Lecture 19: Regulation of Translation I

Regulation of Translation I Side Notes: Regulation of Translation • Bacteria and eukaryotes invoke various strategies - both globally and specifically - to ensure that translation machinery operates correctly and that the correct end-products are made • Translation regulation allows cells to respond to developmental and environmental changes Translation is regulated globally upon amino acid starvation • Protein rates need or be high to support growth under nutrient rich conditions, but low during nutrient deprivation. Amino acids act as the precursors to building proteins • Normally, soluble tRNAs are loaded with an amino acid, making them charged • However, under nutrient-poor conditions, there are not enough free amino acids to attach to tRNA (uncharged) • Uncharged tRNA act as a marker of low amino acid levels, prompting the shutdown of protein synthesis • In bacteria, uncharged tRNAs compete with charge tRNAs for binding at the ribosome A site. This unproductive complex 1) impedes translation and 2) recruits protein RelA • RelA synthesizes high levels of (p)ppGpp (pentaphosphate guanine nucleotide alarmone, “magic spot”) from GTP/GDP and ATP precursors • (p)ppGpp effects transcription: decrease in stable RNA synthesis (rRNA, tRNA) and increase in synthesis of stress response factors (“stringent response”) • In eukaryotes, uncharged tRNA binds instead of Gcn2, whose kinase domain activates and phosphorylates the translational initiation factor eIF2 • eIF2 normally guides the initial Met-tRNA(Met) to the site, but phosphorylated eIF2 binds strongly to eIF2B, which prevents eIF2 from being used in initiation, thereby shutting down translation • Eukaryotic cells also undergo a stress response and induce other translational and transcriptional changes that restore amino acid levels Global translational control involves modification of translational factors • For example, phosphorylation of eIF2 affects the equilibrium between free eIF2 and eIFWB:eIF2, where phosphorylation of eIF2 leads to constitutive binding of eIF2-GDP to eIF2, sequestering free eIF2 from participating in initiation • Several kinases have been identified that phosphorylate serine 51 in response to specific forms of stress Global translation can be regulated through the mRNA-bound complex • mRNAs are more abundant than the translation machinery and thus compete for translation, providing another opportunity for translational regulation • Regulation of eIF4E, PABP, and eIF4G in mRNA bound cap includes: ◦eIF4E is regulated at the transcriptional level in response to external stimuli ◦eIF4E binds proteins 4E-BPs (eIF4E - binding proteins) that compete with eIF4G for binding eIF4E, preventing assembly of closed loop and thus reducing initiation ◦eIF4E is phosphorylated in response to external stimuli, decreasing affinity for mRNA 5’ cap Specific regulation of translation initiation • Translation can be regulated specifically - on the individual level - depending on mRNA sequence, and thus structure, features Regulation of Translation I Side Notes: • Cells can rapidly respond to stimuli by controlling rates of translation initiation from already available mRNA • mRNAs assume different shapes in different conditions - and these affect translation levels ◦In bacteria, mechanisms involve direct obstruction of shine-Dalgarno sequence ◦In eukaryotes, mechanisms involve both 5’ UTRs and 3’ UTRs of mRNA Bacteria regulate translation through Shine-Dalgarno sequestration: conformational changes • The Shine-Dalgarno sequence is needed upstream of the AUG start site for translation initiation • If the SD sequence is not accessible to form interactions with the small ribosomal subunit, initiation cannot occur • Bacteria can sequester the SD sequence with conformational changes, small regulatory RNAs, RNA-binding proteins, and small molecules to inhibit translation • For example, in bacterium listeria monocytogenes, translation of PrfA is temperature sensitive ◦At low temperatures (30 C) outside of the host, the SD sequence of PrfA mRNA is locked, inaccessible to ribosome ◦At high temperature (37 C) within a host, the PrfA mRNA restructures, making the SD sequence accessible and translation can commence Bacteria regulate translation through Shine-Dalgarno sequestration: RNA-binding proteins • SD can be regulated by an RNA-binding protein as seen in the translation of threonine tRNA synthetase in E. coli ◦Threonine tRNA synthetase (ThrRS) is needed to aminoacylate tRNA(thr) ◦Demand for ThrRs is therefore high when protein synthesis levels are high ◦ThrRS can also bind to its own mRNA, block the SD sequence, and inhibit its own translation ‣ ThrRS mRNA and tRNA(Thr) have similar molecular features, which allow ThrRs to bind either RNA, whichever is more available ‣ deacylated tRNA(Thr) and mRNA thus compete for binding to ThrRS and this competition influences its protein synthesis Bacteria regulate translation through Shine-Dalgarno sequestration: riboswitches • The SD sequence can also be of structure by riboswitches: riboswitches are RNA elements (typically in the 5’ UTR) that control their own synthesis, typically through the binding of small-molecule metabolites that cause conformational changes • One riboswitches example is that controlling thiamine synthesis ◦The 5’ UTRs of thIM and thiC mRNAs (thiamine synthesis genes) bind an intermediate of thiamine synthesis, thiamine pyrophosphate (TPP) ◦The TPP bound 5’ UTR blocks the SD sequence and stops translation ◦When thiamin (and TPP) are low, no TPP binds and the 5’ UTR structure no longer blocks SD, allowing translation Specific regulation of translation initiation • Translation can be regulated specifically - on the individual level - depending on mRNA sequence, and thus structure, features • Cells can rapidly respond to stimuli by controlling rates of translation initiation from already available mRNA Regulation of Transcription I Side Notes: • mRNAs can assume different shapes in different conditions - and these affect translation levels ◦In bacteria, mechanisms involve direct obstruction of Shine-Dalgarno sequence ◦In eukaryotes, mechanisms involve both 5’ UTRs and 3’ UTRs of mRNA Eukaryotes can regulate translation by blocking access to the ribosome-binding site • 5’ UTR regulation of translation is less common in eukaryotes, but there are examples for iron regulation • iron is an essential nutrient, but must be tightly regulated as too much free iron is toxic. iron is tightly bound to storage protein ferritin: the higher the iron concentration, the more ferritin is needed • The 5’ UTR of ferritin mRNA has a stem loop structure called Iron response element (IREs) that bind to Iron regulatory proteins (IRPs) • When iron is scarce, IRPs bind to IREs and stop translation by preventing ribosomes from accessing AUG • When iron is plentiful, iron binds to IRPs, which then cannot bind IREs, and translation of ferritin proceeds Upstream ORFs regulate the synthesis of Gcn4 protein in yeast • Gcn4 is a transcriptional activator of amino acid biosynthetic genes in yeast • Translation is globally repressed during cellular starvation, but Gcn4 translation is specifically increased to activate synthesis of amino acids • Gcn4 regulation depends on multiple upstream open reading frames (uORFs) on polycistronic Gcn4 mRNA ◦Orf1 is always translated, and the 40S ribosome remains attached after first stop codon, scanning the remaining mRNA Instead of recycling ◦WHEN amino acids are abundant, eIF2is unphosphorylated and re-initiation occurs readily at Orf4 (and possibly Orf2 and Orf3), but sequence context of Gcn4 ORF does not favor re-initiation at Gcn4 ORF, preventing its translation