1. 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

2. Introduction
All viruses must use the host translational machinery to make viral proteins.
Translation is the primary battleground between virus and host cells.

3. 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.

4. 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.

5. 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

6. Eukaryotic protein synthesis
Eukaryotic mRNAs
Monocistronic
5’ 7MethylGppp caps
3’ polyA tails
Spliced
Fig 11.1 top

7. 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

8. Accessory proteins (See Table 11.1)
Called “factors”.
Required for the 3 stages of translation:
Initiation (eIF)
Elongation (eEF)
Termination (eRF)

9. Translation initiation (Fig. 11.3)
Multiple eIF factors recognize and bind to 5’ 7MethylGppp cap structures
These interact with polyA tail
Form a ‘closed loop’ structure: translation competent
40S + initiator tRNA + an eIF (‘ternary complex’) recognizes and binds to closed loop only
Ternary Complex “scans” downstream (3’) to AUG in “good” context.
60S joins up to make 80S ribosome.
eEF’s bring tRNAs to ribosome, and aid translocation

10. Translation initiation
(Fig. 11.3)

11. Translation elongation
(Fig. 11.7)
eEF1 complex brings aminoacyl-tRNA (aa-tRNA) to ribosomal A-site
Correct codon:anticodon fit induces GTP hydrolysis. aa-tRNA locked in
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

12. Translation termination
Termination codon enters A-site
No cognate tRNA
eRF3/eRF1 complex enters A-site
Stimulate peptide bond hydrolysis from peptidyl-tRNA
No acceptor in A-site
Peptide released from ribosome.
(Fig. 11.8A)

13. 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

14. 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.

15. 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.

16. 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
Sendai Virus P/C gene

17. 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

18. 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

19. 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

20. Advantage of 5’ end independent translation
Virus encoded factors can knock out cap-dependent initiation. Examples include:
Viral proteases cleave eIF factors.
Viral kinases/phosphorylases alter phosphorylation of eIF factors
Shut down translation of host mRNAs
Only viral mRNAs get translated.
(Fig C)
Timecourse assay of protein synthesis after Poliovirus infection

21. 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.

22. 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 : Poliovirus proteins)

23. 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 )

24. Mechanism of -1 Ribosomal Frameshifting
Ribosome pauses
at RNA pseudoknot OH
XXY YYZ
X XXY YYZ
tRNAs slip 1 base in -1 (5’) direction. Re-pair non-wobble bases OH
XXY YYZ
XXX YYY Z OH
YYZ
XXX YYY ZNN
Pseudoknot melted out, translocation occurs, translation resumes in -1 frame

25. Viruses and Termination
Termination bypass
Genome condensation strategies
Keeps ribosomes on viral mRNA after termination
This allows them to translate additional open reading frames.

26. 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)

27. 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