Class Note for BIOC 461 at UA
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Date Created: 02/06/15
FiguresNotes for Chapter 30 Page 1 of 20 Biochemistry 461 Fall 2007 CHAPTER 30 PROTEIN SYNTHESIS LECTURE TOPICS o TRANSLATION mRNA o protein COMPLEXITY STAGES o tRNAs and AMINO ACID ACTIVATION o tRNA re GENETIC CODE AND TRANSLATION o RIBOSOME STRUCTURE o TRANSLATION PROCESS o PROCARYOTIC EUCARYOTIC INHIBITORS KEY CONCEPTS 0 Translation the process whereby the genetic code represented in a mRNA is translated into an amino acid sequence ofa polypeptide 0 Once again E coli is the best understood system and will be discussed in detail but some features ofeucaryotic protein synthesis will be discussed 0 Translation involves over 100 different macromolecules making it much more complex than those for replication and transcription 0 Aminoacyl tRNAs aatRNAs are activated amino acid donors for protein synthesis The amino acid is covalently linked by its COOH group to the 339 or 239OH terminus of its cognate tRNA 0 aatRNAs are synthesized by aatRNA synthetases at least one kind per amino acid in reactions which require the energy form ATP hydrolysis 0 Errors occur in the process about once in 110000 aa s incorporated into proteins Several proofreading mechanisms contribute to assure this accuracy 0 Basepairing matches mRNA code words codons with recognition sequences anticodons for each amino acid 0 Protein synthesis occurs in the amino to carboxy terminal direction Amino acids are added to the COOH end of the growing polypeptide chain mRNA codons are read from 5339 direction 0 The ribosome is a ribozyme involved in catalysis of peptide bond formation Page 2 of 20 THERE ARE THREE DISTINCT STAGES OF PROTEIN SYNTHESIS Initiation Initiator tRNA binds to a start signal on mRNA and basepairs with the AUG codon The complex occupies the ribosome P peptidyl site Basepairing omeNA sequences nearthe start signal with 16S RNA in the small ribosome subunit is also required Requires GTP hydrolysis for energy Elongation begins when an aatRNA binds to the ribosome A amino acyl site Then a peptide bond is formed between the COOH group ofthe amino acid of the aatRNA in the P site with the NH2 group ofthe amino acid on an aatRNA in the A site The resultant dipeptidyltRNA is moved translocated from the A to the P site displacing the tRNA from the Psite to the E site exit site The process repeats powered by GTP hydrolysis until the stop codon is reached 23S rRNA is a ribozyme finvolved in catalysis of peptide bond formation Termination occurs when specific protein release factors for each stop codon cause hydrolysis of the nascent polypeptide chain from the peptidyl tRNA and its release from the ribosome THE CAST OF CHARACTERS INVOLVED IN PROTEIN SYNTHESIS See handout table FLOW OF GENETIC INFORMATION PROCESS RATES RNA and protein synthesis rates in e cell are more or less synchronized PROCESS quotMACHINERYquot RATES DNA REPLICATION DNA Polymerase l 10 basessec DNA Polymerase Ill 1000 basessec TRANSCRIPTION RNA Polymerase 50 basessec synthesis of mRNA for 200 aa protein takes 200 X 350 12 sec TRANSLATION Ribosomes 20 aa39ssec synthesis of a 200 aa protein takes 20020 10 sec Page 3 of 20 tRNA molecules have common features such as Fig 2936 7 to 15 modified bases per molecule these are usually not basepaired 339end is CCAOH The 339A is the amino acid attachment site About 50 of nucleotides are basepaired except for 3 loops and the extra TRANSFER RNA 0 1 73 to 93 bases long 2 3 539end is usually pG 4 5 arm 6 The anticodon loop has 7 unpaired bases with two modified bases 339to the anticodon 7 The 3D structure is L shaped with two major segments of double helix and bases in nonhelical regions are involved in tertiary Hbonding interactions The 339CCA is at the end of one arm of the L and the anticodon loop is at the other end 8 tRNA genes are often arranged in clusters in bacterial and organelle chromosomes VAnticodon J 1 loop terminus 5 OH t Amum mi attachmenr sire pimprmwiared 539 terminus gt5 p DHU loop nticodon loop AMI NO ACID ACTIVATION Formation of peptide bonds between amino acids requires energy that is provided by an activated intermediate an amino acid ester COOH group linked to 239 or 339OH of the ribose of the terminal 339A of a tRNA Fig292 Fig 34 7 tRNA is an quotadaptorquot which lets amino acids be recognized by specific codon m RNA anticodon tRNA interaction Page 4 of 20 L39Fransfer Fl NA Adenine NH 0 R H NH C70 tRNA Page 5 of 20 Fidelity of grotein synthesis One mistake in 104 amino acids is maximum allowable Accuracy relies mostly on the speci city of amino acid cognate tRNA recognition by aminoacyl tRNA synthetases Additional proofreading occurs when aatRNAs are delivered to ribosomes that are actively translating mRNA I TABLE 19l Accuracy of protein synthesis Probability of synlhesizing an errorfree protcin Fm ucnc of r o q 3 Number 01 ammo acid residues mscmng an Incorrect amino acid 100 300 1000 m 3 0366 0 049 0000 10 3 0905 0 m o 3wa 10 4 n 990 0070 0005 Wis 419 0997 UNI AminoacyltRNA synthetases catalyze ATPdependent aminoacylation oftRNA Fig2978 Aminoacyladenylate enzymebound is an intermediate and the reaction is driven by hydrolysis the reaction product PPi p815 There is at least one tRNA and one aminoacyltRNA synthetase per amino acidAminoacy tRNA synthetases differ in size and subunit structure Two classes ofthese enzymes recognize different faces oftRNA molecules Fig2914 p822 CLASS I CLASS tRNA Complex tRNA Complex Page 6 of 20 AminoacyltRNA synthetases have specific interactions with the anticodon and the 339CCA arm of tRNAs Fig291 1 ThrtRNAT 39synthetase specificity and fidelity an example The enzyme actually does couple Serine to tRNATh l in 100 to l in 1000 activations mreonine Valine Thr T 7 Val V But quotproofreadingquot occurs by Figs29911 1 quotFitquot of an amino acid in aminoacyl tRNA synthetase catalytic site 2 Editing by hydrolysis of incorrectly coupled amino acid from the bound aminoacyl tRNA Editing site Ionp Page 7 of 20 CODONANTICODON RECOGNITION 0 Codon recognition is specified by the tRNA anticodon not by the actual amino acid on the tRNA Chemically modified CystRNACys to AlatRNACys resulted in incorporation of Ala where Cys codons were present in hemoglobin mRNA This example shows one way that ieincorrectly aminoacylated wrong amino acid can be misincorporated into proteins p831 H30 2 P 395H Vquot H ATP AMP 39 H H H H C Meme tRNA l O39 r J39 O r Y LysreinylrtRVR 39HN quot tRNAquotquotquot Rang nickel st tRNAw sy ntllr tase u 0 CysrtRNAr AIa tnNA V 0 Base change mutations can alter codons so as to code for a different amino acid or to code for a stop signal Proteins are then synthesized that are structurally or functional altered or are prematurely terminated and often not functional 0 Wobble basepairing between the 339 base of codons and the 539base of anticodons allows multiple codon recognition code degeneracy by one tRNA p 832 and Table 283 a Codons differing in the first two bases are recognized by different tRNAs b The anticodon 539 base determines how many codons a tRNA recognizes 39 TABLE 293 Allowed pairings at the third base of the codon according to the wobble c d39 hypothesls quot LE FE 1 First base of Thlrd base of quotlziw anticodon cndon hr H C G Adenosll ieJanm quot A U has pair U A or C quotchclrquot H G U or C 39 E 7 I U C or A Undlnerinosine lt base Pair Page 8 of 20 RIBOSOME STRUCTURE 0 Ribosomes of E M are 708 and consist of a large 508 and small 308 subunit Figs 291516 3418 Each ribosome component occurs in unit stoichiometry except 2 molecules each of large subunit proteins L7 and L12 rRNAs are derived from precursors by RNA processing see Ch28 notes rte 30 subunit Peptidy GT 58 39ztransferase 23S RNA 55 RNA slt39 4K 505 subunit 34 proteins O 503 subunit 70 ribosome w 16 RNA 705 ribosome 30 subunit 21 DfOlelHS 505 subunit 705 ribosome SOS subunit Page 9 of 20 There are 20000 ribosomesE ci cell are 25 ofmass of the cell Ribosomes selfassemble from mixtures ofthe rRNA and proteins Bases in rRNA molecules are over 60 basepaired 235 rRNA functions as a ribozyme in peptide bond synthesis Small nbosome subunit 16 rRNA baspairing and folding In three dimensions rRNAs are extensively folded and in close contact with ribosomal proteinsFig 29 17 Page 10 of 20 TRANSLATION PROTEIN SYNTHESIS 0 Protein synthesis is in NH2 to COOH direction as shown by labelling experiments and the mRNA is read from the 539 to 339 direction 0 Polyribosomes are complexes of mRNA and ribosome 1 ribosome80 RNA bases maximum density in which each successive ribosome on mRNA from 539 to 339 endis nearer completion of protein synthesis as it reaches the end of the message Figs 2919 3425 Nearly completed protein Beginning of protein e was OVERVIEW of PROTEIN SYNTHESIS 2 major functions decoding and peptide synthesis A Page 11 of 20 Three tRNA binding sites on the 708 ribosome are Figs292122 A aminoacyl tRNA site P peptidyl tRNA site E exit site 3 mRNA A site F site P site The growing polypeptide chain exits the ribosome while bound to the P site and passes through a channel a hole in the 508 ribosome Protein exit channel in 505 subunit Growing protein passes through channel Protein doesn t fold until it s out of exit channel 505 cut in half and opened like a book Page 12 of 20 RNA movements and polypeptide chain growth The growmg po ypepude 5 passed from the RNA at me P sue to the RNA at me A sue dunng pepude bond Synthema The RNA at the A sue then 5 trans ocated moves to the P sue The RNA 015th at me E sue 5 re eased WhHe me tRNAfrom me P sue stranmocated to me E sue ng 29 24 0 Wwwm pepuaewm hmdmg ruxmanou m Tvanslammm angnmu mm p cm m mm msmmm Page 13 of 20 TRANSLATION CYCLE THE PROCESS INITIATION 0 The AUG start signal is preceded by a conserved ShineDalgarno sequence Figs 2920 3428 that base pairs with the 339end of 168 rRNA to align the mRNA and the 308 ribosome subunit CCACo AAAucquuccAACGCUA E colirrpA UUUGGAUUGAAACGAUGGCGAUUGCA E coliaraB GGUAAC AACAACCAUGCGAGUGUUG E colithrA CAAUUCAGMGEGAAUGUGAAACCAGUA EcoIiacl AAUCUUCUUUUUUA39UCGUUCGU ucu x174 phageA protein U AA CU LZ5 1IEUGAA AU GCAUGU CUAAG ACA QB phage repiicase R17 phage A protein A phage cro 3 H g 339 and of 1 8 RNA A U 6 r U C U C CH if IA 5 3 GALJ uccu rsm ieu uquccuiAuobGAEG cuiu UUiAGU Messenger FINA W191quot Arg 397 Aia 1 F heLScr Polypsmude 0 The initiation codon AUG base pairs with the anticodon on fMettRNAquotVlet Fig 3428 in the P site and in the presence of GTP and 3 protein initiation factors forms a M 5 initiation complex 308 subunit mRNA fMet 0 gt7 Mquot tRNAf39V39e Fig 2927 mm mm mm Ynmwi ierraiwdwiuialo mmmmwrasp Tmmmrmm o H H s N CHS olt H i rmmylmmnlnnynmp IRMA Imemnmw Page 14 of 20 The 308 initiation complex combines with a 508 subunit to yield a m initiation complex GTP hydrolysis to GDP and Pi and release of F1 and F2 also occurs Fig 2927 305 ribosomal subunit k Initiation fa ETHI S S OSIFllFS F7 GTPjgtfMel IRMA mRNA IFS fMet ms initiation complex 50395 subunit H20 m IFquot GDP pi 105 initiation to triple Page 15 of 20 ELONGATION See also Fig2924 Elongation factor protein EFTu delivers aminoacyltRNAs to the ribosome to the A site with codonanticodon basepairing The EFTuaatRNAGTP complex dissociates with GTP hydrolysis and release of EFTuGDP The EFTu is recycled to the GTPform which binds a new aatRNA for delivery to the ribosome Fig3430 EF Tu doesn t bind to fMel tRNAWe but MettRNAMe does bind EF Tu and is used for all internal methionines Also lF2 is only used for fMet tRNAWe recognition EfTu is a Gprotein with an amino terminal P loop NTPase domain Fig 2928 Proofreading by EFTu GTP hydrolysis The GTPase rate of EFTu is rate limiting for protein synthesis and acts as a proofreading step Mistakes are reduced by increasing the time in msec allowed for checking out the codon anticodon match Proofreading occurs before and after hydrolysis of the EFTu bound GTP Error rates are about 1X1 0 4 giving synthesis of mostly functional of incorrect aaRNA proteins Fig 3432 H20 P EFTuGDP EFTuGTP E Z EFTuGDP if aatRNA a Peptidebond aatFlNA aatFlNA formation Dissociation Dissociellun of incorrect aaetRNA Page 16 of 20 Peptidyl transferase peptide bond formation is catalyzed by a ribozyme activity of the 503 ribosome subunit called peptidyl transferase A peptide from the P site is transferred with peptide bond formation to the amino acid of the aatRNA in the A site Adenine 2451 of 238 rRNA catalyzes the peptide bond formation reaction P slle A site P size A site H 0f if H o H 1439 aquot f Papiidvi 04f N NH quotmien H i I A i n fiH R H Rr H oc 0 0 i i W iRNA RNA IRNA RNA ranNA Aminouyl Dlaqland VnptidylrlRNA mNA mun 505 Peptidyl transferase A ribozyme activity A site Green P sitered 23 RNAcatalyzed peptide bond formation A Site Page 17 of 20 Translocation The uncharged tRNA in the P site is released and the mRNA moves 3 bases in the nbosome thus shifting the peptidyltRNA to the P site This process is called translocation Then a new aatRNA occupies the A site and the process repeats Translocation requires GTP hydrolysis and a protein factor called EFG that is also a GTPaseThis G protein is a mimic ofthe EFTuaa tRNA complex its carboxy terminal region with Bsheet domains shaped like a tRNA molecule and its amino terminal domain similar to EFTu Fig2929 30 Guanine nucleotide m I E z gt 39 O lz Page 18 of 20 Release RF factors Protein synthesis stops when a stop codon is located in the A site These codons are recognized by release factor RF proteins RF1 recognizes UAA and UAG RF2 recognizes UAA or UGA After RF binding peptidyltransferase is activated and the nascent peptide is hydrolyzed from the tRNA followed by release of the tRNA mRNA and ribosome subunit dissociation After binding lF 3 the 308 subunit can participate in another translation cycle A eucamotic release factor has a tRNA fold that mimics the acceptor stem GlyGlyGln binds a water molecule that can participate in hydrolysis of the peptidyltRNA bond at the peptidyl transferase center catalyzed by the 508 ribozyme catalytic activity center Fig2932 Ribosome release factor RRF procaryotic has 0 helices that mimic tRNA conpared with the Bstrand nature of the tRNA mimic in EF G RRF is required along with GTP hydrolysis catalzed by EF G for release of the mRNA and the last tRNA from the ribosome EUCARYOTIC PROTEIN SYNTHESIS Page 19 of 20 Translation process and machinery are similar to procaryotes in concept but components of ribosomes are larger and the process involves more nonribosomal proteins Eucaryotic ribosomes are 808 with 408 and 608 subunits containing 188 and 58 288 and 588 r RNAs respectively and about 100 proteins Fig 3436 Initiation tRNA uses Met not fMet and is a special tRNA Start signal is usually the AUG nearest the 539end of mRNA 408 subunits attach to the 539 cap and then search 539a339 sliding for the first AUG in an ATP hydrolysisdependent process mRNAs code for only one protein ie one continuous polypeptide chain Initiation complexes more initiation factors elFs Elongation EF10 and EFB function like Ef Tu and Ef Ts EF2 drives translocation GTP hydrolysis required Termination one release factor Eef2 605 subunit mm 185 RNA M 405 subunit 50 nm 500 A mRNA Cap Initiation fartnr 4 me Me tRNA 4CIS subunit NW1quot ms subunit with imitation 39 components n ATP in ADP Fl tMPr E505 subunit lanI TlUn tarmrq EDS initiation complex Page 20 of 20 INHIBITORS OF TRANSLATION Table 344 0 Numerous antibiotics inhibit different stages of protein synthesis Table 2924 0 Puromycin looks like an animoacyItRNA It binds to the A site forms a peptide I TABLE 294 Antibiotic inhibitors of protein synthesis Antibiotic Action Streptomymn and 011191 inhibit initiation and cans misreading oi niRNA aminogiycosid s iprokaryctcsi Tetracycline Binds to the Su subunit and inhibits binding of aminoacyliRNs prokarymesi tIhloramphcnicoi inhibits the pcptidyl ii axxsib Iibosomai subunit i pmkt 39 Cycloheximidc Inhibits thu peptidyl transfciusc activity of the 705 I ibosomai subunit icukaryotcsr Erythroniycin Binds to he 5 subunit and inhibits transiocalion iprokai39yoies Puromycin Causes premature chain termination by acting as an analog of aminoacyirinA proix39ar39yotas and eukHi39ynlcsi bond with the growing polypetide and falls offthe ribosome So it is an elongation inhibitor also its binding to the A site prevents aminoacyItRNA from binding to the A site Binding of puromycin was used to identify existence of the A site NH N H 0 Haco HN 0H 0 H NHI AminoatyHRNA Fummydn o Streptomycin both inhibits initiation and causes misreading of codons Its site of action is associated with protein 812 and with specific 16S rRNA sequences
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