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) 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

Translation initiation (Fig. 11.3)

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

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)

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 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. 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 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

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