From RNA to Protein Lecture 6

Translation From RNA to Protein Genetic code dictates how mRNA translated into aa sequence of protein Nucleotides read consecutively in grps of 3 (codons) Degenerate genetic code; 64 codons specify 20 aa reading frames

Translation tRNA as an Adaptor Molecule Recognizes and binds to codon and appr. aa ~80 nucleotdies Folds into clover leaf and then into L-shaped structure Two regions of unpaired nucelotdies at ends of L shaped molecule essential to function 1. anticodon= set of 3 consecutive nucleotides pairs w/complementary codon on mRNA 2. short ss region near 3’end where aa attaches

Translation Redundancy/Degeneracy of Genetic Code More than one tRNA for many of the aa Some tRNAs base pair w/ more than one aa; Wobble Position

Translation tRNAs Number and kinds of tRNAs varies across species ie: humans have 497 tRNA genes w/ only 48 diff anticodons represented Transcribed by RNA Pol III Some transcripts are spliced via cut-and-paste mechanism catalyzed by proteins (no lariat) occurs when tRNA is properly folded in cloverleaf conf Extensive modifications > 50 different kinds 1 modification/10 nucleotides Modifications essential to: 1) accuracy of tRNA attaching to correct aa 2) recognition of mRNA codon by tRNA anticodon

Translation Aminoacyl-tRNA Synthetases Couple aa to appr set of tRNAs Aa specific Enzymatic reaction attaches aa to 3’ end of tRNA requires ATP High energy bond produced btwn aa and tRNA later used to covalently link aa to growing polypeptide chain

Translation Editing by tRNA synthetases Selects correct aa in two step process: 1. correct aa highest affinity for active site 2. aa forced into second pocket whose dimensions exclude correct aa Those that enter second editing site are hydrolyzed from AMP- hydrolytic editing Raises overall accuracy of tRNA charging to 1 mistake/40,000 couplings Synthetase must also recognize correct tRNAs

Translation Ribosome Structure: rRNA molecules + 50 different proteins

Translation Ribosome Structure and Function Large and Small Subunits; rRNA sequence highly conserved 66% RNA; 33% protein rRNA responsible for: structure positioning tRNAs on mRNA catalytic activity in forming peptide bond

Translation mRNA Decoded on Ribosomes Ribosomal subunits assemble on mRNA near 5’ end Ribosome translates mRNA in aa sequence using tRNAs as adaptors to add aa in correct sequence to end of growing polypeptide chain aa linked by peptide bond formation 2 aa added/sec eucaryotic cell; 20 aa/sec added by bacteria 4 impt ribosomal binding sites

Translation Major Steps in Translation Ribosomal Assembly and Initaition Elongation Termination and Release of Nascent Polypeptide

Translation Ribosomal Assembly Initiation tRNA-Met & eIFs bind sm rRNA subunit Sm rRNA subunit binds to 5’ end of mRNA recognizing CAP and 2 eIFs (eucaryotes) Sm rRNA scans for AUG start eIFs dissociate and lg rRNA binds Initiator tRNA-met now in P-site leaving A-site vacant for incoming aminoacyl rRNA to start protein synthesis

Translation Procaryotes have Shine Delgarno Sequence Specific sequence few nucleotides upstream of AUG start AGGAGGU Binding site positions sm rRNA subunit at AUG start Bacterial mRNAs can be poly-cistronic; eucaryotes monocistronic

Translation Elongation tRNA carrying next aa in chain binds to ribo A-site base pairing w/ mRNA codon Peptide Bond Formation= carboxyl end released from tRNA at P-site and joined to free amino grp of aa linked to tRNA at A site forming new peptide bond Translocation= conformational chgs move mRNA 3 nucleotides along ribo resetting ribo for next acyl tRNA

Translation Two Impt Elongation Factors Drive Translation EF-Tu Mechanism- resp. 99% accuracy a. Chged tRNA enters ribo bound to GTP form b. Codon recognition triggers GTP hydrolysis c. EF-Tu dissociates from ribo w/out tRNA d. Introduces 2 short delays btwn codon- anticodon pairing and chain elongation EF-G binds in or near A site hydrolyzes GTP whose energy accelerates movement of bound tRNAs in A/P and P/E hybrid states

Translation Termination Stop codons: UAA, UAG, UGA Stop codons recognized by “release factor” proteins Release factors cause peptidyl transferase to catalyze addition of water to peptidyl tRNA Hydrolysis frees COOH end of polypeptide chain from attachment to tRNA Ribosomal dissassembly

Translation Proteins are Made on Polyribosomes Syn of avg protein varies 20 sec to 2-3 min mRNAs translated in form of polyribosomes w/ ribos spaced > 80 nucleotides Transcription and translation occur simultaneously in procaryotes

Translation Quality Control of Translation Accuracy ~1 mistake/104 aa Speed of translation 20 aa incorporated/sec in pro. vs 2 aa/sec euk. Protein synthesis consumes more energy than any other biosynthetic process; 4 ATP equivalents/ aa Quality control mech to ensure mRNA is complete recognition of Cap and poly A tail by initiation complex

Translation Antibiotics Inhibitors of bacterial protein synthesis are effective antibiotics Specificity of antibiotics useful for molecular cell biology studies

Life and Death of Proteins Protein Maturation Protein folding begins while protein is being synthesized Maturation: unique 3d structure bind sm molecules modifications assemble w/ other proteins

Life and Death of Proteins Protein Folding Steps in protein folding: 1. molten globule 2. slow phase Most of folding complete by time released from ribosome Molecular chaperones held guide folding of many proteins

Life and Death of Proteins Molecular Chaperonins Two major families: hsp60 & hsp70 Affinity for exposed hydrophobic patches Massage protein into folded conformation via ATP hydrolysis Hsp70 acts early in life of protein; binds to stretch of 7 hydrophobic aa before protein leaves ribo Hsp60 acts later in proteins life, forms barrel into which proteins fed

Life and Death of Proteins Proteosome Protein disposal apparatus dispersed throughout cytosolo Hollow cylinder of proteases form stack of 4 heptameric rings Ends composed of protein complex of ~20 polypeptides, 6 of which hydrolyze ATP to unfold proteins and move them into proteosome Acts on proteins marked by ubiquitin

Life and Death of Proteins Ubiquitin conjugating system Multiubiquitin chain on target proteins recognized by receptors on proteosome Targeted proteins: misfolded, oxidized, or other abnormal aa Linear series of ubiquitin conjugates linked via lysine on target protein

Life and Death of Proteins Regulated Destruction Activation of compments in proteolytic pathway E1, E2, E3 Degradation signals created in response to intracellular or extracellular signals All six pathways are used by cells to move protein into proteosome