UMD BSCI 437 - Lecture 17 Translational control of gene expression

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Lecture 17. Translational control of gene expressionIntroductionTheory 1Theory 1 (con’t)Theory 2Eukaryotic protein synthesisSlide 7Accessory proteins (See Table 11.1)Translation initiation (Fig. 11.3)PowerPoint PresentationTranslation elongationTranslation terminationThe closed loop modelTHE DIVERSITY OF VIRAL TRANSLATION STRATEGIESViral initiation strategiesSlide 16Slide 17Slide 18Slide 19Advantage of 5’ end independent translationElongationViral elongation strategiesSlide 23Slide 24Viruses and TerminationSlide 26Slide 27Lecture 17. Translational control of gene expression Flint et al., Chapter 11Outline•Introduction•Eukaryotic protein synthesis•Viral translation strategies•Regulation of translation during viral infectionIntroduction•All viruses must use the host translational machinery to make viral proteins.•Translation is the primary battleground between virus and host cells.Theory 1Eukaryotes 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 mRNAEukaryotic protein synthesisEukaryotic mRNAs•Monocistronic•5’ 7MethylGppp caps•3’ polyA tails•SplicedFig 11.1 topEukaryotic protein synthesisRibosomes •2 subunits: 60S + 40S = 80S•Composed of rRNA + proteins. •Catalytic activity in rRNA.•tRNAs: adaptors between genetic code and protein sequence Fig. 11.2Accessory 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 structures2. These interact with polyA tail3. Form a ‘closed loop’ structure: translation competent4. 40S + initiator tRNA + an eIF (‘ternary complex’) recognizes and binds to closed loop only5. 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 translocationTranslation initiation(Fig. 11.3)Translation elongation1. eEF1 complex brings aminoacyl-tRNA (aa-tRNA) to ribosomal A-site2. Correct codon:anticodon fit induces GTP hydrolysis. aa-tRNA locked in•Incorrect fit…no hydrolysis…aa-tRNA drifts off (proofreading)3. Peptidyltransfer occurs in large subunit: rRNA catalyzed4. eEF2 promotes translocation via GTP hydrolysis5. Ribosome moves precisely 1 codon downstream6. Elongation cycle starts anew(Fig. 11.7)Translation termination1. Termination codon enters A-site2. No cognate tRNA3. eRF3/eRF1 complex enters A-site4. Stimulate peptide bond hydrolysis from peptidyl-tRNA5. No acceptor in A-site6. 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 mRNPFig. 11.8CTHE 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 strategies5’ 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. 11.11Sendai Virus P/C geneViral initiation strategies5’ 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 virus5’ 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 downstreamViral initiation strategiesViral 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 IRESAdvantage 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 infectionElongation•Cellular mRNAs are monocistronic:


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