

• The stop codons are UAA, UGA and UAG

• Because no tRNA can recognize the stop codon, releasing

factors (RFs) present in the A site recognize the stop
codon.

• The releasing factor (RF)1 forms a complex with RF3 and

GTP

25
 Termination (2)
• In the presence of a peptidyl transferase, the complex
hydrolyzes the bond between the peptide and the tRNA
occupying the P-site

• This results in release of the completed protein and the

tRNA from the P-site

• The 80s then dissociates into its component sub units to

be used in another round of translation

26
 Assembly of amino acids
• The process of translation involves use of specific tRNA
molecules to read the codon on mRNA and match it with
an appropriate amino acid

• The T ψC arm of the tRNA allows for binding of the amino

acyl tRNA to the ribosomal surface

• The D-arm is need for proper recognition of the tRNA

molecule by a specific amino acyl tRNA synthetase

• There over 20 amino acyl tRNA synthetases in the cell

each of which is specific for a particular tRNA
27
 Assembly of amino acids (2)
• The acceptor arm is the site for attachment of the specific
amino acid

• The anticodon arm binds to the codon on mRNA to

indicate assembly of a specific amino acid as determined
by the genetic code

• The anticodon consists of 7 nucleotides and recognizes 3

letter codons on mRNA.

• The anticodon is read in the 3’ to 5’ direction as the codon

is read in the 5’ to 3’ direction (NB. The anticodon- codon
28
pair are complementary)
 The genetic code and the wobble
hypothesis
• Nucleotides are organized into 3 letter sets called codons

• A collection of codons forms the genetic code ( in Slide 30 )

• The genetic code provides the set of rules used to specify

assembly of amino acids and has the following
characteristics .

I. Universality IV. Unambiguous

II. Degeneracy V. Non-punctuated
III. Redundancy VI. Non-overlapping
29
30
 Properties of the genetic code
i. Universality
The genetic code is the same for all organisms

ii. Degeneracy
Each amino acid is specified by multiple codons

iii. Redundancy
Of the several codons for each amino acid, only one is used
at a time during protein synthesis, making the others
redundant

iv. Unambiguous
For any specific codon, only a single amino acid is indicated
31
 Properties of the genetic code
V. Non-punctuated
The genetic code does not involve any overlapping of the
codons. It is read in 3 letter codons until the stop codon is
reached

VI. Non-overlapping
The message is read in a continuous sequence of nucleotide
triplets until a translation stop codon is reached

32
 The wobble hypothesis
• The degeneracy of the genetic code is exhibited mainly in
the 3rd nucleotide

- Example; codes for serine are UCU, UCC, UCA, UCG (the
change is seen in the 3rd base)

- Refer to the genetic code for other examples

• The fact that a single tRNA is indicated for each amino

acid, it means that the pairing of the 3rd nucleotide to the
corresponding anticodon is not strictly done by the Watson
and crick rule of complementary base pairing

• As such the pairing of the codon and anticodon wobbles

(shakes) at this site. Hence ‘the wobble hypothesis’ 33
 Regulation of protein synthesis
• In eukaryotes, protein synthesis is regulated by controlling
the rate of initiation.

• This is done at the level of

1) formation of the 43s pre-initiation complex and 2) the
48s initiation complex

1) Formation of the 43s pre-initiation complex

• Initiation of protein synthesis is regulated at the eIF-2
level.

• eIF-2 comprises of α, β and ᵞ subunits. The α subunit

becomes phosphorylated when the cell is under stress and
when the energy cost for protein synthesis is great 34
 Regulation of protein synthesis (2)
• This occurs during
- amino acid and glucose starvation
- viral infections
- misfolded proteins
- serum deprivation
- heat shock

• Under such circumstances, phosphorylated eIF-2α binds

tightly to and inactivates the GTP-GDP recycling protein
needed for formation of the 43s pre-initiation complex

• This results in failure of formation of the 43s pre-initiation

complex and blocking of protein synthesis
35
 Regulation of protein synthesis (3)
2) Formation of the 48s initiation complex
• Control of the rate of initiation is also done through
regulation of the eIF-4E control, by a phosphorylation
mechanism

• eIF-4E is required for recognition of the mRNA cap

structure

• Insulin and mitogenic growth factors lead to

phosphorylation of eIF-4E.

• When phosphorylated, eIF-4E binds to the mRNA cap

more strongly thereby enhancing the rate of initiation
36
 Regulation of protein synthesis (4)
• Thus, insulin causes a remarkable post transcriptional
increase of protein synthesis as seen in the liver, adipose
tissues and muscles

• Some proteins such as protein BP1, bind to and inactivate

eIF-4E.

• However, the phosphorylation of eIF-4E by insulin and

other growth factors leads to dissociation of the protein
BP1 from eIF-4E

• This leads to activation eIF-4E and initiation of protein

synthesis
37
 Post translational processing
• Many ribosomes can translate the same mRNA molecule
simultaneously.

• This assembly is called a polysome or polyribosome

• Proteins synthesized by the attached polysome are

extruded in the cisternal space between the sheets of the
endoplasmic reticulum and then exported

• The process of protein processing and packaging occurs

in the endoplasmic reticulum and golgi apparatus

38
 In prokaryotes
• Some animal viruses such as HIV, polio and hepatitis A
synthesize long polycistronic proteins from one long
mRNA molecule

• These proteins are subsequently cleaved/cut at specific

sites to provide the several specific proteins needed for
viral function

39
 In eukaryotes
• In animals cells, proteins undergo several post-
translational processing to form mature proteins. The
processing includes;

1) Removal of amino terminal amino acid residues by

specific aminopeptidases

2) Hydroxylations, oxidations to provide cross links of

greater stability (e.g. in collagen)

3) Covalent modifications through acetylation,

phosphorylation, methylation, ubiquitinylation and
glycosylation
40
 Mechanisms of viral evasion (1)
• Several viruses replicate using the host protein synthetic
machinery

1) Some viral mRNAs are translated much more efficiently

than the host mRNAs and so have a competitive
advantage over the host cell for the limited translation
factors.

2) Others inhibit host cell protein synthesis by preventing

the association of mRNA with the 43s ribosome

- Some viruses (e.g. polio virus) disrupt the function of the

cap binding proteins, disabling cap recognition, therefore the
host mRNA can not be directed to the ribosome
41
 Mechanisms of viral evasion (2)
- Yet, viruses do not have a cap structure; they use a
ribosomal entry site to direct their mRNAs to the ribosome.

• This means while host translation is stopped, the viral

mRNA continues

3) Other viruses promote de-phosphorylation of protein BP1,

there by decreasing cap-dependent translation

42