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This 9 page Class Notes was uploaded by nicin88 on Monday January 25, 2016. The Class Notes belongs to BMB401 at University of Miami taught by T. K. Harris in Spring2015. Since its upload, it has received 48 views. For similar materials see Biochemistry for the Biomedical Sciences in Biochemistry at University of Miami.
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Date Created: 01/25/16
Lecture 10 Protein Translation Reading LIR Biochem6th Ch 31 I V Cell Factor Function lF2GTP Bring charged initiat elF2 GTP ing tRNA to P site lF 3 Prevent association elF39393 of subunits v 7 Bring all other H EF TuGTP f I f EF1 WGTP charged tlFlNAs to ASlte EFTs Guanine nucleotide E E F t 3y exchange factor B EFGGTP gt i EF2GTP Translocation n l l t item a HI nF1 2 Recognize quotstopquot eRF Godons RFSGTP Release of other RFS eRFSGTP Copyright2014Wolters Kluwer Health Lippincott WilliamsampWilkins Protein factors in the three stages of translation P prokaryotes E eukaryotes tRNA transfer RNA IF initiation factor EF elongation factor RF release factor 1 Describe how aminoacyl tRNA is generated 393 AminoacyltRNA Synthetases This is the essence of faithful translation of the genetic code and this one translated nucleic acid sequence into proteins by conjugating amino acids into tRNA gt aatRNA synthetases catalyze attachment of a single given amino acid to the 339 CCAOH of a tRNA molecule as specified by its anticodon i In both prokaryotes and eukaryotes there is a specific enzyme for each amino acid that correctly matches it With a tRNA that has a corresponding anticodon sequence ii The quotextremequot specificity in recognizing and conjugating cognate amino acids and tRNA ensures faithful translation of the genetic code 3 Reaction Mechanism Which part of the amino acids are conjugated together It is the carboxyl group that is conjugated to the 5 phosphate of ATP The beta gama phosphate are released as pyrophosphate 3 hydroxyl is the nucleophile that attacks the amino acids and AMP leaves gt activation First the amino acid aa is quotactivatedquot by reacting with ATP to form aminoacylAMP and pyrophosphate PPi The reaction is driven to completion by subsequent hydrolysis of PPi to 2Pi aa ATP lt gt aaAMP PPi gt Pi Pi gt transfer Second the amino acid is quottransferredquot to the 339CCA of tRNA to form aminoacyltRNA with release of AMP aaAMP tRNA lt gt aatRNA AMP 2 Know the components of prokaryotic versus eukaryotic ribosomes Prokaryotic 70S gt 50S 23S rRNA 5S rRNA and 34 proteins LlL36 gt 30S 16S rRNA and 21 proteins SlS21 Eukaryotic 80S gt 60S 28S rRNA 5S rRNA 58S rRNA and 50 proteins LlL50 gt 40S 35 proteins S1S35 3 Know the components and understand the steps in prokaryotic protein synthesis including i initiation ii elongation and iii termination Initiation 0 ShineDalgarno Sequence Not AT rich and not GC rich becaue its purine rich arginine and guanine gt ShineDalgarno sequence The quotShineDalgarnoquot sequence SD is a purine rich sequence located 610 nt 539upstream of the AUG start codon This region base pairs with 30S 16S rRNA near its 339 region which guides the quotAUGquot start codon to the quotPsitequot on the ribosome gt P site The quotpeptidyl sitequot is the tRNA binding site where peptide elongation occurs gt A site The quotaminoacyl sitequot is the site where the new incoming aminoacyl tRNA binds gt E site The quotexit sitequot is the site from which tRNA is released after it donates its amino acid to the growing peptide chain O fMettRNAf AUG encodes for Met its chemical modified This tRNA has the ability to recruit transformylase to formulate the amino group of Met gt tRNA quotformylmethionyl tRNAquot is a special tRNA that brings a modified form of Met to the ribosome to initiate protein synthesis i MettRNA synthetase catalyzes attachment of Met to the 339 CCAOH of tRNAf as well as the regular MettRNA molecule ii However only MettRNAfis recognized by quottransformylasequot which catalyzes transfer of a formyl group from N10formylTHF to the amino group of Met attached to tRNA f 0 Formation of 708 Initiation Complex gt quotInitiation factor3 quot IF3 binds the small 30S ribosomal subunit which i promotes mRNA binding and ii blocks premature association with the 50S ribosomal subunit gt mRNA binds 30S and the quotAUGquot start codon is guided to the quotPsitequot by the quotShineDalgarno SDquot sequence SD is a purine rich sequence located 610 nt 539 upstream of the AUG start codon which base pairs with a region of 16S rRNA gt IFl binds 30S quotAsitequot which blocks binding of fMet tRNA at that site and guides it to correct quotPsitequot gt IF2GTP binds fMet tRNA and loads it into the quotPsitequot IF2 is a quotGTPasequot that can effect structural changesby hydrolyzing GTP to GDP and Pi gt After proper loading IF2GTP interacts with aquotGTPase activating region GAR quot on 30S that stimulates GTP hydrolysis The resulting IF2 conformational change allows binding of SOS to form the quot705 initiation complexquot while simultaneously releasing IFl IF2 IF3 GDP and Pi Elongation 0 Delivery of aatRNA to A Site With different charged tRNAs keeps sampling but when the correct one goes in those Hydrogen bonds form so get complementary base pair in all 3 This factor with GTP binds recognizes that and its like trying different keys not working until the right one does When it does it signals EF Tu to hydrolyze GTP to GDP You do not phospholyrate GDP to make it GTP and GDP must come off and a new GTP comes on catalyzed by EFTs and its temperature sensitive 1 quotElongation factorTu with bound GTPquot EFTuGTP they tend to be temperature sensitive and unsensitive binds aatRNA and loads it into the quotA sitequot Like IF2 EFTu is a quotGTPasequot that can effect structural changes by hydrolyzing GTP to GDP and Pi 2 After proper loading EFTuGTP interacts with a quotGTPase activating region GAR quot that stimulates GTP hydrolysis resulting in the release of Pi and EFTu GDP 3 EFTs binds EFTuGDP and catalyzes exchange of bound GDP for GTP to regenerate EFTuGTP for next round of elongation EFTs acts as a quotguanine nucleotide exchange factor GEFquot Note Before delivery of next aatRNA can occur two additional steps must occur i a quotpeptide bondquot is formed between the two adjacent amino acids and ii the ribosome is quottranslocatedquot along the mRNAin the 339 direction to the next codon EF T u is the GTP binding protein that delivers the amino acid EFTs is the guanine nucleotide exchange factor that binds EF Tu and makes it let go of GDP to bond to another GTP Peptide Bond Formation gt After delivery of aa tRNA to A site fMet is transferred from tRNAf in the P site to the amino group of aatRNA in the A site gt quotPeptidyltRNAquot resides in the A site and quotdeacylated tRNAquot resides in the P site Peptidyl Transferase Center In the A site there is that free amino group that s the new nucleophile that attacks the carbonyl on the ester and the bond will break and the amino acid of P site transfer to the amino acid in the A site 1 Peptide bond formation is catalyzed by the quotpeptidyl transferase center PTCquot which is comprised of nucleotides from 23S rRNA in a cleft in the SOS subunit 2 Since no atoms from ribosomal proteins are in or near the reaction center PTC is a quotribozymequot Translocation 1 quotElongation factorG with bound GTPquot EFGGTP binds near the quotAsitequot Like IF2 and EF TuEFG is a quotGTPasequot that can effect structural changes by hydrolyzing GTP to GDP and Pi 2 EFGGTP hydrolyzes GTP and the resultingconformational change causes the 308 and SOSsubunits to rotate in opposite directions causing the mRNAtRNA pairs in the P and A sites to shift in a quotratchetlikequot manner Slte i deacylated tRNA shifts from P gtE siteii peptidyltRNA shifts from AP site 3 The elongation processi delivery of aatRNA to the A siteii transfer of growing peptide in P site to the aa tRNA in A site andiii tranlocationis repeated until a stop codon enters the A site 1 The growing peptide chain passes through a long quotexit tunnelquot in the SOS subunit and deacylated tRNA is released from the E site 2 The Nterminal fMet residue is usually removed before translation is completed Termination When a stop codon UAA UAG or UGA enters the A site it is recognized by quotrelease factorsquot RFl recognizes UAA and UAGRF2 recognizes UAA and UGA Both RFl and RF2 are proteins that resemble tRNA in size and shape RF3GTP is a GTPase that promotes binding of RF12 to the A site Hydrolysis of RF3GTP causes release of RF12 and the PTC is converted to an quotesterasequot The peptidyltRNA is hydrolyzed and the newly synthesized protein is released Following release of protein from the P site the mRNA and tRNA are also released and the 708 subunit binds the quotribosome recycling factor RRFquot RRF binds to the A site and recruits EFGGTP Hydrolysis of GTP causes dissociation of 708 to the individual 308 and SOS subunits IF3 binds to 308 and prevents reassociation withSOS and promotes binding of 308 with another mRNA 4 Know the components and understand the steps in eukaryotic protein synthesis including i initiation ii elongation and iii termination Initiation O 438 PreInitiation Complex Prokaryotes have a ShineDalgarno sequence that loads mRNA and there is NO mRNA IN EUKARYOTES 1 quotEukaryotic initiation factors quot eIFl eIFlA eIF3 and eIFS bind the small 408 ribosomal subunit eIF 3 blocks premature association with 608 eIFIIA block the quotAsitequoteIF 5 functions as a quotGTPase activating protein GAP quot specific for the eIF2 GTPase that delivers MettRNAi 2 eIF2GTP binds MettRNAi and loads it into the quotPsitequot which completes assembly of thequot43S preinitiation complexquot 0 488 Initiation Complex the mRNA is recognized by the cap binding complex so eIF4E binds this m7GTP Eukaryotes mRNA have lots of hairpins that prevents the ribosome from finding the Met the start codon 1 The quotcap binding complex CBC or eIF 4F quot binds to the mRNA 539 cap region The components of this complex include the following eIF 4E 539 cap binding proteineIF 4G a scaffold proteineIF4AB mRNA helicase ATPdependent 2 Multiple copies of quotpolyAbinding protein quot PABP bind the mRNA 339 polyA tail and tether it to eIF4G to form quotcircular mRNAquot which associates with 438 to complete assemblyof the quot48S initiation complexquot 0 488 Initiation Complex 1 eIF4AB helicase unwinds mRNA secondary structure in the 539UTR 2 eIFllA helps 488 complex to scan the mRNA in search of the first AUG start codon 3 Correct base pairing between MettRNAi in the P site and the AUG start codon of bound mRNA triggers the GAP function of eIF5 to cause hydrolysis of eIF2 bound GTP and its subsequent release 0 SOS Initiation Complex 2 GTP WERE HYDROLYZED TO FORM THE SOS INITIATION COMPLEX eIFSBGTP binds and promotes joining of the 608 subunit thereby releasing eIFllA eIF3 and eIFS Hydrolysis of GTP on eIF5B causes its dissociation leaving the quotactivequot 805 initiation complexquot Elongation gt gt 0 Delivery of aatRNA to A Site 1 eEFlOtGTP binds aatRNA and loads it into the quotAsitequot 2 After proper loading eEFlOtGTP interacts With a quotGTPase activating region GARquot that stimulates GTP hydrolysis resulting in the release of Pi and eEFlOtGDP 3 eEFlB binds eEFlOtGDP and catalyzes exchange of bound GDP for GTP to regenerate eEFlOtGTP for next round of elongation eEFlB acts as a quotguanine nucleotide exchange factor GEFquot Note Peptide bond formation and translocation must occur before delivery of the next aatRNA 0 Peptide Bond Formation gt After delivery of aa tRNA to A site Met is transferred from tRNAi in the P site to the amino group of aa tRNA in the A site gt quotPeptidyltRNAquot resides in the A site and quotdeacylated tRNAquot resides in the P site 0 Peptidyl Transferase Center Peptide bond formation is catalyzed by the quotpeptidyl transferase center PTCquot Which is comprised of nucleotides from 288 rRNA in a cleft in the 608 subunit Since no atoms from ribosomal proteins are in or near the reaction center PTC is a quotribozymequot Translocation 1 eEF2GTP binds near the quotAsitequot 2 eEF2GTP hydrolyzes GTP and the resulting conformational change causes the 408 and 608 subunits to rotate in opposite directions causing the mRNAtRNA pairs in the P and A sites to shift in a quotratchetlikequot manner i deacylated tRNA shifts from P gtE site ii peptidyltRNA shifts from A gtP site 3 The elongation processi delivery of aatRNA to the A siteii transfer of growing peptide in P site to the aatRNA in A site andiii tranlocationis repeated until a stop codon enters the A site 4 The growing peptide chain passes through a long quotexit tunnelquot in the 608 subunit and deacylated tRNA is released from the E site Termination gt When a stop codon UAA UAG or UGA enters the A site it is recognized by quotrelease factorsquot eRFl recognizes UAA UAG and UGA and it is a protein that resembles tRNA in size and shape gt eRF3GTP is a GTPase that promotes binding of RFI to the A site Hydrolysis of RF3GTP causes release of RFI and the PTC is converted to an quotesterasequot The peptidyltRNA is hydrolyzed and the newly synthesized protein is released gt Following release of protein from the P site the mRNA and tRNA are also released gt Dissociation of ribosomal subunits is mediated by eIFl 1A and eIF3 and re initiation by eIF5 Since the mRNA is quotcircularizedquot the SOS initiation complex is easily reassembled after termination near the polyA tail which is bound to the capbinding complex CAP 5 Know the components and understand down regulation of protein translation by i eIF and eEF phosphorylation eIF2 Phosphorylation In initiation blocks charged initiating tRNA to P site gt eIF 2 phosphorylation eIF2 is phosphorylated by a number of kinases that respond to different stress conditions This increases eIF2 affinity for eIF2 GEF and prevents GDPEIGTP which blocks translation initiation gt eIF 2 kinases eIF2 kinases respond to four main types of stress conditions In amino acid starvation In ER stress eg oxidative lin dsRNA e g viral infections relia heavy metal e g low heme eEF2 Phosphorylation Blocks translocation in Elongation gt eEF2 phosphorylation eEF2 is phosphorylated by a single kinase that responds to different stress conditions Similar to eIF2 phosphorylation exchange of GDPDGTP is inhibited which blocks translation elongation gt eEF2 kinase eEF2 kinase responds primarily to different types of nutrient deprivation Under such conditions it is activated by increased calcium levels It seems to be most relevant in neurons as well as any cancer cells As might expected it correlates With down regulation of the mTOR pathway 6 Know the antibiotics that inhibit prokaryotic translation e g streptomycin tetracycline chloramphenicol erythromycin and puromycin Antibiotic Step inhibited Streptomycin Initiation Tetracycline Elongation Chloramphenicol Elongation Erythromycin Translocation Puromycin Not applicable 7 Know the antibioticstoxins that inhibit eukaryotic translation e gcyclohexamide puromycin diphtheria toxin and ricin Antibiotic Step inhibited Cyclohexamide Translocation Puromycin Not applicable Protein Toxin Diphtheria toxin Elongation Ricin Elongation GTP hydrolysis Prokaryotes Eukaryotes Initiation 1 2 Elongation 2residue 2residue Termination 2 1
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