BMS2036 Molecular Biology and Genetics Lecture Notes PDF

BMS2036: Molecular Biology and Genetics: From Genes to Biological Function Week 4: Translation in Eukaryotes Lecture 1 Dr Hannah Burgess 01AW01 [email protected] Dr Hannah Burgess Lecturer and principal investigator I research translation control during virus infection of eukaryotic cells O...

BMS2036: Molecular Biology and Genetics: From Genes to Biological Function Week 4: Translation in Eukaryotes Lecture 1 Dr Hannah Burgess 01AW01 [email protected] Dr Hannah Burgess Lecturer and principal investigator I research translation control during virus infection of eukaryotic cells Office: 01AW01 Email: [email protected] Please post questions to the module discussion board! Translation Lectures 1. Overview of translation (protein synthesis) in eukaryotes and the function of key translation initiation factors (eIFs) Alberts, Molecular Biology of the Cell, 7 th Edition, Chapter 6, from RNA to Protein (p358-376) 2. How these factors facilitate “global” regulation of translation and links to diseases 3. Additional translational control mechanisms Alberts, Molecular Biology of the Cell, 7 th Edition, Chapter 7, Control of Gene Expression (p456-462) Lecture outline - Eukaryotic Translation - General principles • Similarities and differences between eukaryotic and prokaryotic ribosomes (and mRNAs) • Recap of overall translation process • Translation initiation in eukaryotes • What happens to the new protein after translation? Alberts, Molecular Biology of the Cell, 7 th Edition, Chapter 6, from RNA to Protein (p358-376) Learning Objectives • • • • • • • You can outline the differences in composition of bacterial and eukaryotic ribosomes and translation factors You know translation occurs in 3 phases (initiation, elongation, termination) You know the steps of eukaryotic translation initiation, the key factors involved and what they do You know how correct start codon selection is ensured You understand that translation initiation is the rate limiting step and a frequent target of regulation You know how and why nascent proteins are directed to the endoplasmic reticulum You know protein stability contributes to overall protein levels, and an example of how this can be controlled The central dogma transcription messenger RNA Protein translation Genomic DNA Translation – a protein production line ➢ mRNA coding region! ➢ Ribosomes! Translation factors! tRNA! ➢ Amino acid! ➢ ATP and GTP hydrolysis! ➢ Surveillance pathways! • Instructions • Tools • Raw materials • Energy • Quality control mRNA Translation Protein Ribosomes - Composition Prokaryotes 70S 30S Small Large • 16S RNA • 21 proteins 50S • 5S, 23S RNA • 34 proteins Free in the cell Eukaryotes 80S 40S • 18S RNA • 33 proteins 60S • 5S, 5.8S, 28S RNA • 49 proteins In the cytoplasm, ER associated, can be localised to specific intracellular areas Ribosomes - Composition Prokaryotes 70S Eukaryotes 80S Large Small An RNA scaffold coated by proteins Ribosomes - History and Background 1970s Biochemical investigation of translation 2000 onwards 2009 2000 First high resolution structure of the ribosome (x-ray) by Venki Ramakrishnan Dozens of structures of various functional state of the ribosome Rationalization of more than 30yrs of biochemical findings Nobel Prize (Chemistry) to Yonath, Steitz and Ramakrishnan for their structural investigations of the ribosome • Translation pathways deciphered (initiation-elongation-termination) • Antibiotics mode of action unravelled Ribosomes – not so homogenous after all • Many RP paralogs produced in higher eukaryotes • RPs can be post-translationally modified • Variable rRNA modification Variability in composition = “specialized ribosomes” ? Genuth NR, Barna M. The Discovery of Ribosome Heterogeneity and Its Implications for Gene Regulation and Organismal Life. Mol Cell. 2018 Aug 2;71(3):364-374. doi: 10.1016/j.molcel.2018.07.018. PMID: 30075139 Ribosomes - Protein synthesis in 3 steps Prokaryotes Eukaryotes IF1, 2, 3 eIF1, 1A, 2, 3, 4A, 4B, 4E, 4G, 5, 5B EF-Tu, EF-G eEF1, 2, 3 RF1, 2 eRF1, 2, 3 initiation elongation termination mRNA features that influence eukaryotic translation 5’ untranslated region (5’UTR) 5’ cap m7G structure AUG open reading frame (ORF) 3’ untranslated region (3’UTR) UGA poly(A) 3’ poly(A) tail Translation initiation: where do we start? Translation initiation: cap binding eIF4F complex eIF4G scaffolding subunit eIF4A helicase 4G 4E eIF4E cap binding protein 4A Translation initiation: cap binding eIF4F complex Poly(A) binding protein Crosstalk between 5’ and 3’ end eIF4G scaffolding subunit eIF4A helicase 4G 4E m7G eIF4E cap binding protein PABP poly(A) UGA 4A AUG Translation initiation: ribosome recruitment 43S ‘pre-initiation complex’ 40S small ribosomal subunit eIF3 bridges 4F and ribosome eIF5 GTPase – activating protein 3 40S 5 Met 2-GTP 1 1A eIF2-tRNAi-GTP eIF1+1A proofreading AUG selection eIF2:GTPase Ternary complex (ie made of 3 things!) tRNA delivery Translation initiation: ribosome recruitment 43S ‘pre-initiation complex’ 40S small ribosomal subunit eIF3 bridges 4F and ribosome eIF5 GTPase – activating protein 3 4G 4E m7G 4A 40S 5 Met 2-GTP AUG 1 1A eIF2-tRNAi-GTP eIF1+1A proofreading AUG selection eIF2:GTPase Ternary complex tRNA delivery UGA poly(A) Finding the translation start codon Scanning 43S Translation initiation: scanning 43S ‘PIC’ 5’-3’ scanning to find AUG requires: • Unfolding of 5’UTR • ATP hydrolysis (helicase) 40S small ribosomal subunit eIF3 bridges 4F and ribosome 3 4G 4E m7G 4A 40S 5 Met 2-GTP AUG 1 1A eIF2-tRNAi-GTP eIF1+1A proofreading AUG selection Ternary complex tRNA delivery UGA poly(A) Translation initiation: scanning 48S complex 3 4G 4E m7G 4A 40S 5 Met 2-GTP AUG 1 1A AUG recognition! codon/anticodon pairing UGA poly(A) Translation initiation: AUG recognition 2-GDP 5 3 4G 4E m7G 40S Met 4A AUG 1 1A GTP hydrolysis tRNA delivery UGA poly(A) Translation initiation: AUG recognition J Rabl et al. Science 2011;331:730-736 eIF1 polices accurate codon:anti-codon matching Translation initiation: 80S assembly 80S ribosome 60S large ribosomal subunit peptidyl transfer center 2-GDP 5 3 1 40S 4G 4E m7G Met 4A AUG 1A 60S UGA poly(A) 5B eIF5B GTPase Bridges the 60S to the 40S eIF release large subunit joining Translation initiation complete 80S ribosome 2-GDP 3 5 40S 4G 4E m7G Met 4A AUG 1 1A 5B UGA poly(A) 60S GTP hydrolysis (2nd) eIF release Further mechanistic details of translation initiation: https://www.youtube.com/watch?v=7EZ87bIvCOM Translation elongation Alberts p368 ➢ elongation factors (eEF1, eEF2) accelerate translation elongation and ensure accuracy of codon:anticodon matching mRNA translation https://www.youtube.com/watch?v=TfYf_rPWUdY translation termination Alberts p375 ➢ release factors (eRF1, eRF3) resemble tRNAs, recognise stop codons and terminate translation Prokaryotic and eukaryotic translation apparatus is v similar Melnikov, S., Ben-Shem, A., Garreau de Loubresse, N. et al. One core, two shells: bacterial and eukaryotic ribosomes. Nat Struct Mol Biol 19, 560–567 (2012). Efficiently translated mRNAs associate with polyribosomes The number/density of ribosomes associated with an mRNA indicative of its translation efficiency Alberts p375 polysome profiling Ribo-Seq / ribosome profiling The ribosome coordinates folding and assembly of new proteins NC = protein nascent chain Liutkute, M.; Samatova, E.; Rodnina, M.V. Cotranslational Folding of Proteins on the Ribosome. Biomolecules 2020, 10, 97. https://doi.org/10.3390/biom10010097 For further reading also see Alberts p380-383 ➢ Speed of translation can influence proper folding Targeting the nascent protein to the ER • Proteins to be secreted are directed to the ER by an Nterminal signal sequence • Recognized by Signal Recognition Particle (SRP) • SRP = 1 RNA + 6 proteins • Escorts nascent protein to ER and docks to SRP receptor Alberts p703 For further reading see Alberts p703-705 Control of protein stability – the proteasome • Proteins (like mRNAs) have different rates of turnover • Turnover of damaged or unfolded proteins, and those targeted for destruction is largely achieved by the proteasome Alberts p386 • Ubiquitin ligases tag proteins with lysine 48-linked ubiquitin molecules for proteasomal destruction Further reading on the proteasome Alberts p386-388 - additional mechanism autophagy and lysosomal degradation p799-807 In the next two lectures we’ll discuss… Regulation of translation: • Mechanisms to control translation of all (or most) mRNAs simultaneously = GLOBAL control • Mechanisms to control the translation of single/select mRNAs = SPECIFIC control Alberts p388 Translational control and disease Rate limiting steps are key targets for regulation… and dysregulation! 4G 4E m7G 4A Cap recognition eIF4E sequestration eIF4E phosphorylation eIF4E levels eIF4G cleavage Met 2-GTP Ternary complex formation eIF2-GDP recycling eIF2 phosphorylation Take home messages! • Initiation is the most regulated step • Key steps during translation initiation are cap recognition, scanning and AUG (start codon) recognition • Roles of key initiation factors • Role of ribosome in protein folding/targeting • Protein turnover is also regulated Recommended video! translation initiation explained by Prof Rachel Green: https://www.youtube.com/watch?v=7EZ87bIvCOM Learning Objectives Now….. • • • • • • • You can outline the differences in composition of bacterial and eukaryotic ribosomes and translation factors You know translation occurs in 3 phases (initiation, elongation, termination) You know the steps of eukaryotic translation initiation, the key factors involved and what they do You know how correct start codon selection is ensured You understand that translation initiation is the rate limiting step and a frequent target of regulation You know how and why nascent proteins are directed to the endoplasmic reticulum You know protein stability contributes to overall protein levels, and an example of how this can be controlled