Lecture 17.
Translational control of
gene expression
Flint et al., Chapter 11
Outline
• Introduction
• Eukaryotic protein
synthesis
• Viral translation strategies
• Regulation of translation
during viral infection
Introduction
• All viruses must use the
host translational
machinery to make viral
proteins.
• Translation is the primary
battleground between virus
and host cells.
Theory 1
Eukaryotes evolved monocistronic
mRNAs and nuclei as ways to
compartmentalize mRNA
production from protein translation
as an antiviral mechanism.
In the Nucleus:
• pre-mRNA transcription
• 5’ end capping
• 3’ end tailing
• Splicing (and marking of mRNAs
with proteins)
In the Cytoplasm:
• Translation of mature mRNAs.
Theory 1 (con’t)
• Eukaryotes evolved monocistronic
mRNAs and nuclei as ways to
compartmentalize mRNA production
from protein translation as an antiviral
mechanism.
Viruses (RNA especially)
• Enter cell via cytoplasm
• mRNA = genome
• Separating mRNA production from
translation provides a way for cells to
mark mRNAs as their own.
• Of course, viruses have evolved within
these parameters, and have actually
figured out how to use this to their own
advantage.
• Cells have in turn responded…and the
battle continues.
Theory 2
In the pursuit of genome minimization,
viruses have evolved unique
translational mechanisms
• Viral genomes are limited in size by
volume constraints of capsids
• Viral genomes evolve toward the small
• Can shrink genomes by overlapping or
nesting genetic information
• Translational recoding:
– Allows cellular translational apparatus to
decode overlapping open reading frames
– Allows regulation of viral protein
stoichiometries
• Polycistronic mRNAs: allow multiple
proteins to be synthesized by a single
mRNA
Eukaryotic protein
synthesis
Eukaryotic mRNAs
• Monocistronic
• 5’ 7MethylGppp caps
• 3’ polyA tails
• Spliced
Fig 11.1 top
Eukaryotic protein
synthesis
Ribosomes
• 2 subunits: 60S + 40S = 80S
• Composed of rRNA +
proteins.
• Catalytic activity in rRNA.
• tRNAs: adaptors between
genetic code and protein
sequence
Fig. 11.2
Accessory proteins (See
Table 11.1)
• Called “factors”.
• Required for the 3 stages of
translation:
• Initiation (eIF)
• Elongation (eEF)
• Termination (eRF)
Translation initiation
(Fig. 11.3)
1. Multiple eIF factors recognize and
bind to 5’ 7MethylGppp cap
structures
2. These interact with polyA tail
3. Form a ‘closed loop’ structure:
translation competent
4. 40S + initiator tRNA + an eIF
(‘ternary complex’) recognizes and
binds to closed loop only
5. Ternary Complex “scans”
downstream (3’) to AUG in “good”
context.
6. 60S joins up to make 80S
ribosome.
7. eEF’s bring tRNAs to ribosome,
and aid translocation
Translation
initiation
(Fig. 11.3)
Translation elongation
1.
eEF1 complex brings
aminoacyl-tRNA (aatRNA) to ribosomal
A-site
Correct
codon:anticodon fit
induces GTP
hydrolysis. aa-tRNA
locked in
2.
•
3.
4.
5.
6.
Incorrect fit…no
hydrolysis…aa-tRNA
drifts off
(proofreading)
Peptidyltransfer
occurs in large
subunit: rRNA
catalyzed
eEF2 promotes
translocation via GTP
hydrolysis
Ribosome moves
precisely 1 codon
downstream
Elongation cycle
starts anew
(Fig. 11.7)
Translation termination
1. Termination
codon enters
A-site
2. No cognate
tRNA
3. eRF3/eRF1
complex enters
A-site
4. Stimulate
peptide bond
hydrolysis from
peptidyl-tRNA
5. No acceptor in
A-site
6. Peptide
released from
ribosome.
(Fig. 11.8A)
The closed loop model
• 5’ and 3’ ends of mRNA are
linked by interactions between
protein factors to form a
translationally competent
mRNP
Fig. 11.8C
THE DIVERSITY OF
VIRAL TRANSLATION
STRATEGIES
Initiation.
• General points
• Initiation is a double edged sword
• By requiring mRNAs to have
special properties in order to be
translated, cells have made the job
harder for viruses.
• However, in evolving to circumvent
these requirements, viruses have
opened up new vulnerabilities for
cells.
Viral initiation
strategies
• 5’ end dependent
• Viral mRNAs can obtain 5’ caps
– By nuclear transcription (e.g.
Retroviruses)
– By cap-stealing in the cytoplasm (e.g.
Influenza)
• Viral mRNAs can have cap mimics
– e.g. Picornaviruses covalently attach a
protein (VPg) to the 5’ ends of their
mRNAs. VPg can interact with eIF
factors, fulfilling the function of the
cap.
Viral initiation strategies
5’ end dependent
• Alternative translational start site
selection
• “Leaky scanning”: High frequency of
ribosomal bypass of first AUG codons
placed in poor contexts. Enables initiation
at more than one open reading frame.
Increases coding potential. Fig. 11.11
Sendai Virus P/C gene
Viral initiation strategies
5’ end dependent
• Alternative translational start
site selection
• Methionine-independent initiation:
viral mRNA contains a tRNA like
structure that interacts with the
ribosome. Directs ribosome to initiate
at a specific location within the mRNA
(Fig. 11.4B).
tRNA like structure in Turnip Yellow Mosaic virus
Viral initiation strategies
5’ end dependent
• Alternative translational start
site selection
• Ribosome “shunting”: although
ribosome binds to the 5’ end,
strong mRNA secondary
structures make the ribosome
bypass or shunt around the first
AUG to initiate further
downstream
Viral initiation strategies
• 5’ end independent initiation:
Internal Ribosome Entry Site
Elements (IRES elements) Special
secondary structures in viral mRNAs
can interact with ribosomes, directing
them to initiate internally on the
mRNA.
• Come in all shapes and sizes.
•(Fig. 11.4A)
Cricket Paralysis Virus IRES
Advantage of 5’ end
independent translation
• Virus encoded
factors can knock
out capdependent
initiation.
Examples include:
– Viral proteases
cleave eIF factors.
– Viral
kinases/phosphor
ylases alter
phosphorylation of
eIF factors
• Shut down
translation of host
mRNAs
• Only viral mRNAs
get translated.
(Fig. 11.18C)
Timecourse assay of protein synthesis after Poliovirus infection
Elongation
• Cellular mRNAs are
monocistronic: 1 gene, 1
protein.
• One way to increase
information content of mRNAs
is to encode multiple proteins:
polycistronic.
• Viruses have evolved many
strategies.
Viral elongation
strategies
Polyprotein synthesis Viral mRNA
encodes many proteins in one long
open reading frame
• Polyprotein cleaved by viral
proteases into many smaller
proteins.
(Fig. 11.10: Poliovirus proteins)
Viral elongation
strategies
Ribosomal frameshifting Viral
mRNA contains overlapping
reading frames
• Sequences and structures on viral
mRNA make ribosome slip.
• Resulting shift in reading frame
directs ribosome into downstream
open reading frame
Type A, B, and D Retroviruses
(Fig. 11.14)
Mechanism of -1 Ribosomal
Frameshifting
Ribosome pauses
at RNA pseudoknot
OH
XXY
X XXY
YYZ
YYZ
OH
XXY
XXX
tRNAs slip 1 base in 1 (5’) direction. Repair non-wobble bases
YYZ
YYY Z
Pseudoknot melted out,
translocation occurs,
translation resumes in -1
frame
OH
YYZ
XXX YYY ZNN
Viruses and
Termination
• Termination bypass
• Genome condensation
strategies
• Keeps ribosomes on viral
mRNA after termination
• This allows them to translate
additional open reading
frames.
Viruses and Termination
Termination of suppression
• Viral genome has back to back
genes separated by a
termination codon.
• Viruses have evolved mRNA
sequences and structures that
fool ribosomes into reading
through termination codons at
set rates.
Moloney murine leukemia virus (Type C retrovirus)
Viruses and
Termination
Translation reinitiation (“Stop/Go”)
• Ribosomes normally fall off of
mRNAs at termination
• Viruses have evolved strategies to
prevent this.
• Allow ribosomes to stay on mRNA
and initiate again downstream
Influenza B virus segment 7