MBG 2024 RG Lectures 1 & 2 on Protein Synthesis - PDF

Regulation of protein synthesis I November 19, 2024 The central dogma of biology General thoughts on regulation What is regulation? Often just changes in thermodynamics and kinetics of the core steps Usually not a whole new way of doing things What dynamic range is relevant in a...

Regulation of protein synthesis I November 19, 2024 The central dogma of biology General thoughts on regulation What is regulation? Often just changes in thermodynamics and kinetics of the core steps Usually not a whole new way of doing things What dynamic range is relevant in a cell? Sometimes on/off is what happens But 2-fold changes can be very relevant Which steps in translation are likely regulated? Initiation -- sort of obvious Elongation -- less common but certainly happens Termination/recycling -- quality control / mRNA surveillance Initiation Elongation Termination/ Recycling Decoding Peptide bond formation Translocation The basics of translation elongation Peptidyl Exit Aminoacyl Some things to keep in mind … mRNA structure Bacterial translation initiation … the Shine Dalgarno Eukaryotic translation initiation … the scanning model In vivo: translation on polysomes What tools can be used to study translation? What do we learn from this experiment? How else can we manipulate the gradients? Purple trace is “regulated” and blue trace is “unregulated”. Gray trace is actin. What mechanism might you think is at play? 35 (a) General amino acid starvation 30 (b) Elongation arrest (c) Initiation block 25 % mRNA (d) Termination defect 20 Actin (e) mRNA decay 15 FLP SYN 10 5 0 1 2 3 4 5 6 7 8 9 10 11 Another way to look at ribosomes … in vivo Meta (or average) gene analysis What are some predictions for data? How to first assess whether it works? Improvements in ribosome profiling ANS Profiling data reflects anticipated stalling Gamma toxin – targets tRNAGlu (UUC) 3-AT histidine starvation Ribosome profiling in E. coli … stoichiometries Li … Weissman, Cell 2014  What is going on in this single gene? … this texture can tell much about specifics of elongation More typical view of “pausing” during elongation Single gene analysis Meta (average) gene analysis Another example of site-specific pausing Profiling can find ribosomes in new places Here is an example … in the 3’ UTR WT dpm1 Dom34 cpr5 ORF 3’UTR Poly(A) A “run on” experiment is powerful … kinetics Harringtonine (trap) identifies start sites Canonical initiation sites What about translational “control”? Ribosome occupancy is an important metric a) Promotes initiation b) Promotes elongation c) Promotes termination d) Promotes recycling Regulation of protein synthesis II November 21, 2024 Broad classes of regulation Global – Bacterial What conditions would – Eukaryal lead to such regulation? Gene-specific Which genes would be – Bacterial subject to such – Eukaryal regulation? RelA is central regulator of translation in bacteria Loveland et al. eLife 2016 Arenz et al. NAR 2016 Global translational control in eukaryotes eIF2 phosphorylation (the ISR) Cap dependent regulation (connections to TOR) Diverse stress inputs activate the ISR Global reduction in translation … but we know there are privileged mRNAs … how GCN2 is activated is work in progress The GCN4 story in yeast … uORFs Alan Hinnebusch and Thomas Dever What happens to GCN4 expression if ORF4 AUG is mutated in rich media? (a) GCN4 expression goes up (b) GCN4 expression goes down (c) GCN4 expression stays the same What happens if the strength of the AUG start context of ORF4 is increased under amino acid starvation conditions? (a) GCN4 expression goes up (b) GCN4 expression goes down (c) GCN4 expression stays the same What does profiling data look like for GCN4 under amino acid starvation? How to test the model post ribosome profiling? ATF4 in mammals regulated in similar manner  Drug screen to block the ISR … ISRIB 100,000 compounds to 400 to 28 to 1 Another screen identified eIF2B (the GEF) as critical to ISRIB mode of action Tsai et al. Science (2018) Rheostat for eIF2B activity Low amounts staples together eIF2B and enhances activity Too much stabilizes tetramer rather than octamer There are multiple ways to target the ISR … effects on health not always predictable The yin and yang of the ISR Global translational control in eukaryotes eIF2 phosphorylation (the ISR) Cap dependent regulation (connections to TOR) Modulation of cap dependent translation events 4E-BPs interact with eIF4E to regulate access to cap Phosphorylation of 4E-BPs and eIF4E promote translation mTOR-mediated regulation of translation TOPs are special mRNAs regulated by mTOR Another way to look at translation … the TOPs Multiple inputs into the cap recognition complex Hyperactivation of these pathways is found in the majority of cancers Siddiqui and Sonenberg, Biochem Soc Trans 2015 Key Points General translational control is a common and critical route to control gene expression in metazoans Regulation is most common at the stage of translation initiation The integrated stress response (ISR) is a major mechanism of translational control downstream of four related kinases (GCN2, PERK, PKR and HRI) mTOR regulation of translation initiation is another major regulatory point with great relevance to cancer Viruses routinely target cap dependent translation … viruses must be able to translate their mRNAs Viruses use internal ribosome entry sites (IRES) Gene specific regulation in bacteria Riboswitches, RNA binding proteins, small RNAs, etc interfere with SD recognition Gene specific regulation in eukaryotes Some examples of 5’ UTR binding proteins Most examples involved 3’ UTR elements that regulate initiation through cap dependent mechanisms 3’ UTR mediated regulation in C. elegans miRNAs typically bind 3’ UTRs for regulation Mechanism not certain but likely cap targeted An elaborate developmental mechanism Consider general 4E regulation … this is a gene specific adaptation Key Points Gene-specific control happens very differently in bacteria and eukaryotes Regulation is most common at the stage of translation initiation both in normal conditions and in cells that are stressed (TOPs and uORFs) IRES’s are critical tools used by viruses to evade general host translational shutdown Much regulation happens through the 5’ UTR of bacterial mRNAs Much regulation happens through the 3’ UTR of eukaryotic mRNAs Nonsense-mediated decay (medically important) PTC = Premature Termination Codon EJC = Exon Junction Complex eRFs = Proteins involved in termination UPFs = Proteins important for NMD NMD modulates phenotypes of genetic disease Human b-globin  Problematic mRNAs are dealt with in all cells Bacteria – tmRNA Eukaryotes – NGD/RQC Key Points Premature stop codons allow for global QC on non- functional mRNAs that escape nuclear QC Stalled ribosomes signal a problem. Mostly they lead to collisions that are recognized in both systems Bacteria and Eukaryotes do same basic thing … target nascent protein and mRNA for decay and ”rescue” ribosomes