329 Class Note for B M B 400 at PSU
329 Class Note for B M B 400 at PSU
Popular in Course
Popular in Department
verified elite notetaker
One Day of Notes
verified elite notetaker
verified elite notetaker
One Day of Notes
verified elite notetaker
verified elite notetaker
verified elite notetaker
This 31 page Class Notes was uploaded by an elite notetaker on Friday February 6, 2015. The Class Notes belongs to a course at Pennsylvania State University taught by a professor in Fall. Since its upload, it has received 43 views.
Reviews for 329 Class Note for B M B 400 at PSU
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 02/06/15
BMB 400 PART THREE V 2 Chapter 14 Translation B M B 400 Part Three Gene Expression and Protein Synthesis Section V Chapter 14 TRANSLATION A reminder mRNA encodes the polypeptide with each amino acid designated by a string of three nucleotides tRNAs serve as the adaptors to translate from the language of nucleic acids to that of proteins Ribosornes are the factories for protein synthesis Figure 351 mRNA is translated into a polypeptide aa 1aa 2aa n1 33 n lt aminoacyltRNAs 5 UGA cap A UC inquot UAGquot zAAUAAAr AAAAAAA 1 UAA Sta 0an for 0an for translation 33 n1 Stop translation aan A tRNAs 1 The transfer RNAs or tRNAs serve as adaptors to align the appropriate amino acids on the mRNA templates 2 Primary structure of tRNAs a tRNAs are short being only 73 to 93 nts long b All tRNAs have the trinucleotide CCA at the 3 end 1 The amino acid is attached to the terminal A of the CCA 2 In most prokaryotic tRNA genes the CCA is encoded at the 3 end of the gene No known eukaryotic tRNA gene encodes the CCA but rather it is added posttranscriptionally by the enzyme tRNA nucleotidyl transferase c tRNAs have a large number of modified bases Over 50 different post transcriptional covalent modifications are known in tRNAs such as dihydrouridine D in which the double bond between C4 and C5 is reduced or pseudouridine 1p in which C5 is replaced with a N providing another endocyclic amino group The modified bases are especially prevalent in the loops EME mm mm THREEV v z chapm u Tnnslaonn A 339 C Accepmx c um gm 352 Secamalysmmm mum a msncmdzrymmmemtRNAsadmalul mm mm annsthhilnaps SeeFxgmi 52 fuxyeas phznylalamm mm m ammn Emmy arm 1 mm by camplzmzmaryhaserpamng between nu ma 7 ms mam am a shun segmzm mar m 3 and Agam thzammacldwmheaddzdm hmnmmllk m D arm end m m D mp ale ammm D camm swam dlhydlmmdmzs whlch m amendml arm era In anoman mp m amwadnms mm m m mm m mp n ma align a a 5 mm m mum nadmg 5 a 3 m val13h mp vamsmsm mmmmm m m enme mm between m 73 m vemls 9 m mm x mm m m val13h mp m 1v armxsnamzdfvnhls hlgmycamewedmnuffnummthz mp BMB 400 PART THREE 7 V Chapter 14 Translation 4 The tertiary structure of tRNA is a quotfat Lquot See Fig 353 a Some nucleotides in the D loop form base pairs with some nucleotides in the TwC loop These and other interactions bring the cloverleaf secondary structure into an inverted L shape with the quotadditionalquot base pairs found mainly at the junction of the inverted L b In the 3D structure two RNA double helices are at right angles One of the double helices is the TwC stem in line with the amino acid acceptor stem The other double helix has the D stem in line with the anticodon stem c The result is that the two quotbusiness endsquot of the tRNA are widely separated in space at the two extremes of the tRNA That is the amino acid acceptor site is maximally separated from the anticodon Figs 353 d The rest of the molecule is a complex sur ce that must be recognized accurately by am inoacylrtRNA synthetases Attach amino acid Anticodon Fig 353 3 D structure of tRNA A chime tutorial on tRNA structure is available from Dr William McClure39s website at Camegier Mellon University httpinfobiocmueduCoursesBiochemMolstRNAiTourLRNAiTourhtrnl BMB 400 PART THREE 7 V Chapter 14 Translation B Attachment of amino acids to tRNA 1 AminoacyltRNA synthetases a Approximater 20 enzymes one per amino acid b Must recognize several cognate tRNAs ie that accept the same amino c acid but recognize a different codon in the mRNA a consequence of the degeneracy in the genetic code Must not recognize the incorrect tRNA 7 ie these enzymes require precise discrimination among tRNAs Two different classes of aminoacylrtRNA synthetases The two classes of enzymes are distinguished by the structure of their tRNArbinding regions The different classes of enzyme approach and bind to different faces of the tRNA but both must recognize the ends as well as any distinguishing features of the their cognate tRNAs Each class has about ten synthetases for ten amino acids Adumsina triphnspllalz ATP Fig 354 3D structure of GlutaminyltRNA synthetase BMB 400 PART THREE V 2 Chapter 14 Translation The two classes of enzymes do not resemble each other much at all in either sequence or 3 D structure leading to the suggestion that they have evolved separately If so this would imply that an early form of life may have evolved using the ten amino acids handled by one class or the other of synthetase 2 Mechanism a Aminoacvl tRNA svnthetase catalvzes a 2 step reaction Fig 355 First the amino acid is activated by adenylylation ie a mixed anhydride intermediate is formed between the COO of the amino acid and the oc phosphoryl of ATP with the liberation of pyrophosphate The intermediate activated amino acid is an aminoacyl AMP In the second step the amino acid is transferred to the 339 or 2 OH of the ribose of the terminal A of tRNA with liberation of AMP The product aminoacyl tRNA retains a high energy bond in an ester linkage a The equilibrium constant is about 1 for each of the two reactions so the high energy of the bond initially between the 0L and 5 phosphoryls of ATP is essentially still present in the ester between the amino acid and the ribose of tRNA b The high energy bond in aminoacyl tRNA provides a driving force for protein synthesis Hydrolysis of pyrophosphate abbreviated PPi to two phosphates provides the free energy to drive synthesis of the aminoacyl tRNA Thus one can consider that the equivalent of 2 ATPs ie two high energy bonds are used to form aminoacyl tRNA but one of the high energy bonds is retained in the product ATP gt AMP PPi PPi gt 2 Pi In both instances the cognate tRNA must be bound before proofreading can occur BMB 400 PART THREE V 2 Chapter 14 Translation Addition of amino acids to tRNAs occurs in two steps catalyzed by aminoacyltRNA synthetase lwl NH2 NH2 0 o o o N N 0 039 N I I q l gt II IN I RCHCO o o olploc O N NJ I R3HC O OCll20 N7 NH 0 o o NH3 0 OHOH HOH aminoacid ATP aminoacylAMP a mixed anhydride PPi NH2 0 o O N N H N IV 39o IPoc e l 739 Fl3HC O ITOC O N H 0 N N o NH3 0 HOH AMP N gt lt HOH aminoacylAMP NH2 o N NH tRNA I lt l N r of 0 r tRNAoFIOC O N hr 0 o I OH CO OH OH H NCH tRNA 3 i the terminal A nucleotide is shown R aminoacyltR NA Overall reaction amino acid ATP tRNA I aminoacyltRNA AMP PPi Figure 355 3 Precise discrimination by AAtRNA synthetases a These enzymes must recognize the correct tRNA and the correct amino acid at the initial binding steps b Proofreading is the removal of the incorrect amino acid or tRNA after binding and often after part of the enzymatic reaction has occurred This can occur at either of the two reactions some synthetases will cleave an incorrect aminoacyl adenylate intermediate and otheIs will BMB 400 PART THREE V 2 Chapter 14 Translation add the incorrect amino acid to the tRNA before recognizing the mistake and cleaving off the incorrect amino acid C Anticodon determines speci city The anticodon determines specificity for incorporation into a polypeptide during translation not the amino acid This was shown in the following experiment a Cys tRNACys can be converted to Ala tRNACys by reductive desulfuration H and Raney nickel releasing H28 b The resultant Ala tRNACys retains the ACA anticodon to match a UGU codon in mRNA When tested in cell free translation it causes alanine to be incorporated instead of cysteine Fig 344 c Thus the amino acid on the tRNA did not direct its incorporation into the growing polypeptide chain the anticodon did Figure 356 The anticodon determines specificity for incorporation of amino acids into polypepetides 0 Cquot II CACCIDHCHZSH 99A 39CCI HCHa NH2 NH2 H23 H and Ni C S CysteinyltRNA y AlanyItRNAcyS Test for incorporation into a polypeptide directed by a UGU codon in mRNA normally encodes cysteine Cysteine is incorporated into the polypeptide Alanine is incorporated into the polypeptide BMB 400 PART THREE V 2 Chapter 14 Translation D Special tRNA for intiation of translation 1 Although Met has a single codon two different tRNAs with different functions recognize the AUG codon 1 tRNAfmet often abbreviated tRNAf is used for initiation or translation in bacteria A comparable initiator tRNA called tRNAi is used in eukaryotes 2 tRNAmIIlet is used for elongation Figure 355 I Different methionyItRNAs used for initiation versus elongation I A Different tRNAs o 0 539 CVCACCHCH 2CH 2SCH 3 A CCHCH 2CH 2SCH 3 iH 39 H2 HC 0 met m et formylmethionyItFiNAf methion ytRNAm Used at initiator AUG oodons Used at internal AUG codons B Elongation steg in protein synthesis CH3 SH CH2 0 CH3 nz EH2 II 5 I NH 2 CH c Ace I NHZ CH C NH CH C ACC 539 methionyltRNATnel alanylcysteinyltRNA CV5 peptide bond formation 3 W o o 0 CH3 II IH2 H CH2 u NHZ CH C NH CH C NH CH c Ace met alanylcysteinylmethionyltRNA m BMB 400 PART THREE V 2 Chapter 14 Translation In bacteria a formyl group is added to the amino group on the charged Met tRNAf using 10 formyl tetrahydrofolate as the formyl donor This prevents its use in elongation In bacteria only formylmethionyl tRNAf can bind to the partial P site on the small ribosomal subunit see below to initiate translation at AUG or GUG less frequently or UUG rarely In all three cases the protein starts with formylmethionine The formyl group is removed after the first several amino acids have been incorporated and in about half the cases the methionine is also removed Note that the meaning of AUG and GUG is dependent on the context AUG or GUG at the initiation site encodes formyl Met but when internal to the mRNA they encode Met or Val respectively tRNAf has a different structure from tRNAm and these differences determine their use either in initiation or elongation In eukaryotes Met tRNAi is used for initiation Although it is not formylated the basic process is similar to that in prokaryotes E Ribosomes 1 Role of ribosomes a Ribosomes are the molecular machines that catalyze peptide bond formation between a growing polypeptide and an incoming aminoacyl tRNA The ribosomes insures that the amino acids are added in the order specified by the mRNA b Ribosomes associate reversibly with the mRNA The two subunits of the ribosome form a complex around the mRNA to translate and then dissociate after translation is completed 2 Size and Composition of large and small subunits see Fig356 a Ribosomes quotribonucleic acidquot quotbodiesquot are large complexes of RNA and protein with a roughly 6040 ratio between RNA and protein There are two subunits Similar components are found in both eukaryotes and prokaryotes although their sizes differ Each subunit has one major RNA in bacteria 23S rRNA for the large subunit and 16 for the small subunit and many proteins 31 and 21 respectively for bacterial large and small subunits The large subunit also has a small rRNAs about 120 nucleotides in size SS RNA Eukaryotic large ribosomal subunits have an additional small RNA 588 that corresponds to the sequence of the 5 end of bacterial 23S rRNA BMIB 400 PART THREE 7 V Chapter 14 Tmnslation The bacterial ribosome is composed of three different RNA molecules and more than 50 different proteins arranged in two rmjol39 subunits which join together to form the complete ribosom During protein synthesis the ribosome binds transfer RNA molecules in three different sites In this image the ribosome with transfer RNAs in all three binding sites the large subunit is gray the small subun is violet and the nee mnsfel39 RNA are green blue and fed Image is from the Center for Molecular Biology of RNA 11 clmentsucscedu99000972 7l39ibosomeal1htrnl Figure 153 Images of ribosomes based on 3D structure determination The top view is from the Noller lab at UCSC the bodom is from the Steitz lab and collaborators at Yale The bodom view shows the RNA in silver ribbons and protein as gold coils A green tRNA is at the peptidyl uanxferase site hrage from hnpwww npaciedufeaurres01l0505Us01html BMB 400 PART THREE V 2 Chapter 14 Translation c The rRNAs and subunits were initially characterized by their sedimentation velocity and hence are referred to by their sedimentation value in Svedberg units or S Larger macromolecules and complexes sediment faster and have a higher S value However other factors play a role in sedimentation rate such as shape and the S values for a complex is not the sum of the S values of individual components 3 Shape a The small subunit is fairly elongated and binds mRNA b The large subunit is more spherical and covers the small subunit c The mRNA may thread between the 2 subunits or it may lie outside the ribosome BMB 400 PART THREE 7 V Chapter 14 Translation 4 P peptidyltRNA and A aminoacyltRNA and E exit sites A LRNA interacts with the ribosome at three major sites as it brings in an amino acid has the growing polypeptide chain attached to that amino acid and then nally leaves the ribosome after donating its amino acid a A site or entry site aminoacylr tRNA binds b P site or donor site peptidylitRNA binds ie the nascent polypeptide chain linked to the last LRNA to occupy the A site see below c E site exit of deacylated LRNA after peptide bond formation d Flow of tRNA through the ribsoome is from the A site to P site then exitVia the E site e The next point will become clearer after we discuss the elongation cycle The molecule attached to the 339 end of the tRNA is different at each site 3 sites on ribosome for interaction withtRNAs S H 92 0 a W 0 W cm i 5 wch 7wch 7m 5 mic no Exit site for m f asnycysmmywmcye thmnw mm ree tRNA ms um PeptidyIIRNA aminoacyItRNA Fig 359 BMB 400 PART THREE 7 V Chapter 14 Translation F The polarity of translation is from the amino N terminus to the caboxy C terminus This was demonstrated in a classic experiment by Dintzis 1 Actively translating proteins were labeled with radioactive amino acids for a brief time short relative to the time required to complete synthesis Completed polypeptides were collected digested with trypsin and the amount of radioactivity in tryptic fragments as determined Tryptic fragments from the Crterminal end of the polypeptide had radioactivity at the earliest times of labeling As the period of labeling was increased longer pulse tryptic fragments closer to the N terminus were labeled This shows that the direction of polypeptide growth is from the N teminus to the C terminus ie translation begins at the N terminal amino acid This corresponds to mRNA chain growth in a 539 to 339 direction Note that this experimental protocol is also used to map origins of replication as we covered in Part Two of the course Polarity of translation Polypeptides being Completed Wabaed row synthesized polypeptides abebd prgtem 0 min Digest With trypslri Synthesis lrl presence of V labeled amino adds Smin N c we mm 1 gt N C Label appearslirs in the C terminal tryptic peptides showing that the C terminus is synthesized last Thus the diredion ot translation islrom N to C terminus Fig 3510 BMB 400 PART THREE V 2 Chapter 14 Translation G Initiation of translation 1 mRNA binds to small ribosomal subunit not the whole SOS ribosome in such a way that the initiator AUG is positioned in the precursor to the P site ie ready for the f met tRNAfIIlet to recognize it a The alignment of the initiator AUG in the mRNA with the appropriate place on the ribosomal subunit involves base pairing between the 3 end of 16S rRNA and a sequence that precedes the initiator AUG in mRNA When this portion of 16S rRNA in the 23S subunit is removed by cleavage with colicin an antibiotic the 23S subunit loses the ability to initiate translation Figure 3511 Choice of the correct AUG as the initiator is mediated by basepairing between a ribosome binding site in the 539 untranslated region and the 339 end of 16 S rRNA Pyrimidinerich tract 339 end of 16 S rRNA 339 OH HMACUA 539 539 ends of mRNAs laCZ 5 39 ACACAGGAAACAGCIRUG 3 39 trpA 5 39 ACEAGGGGAAAUCUGAUG 3 39 RNA polymerase 5 39 GAGCUEAGGAACCCIRUG 3 39 ribosomal protein L1 5 39 CCAGGAGZAAAGCUPAUG 339 Purine h tract comprising the Initiation ribosome binding site codon b The ribosome binding site is in the 5 untranslated region just before the initiator AUG It is also called a ShineDalgarno sequence named for the discoverers of the sequence It is a purinerich sequence eg 5 AGGAG that will pair with the pyrimidine rich 3 end of 16S rRNA 539 CCUCCUUA OH 339 c This base pairing insures the choice of the correct AUG as initiation codon as opposed to an internal AUG BMB 400 PART THREE 7 V Chapter 14 Translation 2 Roles of initiation factors and other factors a Translation factors are used at onl one st of the rocess and are not Enhanth subunits of the ribosome They cycle on and off the ribosomes as they do their function They are frequently present in smaller amounts than the ribosomal subunits Initiation mRNA binds to small subunit mRNA Small ribosomal subunit keeps the large and May aSSIst 1 small ribosomal Binding of F2 subunits apart 0 D Figure 3512 b IF3 Initiation Factor 3 1 An antjassociatjon factor prevents axociation between the large and small ribosomal subunits 2 It also must be associated with the small subunit for it to form an initiation complex ie for the small subunit to correctly bind mRNA and fmetrtRNAf 3 It dissociates prior to binding of the large subunit BMB 400 PART THREE 7 V Chapter 14 Translation fmettRNAf binds to small subunitmRNA fmetAc 539 g V 5 me formylmethionyltRNAf l Fig 3513 c IF2 l Brings fmettRNAf to the partial P site on the small subunit 2 At least in eukaryotes it does this in a ternary complexwith IFZ fmetrtRNAf and GTP In bacteria the GTP may bind the initiation complexseparately In some texts such as MBOG p 412 the GTP7 IFZ complex binds to the 303 subunit separately from fmeHRNAf How would you test the differences in these two models 3 IFZ activates a GTPase activity in the small subunit The resulting change in conformation may allow the large subunit to bind BMB 400 PART THREE V 2 Chapter 14 Translation GTP hydrolysis allows dissociation of factors Fig 3514 d GTP Hydrolysis stimulated by IFZ promotes dissocation of IFZ IFl and IF3 from the initiation complex and association of the SOS subunit e IFl role is unknown perhaps it is an assembly factor that assists in the binding of IFZ Binding of large subunit produces ribosome ready for elongation fmetAc 539 1 Large subunit binds Fig 3515 3 Binding of SOS large subunit to initiation complex gives a complete ribosome ready for the elongation phase of translation Note that f met tRNAfmet is positioned at the P site It has recognized the initiator AUG in the mRNA BMB 400 PART THREE V 2 Chapter 14 Translation 4 Identi cation of initiator AUG in eukaryotes a Bases around AUG in uence efficiency of initiation 1 The most important effects are from a purine 3 nt before AUG and a G after it The preferred context is RNNAUGG 2 The consensus sequence for a large number of mRNAs is GCCRCCAUGG but these other nucleotides have little effect in mutagenesis experiments a Modified scanner model 1 The mRNA is quotpreparedquot for binding to the ribosome by the action of eukaryotic initiation factor 4 abbreviated eIF4 Fig 3516 eIF4 is a multisubunit factor it includes a cap binding protein eIF4F that recognizes the 5 cap structure It also includes proteins eIF4A and eIF4B These are RNA helicases which unwind secondary structures in the 5 untranslated region of the mRNA at the expense of ATP hydrolysis The mRNA then binds to the small ribosomal subunit The met tRNAi has already been brought to the small ribosomal subunit by eIFZ in a complex with GTP eIF3 keeps the small ribosomal subunit apart from the large subunit during the process of binding the mRNA 2 The small subunit with associated factors scans along the mRNA until it reaches usually the first AUG Factors eIFl and eIFlA help move the preinitiation complex to the AUG start BMB 400 PART THREE V 2 Chapter 14 Translation Initiation of translation in eukaryotes elF2 GTP MettFiNA i V GTP MettFiNAi 408 ribosomal subunit small subunit elFS keeps 408 subunit free ATP elF4A and elF4B unwind hydrolysis structures at 539 end cap 0 4n GTP 408 ribosomal subunit migrates along mFiNA to 1st AUG with quotKozak consensusll RNNwG elF5 is a GTPase required for joining with large subunit 608 and release of elF2 and elFS Translation intiation complex with MettFiNAi at the AUG and the 2 subunits together Fig 3516 BMB 400 PART THREE 7 V Chapter 14 Translation H The elongation cycle during translation 1 Binding of aminoacyltRNA to the A site Recent review Weijland A and A Panneggiani 1994 TIBS 191887198 Schroeder R 1994 Nature 370597 a Elongation factor EFTu 1 The ternary complex of aminoacylrtRNA EFiTu and GTP brings the aminoacylitRNA to the A site on the 708 ribosome fig 3517 2 After the aminoacylitRNA is deposited at the A site of the ribosome the GTP is cleaved to GDP Pi The binary complex of EFiTu and GDP dissociates from the ribosome 3 This is one of manv examples of guanineinucleotideibinding proteins that are active when GTP is bound and inactive when GDP is bound The general model is that the GTPrbound state of EFiTu adopts a conformation with a high affinitv for aminoacvlrtRNA The conformation shape charge density etc of the resulting ternary complex containing EFiTuGTP and aminoacylitRNA then allows it to bind to the A site of the ribsosome Hydrolysis of GTP to form GDP and inorganic phosphate causes the EFTu to adopt a different conformation The aminoacylitRNA now has a lower affinity for EFA Tu in the GDP bound state and presumably a higher affinity for the A site on the ribosome so it stays on the ribosome when EFiTu in the GDP bound state dissociates both from aminoacylitRNA and from the ribosome Figure 3517 EFTusz cycle for binding aatRNAs Bind l GTPase Pi A 33 fMetAla Sequot lt BMB 400 C d PART THREE V 2 Chapter 14 Translation 4 EF Tu is one of the most abundant proteins in E coli at 70000 copies per cell This is almost equal to the number of aminoacyl tRNAs per cell so most of the aminoacyl tRNAs are likely to be in the ternary complex when the concentration of GTP is suf ciently high GTP 1 Required for binding aminoacyl tRNA 2 Hydrolysis promotes dissociation of the complex EF Tu plus GDP from the ribosome EFTs 1 Aids in the recycling of EF Tu bV GDP GTP exchange 2 EF Ts binds to EF Tu complexed with GDP causing dissociation of GDP GTP can now bind to the EF Tu Ts complex causing EF Ts to dissociate and leaving EF Tu complexed with GTP This latter binary complex is ready to bind another aminoacyl tRNA The antibiotic kirromycin prevents release of EF Tu GDP thereby blocking elongation This demonstrates that one step must be completed before the next can take place and illustrates the importance of the EF Tu GTP GDP cycle BMB 400 PARTTHIREE e V Chapter 14 Translation 2 Peptidyi usinsfersise on the large nbosornai subunit a The peptidyl uansferase reaction occurs via nucleophiiic displacement The amino group from aminoacyHRNA position rt attacks the quotC4enninalquot carboxyl group of peptidylrtRNA position as in the mRNA This results in cleavage of the high energy peptidyHRNA ester linkage thereby providing the free energy to drive the reaction The resulting products of the reaction are deacylated tRNA at the P site and pepti dyHRNA at the A site Figure 3518 Peptidyl tmnsfemse reaction a Elonganon SQ m Elmaquot smmrsis 0H t Psite nauseatinrw me i Ashe CH3 t 5H cg cm at 4M NWch scenecueceic s sunveyeiernmemranvtaui e A site BMB 400 PART THREE V 2 Chapter 14 Translation b Role of rRNA in catalysis It is likelv that rRNA provides the catalytic center for the peptidvl transferase activitv with perhaps some ribosomal proteins aiding in holding the rRNA in the correct conformation for catalvsis This conclusion is supported by several lines of investigation some of which are listed below 1 No protein singly or in combination with other proteins has been shown to catalyze peptide bond formation 2 Specific regions of 16S rRNA in the small subunit interact with the anticodon regions of tRNA in both the A and P sites In contrast 23S rRNA in the large subunit interacts with the CCA terminus of peptidyl tRNA thus placing it in the right location for peptidyl transferase 3 The antibiotics erythromycin and chloramphenicol block peptidyl transferase Some mutations that confer resistance to them map to the 23S rRNA sequence others map to some SOS ribosomal proteins 4 A preparation consisting of 23S rRNA and some remnants of large subunit proteins retains peptidyl transferase activity For more information see Noller et al 1992 Unusual resistance of peptidyl transferase to protein extraction procedures Science 256 1416 1419 5 Ribozyme RNAs can be selected that catalyze peptide bond formation In this experiment the investigators started with a pool of 13 X 1015 different RNAs of 72 nucleotides anked by constant regions They let this large population of RNAs catalyze a peptide bond formation that adds a biotinyl labeled amino acid in a chemical mimic of a P site to an amino acid connected to the RNA in a chemical mimic of an A site The RNAs that successfully catalyzed the reaction were extremely rare but were now covalently attached to a biotin label Thus they could be selected from the population by binding to streptavidin PCR was used to amplify the successful RNAs and the procedure repeated 19 times At this point the investigators characterized 9 RNAs that catalyzed the reaction They found that these RNAs increased the reaction rate by a factor of 106 over the uncatalyzed reaction 6 The three dimensional structure of the ribosome shows that the active site is comprised of RNA The structure of a ribosome crystallized with an active site directed inhibitor has been determined as well as the structure without the inhibitor This allowed researchers to see precisely where the peptidyl transferase active site is within the structure Only RNA is seen around this site The nearest protein is 20 Angstroms away too far to participate in catalysis BMB 400 PART THREE 7 V Chapter 14 Translation 3 Translocation a The translocation step moves the ribosome another 3 nucleotides downstream one codon and moves peptidylrtRNA to the P site position 71 deacylated tRNA exits through the E site and the A site position n1 is vacant for another round of elongation b Elongation Factor G EFG 1 This is another very abundant protein with about 20000 copies per cell which is equivalent to the number of ribosomes 2 EFVGVGTP binds to the ribosome to aid translocation and is released upon GTP hydrolysis GTPase is from some ribosomal component 3 Recent structural studies from A Dahlberg and colleagues show that EFVG in the GTPrbound state has a shape similar to that of the ternary complex of EFrTu GTP and aminoacylrtRNA Like the latter ternary complex EFVG in the GTPrbound state also has a high affinity for the A site on the ribosome This may help drive the movement of the peptidylr tRNA from the A site to the P site replacing it with EFVG GTP in the A site c Hydrolysis of GTP is required for dissociation of EEG after translocation The GTPase is part of the ribosome not EFVG Fig 3520 EFGzGTP for translocation fM el Ala Ser GDP exchange i E P GTP translocation i l GTPase on ribosome fMel Ala Ser 9qu Pi as BMB 400 PART THREE V 2 Chapter 14 Translation d Action of fusidic acid revealed the need for release of EF G GDP In the presence of fusidic acid EF G GTP binds the ribosome GTP is hydrolyzed and the ribosome moves three nucleotides But the ribosome EF G GDP complex is stabilized by this compound and translation is halted Ribosomes cannot bind EF Tu and EF G simultaneously EF Tu must finish its action before EF G can act and EF G must complete its cycle before EF Tu can act again to bring in another aminoacyl tRNA Effect of diptheria toxin 1 The eukaryotic analog to EF G is eEF2 which is also a translocase dependent on GTP hydrolysis It is also is blocked by fusidic acid 2 Diptheria toxin will catalyze the addition of ADP ribose from substrate NAD to eEF2 thereby inactivating it The target for ADP ribosylation is modified histidine found in eEF2 from many species 4 Elongation rate a Bacteria growing at 370 add about 15 amino acids to a growing chain each second b In eukaryotes the elongation rate is much slower about 2 amino acids added per sec BMB 400 PART THREE 7 V Chapter 14 Translation I Termination 1 The three termination codons are UAG amber UAA ochre most common for bacterial genes UGA opal 2 Releasing factors RF are proteins that promote termination of translation and release of the mRNA from ribosomes at those termination codons a Bacteria haVe two releasing factors RF1 recognizes UAG and UAA RF2 recognizes UGA and UAA Figure 3521 Termination leads to dissociation of new protein ribosome and mRNA AaSer His LeuPhe l P l quPPi AaSer His LeuPhe 0 Completed W polypeptide f There are about 600 molecules of releasing factors per bacterial cell or about 1 per 50 ribosomes The releasing factors act when a termination codon is present at the ribosomal A site and peptidyletRNA is at the P site The releasing factors may mimic the aminoacyletRNA in shape but promote hydrolysis of peptidyletRNA rather than transfer to a new aminoacylrtRNA Hence one mechanism for the action of the releasing factors is to cause the ribosome to use H20 as the nucleophile attacking the ester linkage of peptidylrtRNA rather than the oieamino group of the aminoacyletRNa acting as a nucleophile BMB 400 PART THREE V 2 Chapter 14 Translation b Eukaryotes In eukaryotes a single releasing factor eRF has been characterized This protein requires GTP to bind Hydrolysis of GTP probably promotes dissociation of eRF from ribosomes 3 GTP is utilized by eukaryotic releasing factors The theme of using accessory proteins that cycle on and off the ribosome in GTP bound and GDP bound states respectively is seen in initiation at two steps in elongation and now in termination Curiously it appears that the releasing factors in E coli do not require GTP for their action It is not clear that this represents a fundamental difference since it is possible that the role of GTP hydrolysis has been adopted by some other ribosomal component Indeed the overall mechanism of translation has been highly conserved in all major groups of organisms eubacteria archaea and eukaryotes 4 Termination results in dissociation of the entire translation complex This leads to three different releasing events a Release of newly synthesized completed polypeptide from the tRNA which requires hydrolysis of the ester link between the nascent polypeptide and the tRNA b Release of mRNA c Release of the ribosome When free the ribosome is in equilibrium with the dissociated large and small subunits BMB 400 PART THREE 7 V Chapter 14 Translation J Mutant tRNAs can act as suppressors 1 Definition of suppressors a Mutations at a second site that can overcome a missense or nonsense mutation at an original site are suppressors If the original mutation is reversed to wild type this is called genotypic reversion or a backemutation Both suppression and reversion will produce a wild type phenotype at least a partial one from an original mutation 7 the distinction is whether the original mutation is changed genotypic reversion or whether a second site is altered suppression b The suppressor can be either intragenic or extragenic c These second sites that can mutate to suppress an original mutation usually encode a cellular component that interacts with the component encoded by the originally mutated locus Isolation of suppressors can be used to piece together pathways or complex cellular structures 2 Nonsense suppressors a Nonsense suppressors are often mutant tRNAs that still accept an amino acid but whose anticodon has been altered to match a termination codon b E g supE encodes a mutant tRNAgl39n Figure 3521 Mutant tRNAs can act as suppressors Mutate gene for Gm tRNA 939 G g Anticodon lt U0 5 Anticodon UC 5 Codon 5 CAGgt Codon 5 UAGgt Encodes Gln Encodes STOP The mutant GlntRNA 939 will insert a Gln at a UAG lfthe UAG were a premature stop ie nonsensemutant then the mutant tRNA would suppressthe nonsense mutant BMB 400 PART THREE V 2 Chapter 14 Translation c The quotdown sidequot to nonsense suppression is that the suppressor tRNA can act at any amber codon Therefore it competes with the releasing factors in recognizing the normal termination codons When the suppressor tRNA is used instead of releasing factors translation proceeds further down the mRNA than it is supposed to leading to production of aberrant proteins Suppressor strains of E coli can be pretty sick ie they don t grow as well as wild type strains d Two other amber suppressors are encoded by the supD gene which encodes a tRNA that will insert Ser at a UAG and supF which will insert Tyr 3 Missense suppressors These are mutant tRNAs that lead to the insertion of an amino acid that is compatible with the wild type amino acid altered by the original mutation 4 Frameshift suppressors These are mutant tRNAs whose anticodon has been expanded or contracted to match the length altering mutation in the mRNA Eg Consider an original mutation 539GGG gt 5 GGGG insert a G A frameshift suppressor would also have an additional C in the anticodon wt tRNA anticodon 3 CCC gt suppressor tRNA 339CCCC BMB 400 141 142 143 144 145 PART THREE V 2 Chapter 14 Translation Questions on Chapter 14 Translation FOB Methionine Has Only One Codon Methionine is one of the two amino acids having only one codon Yet the single codon for methionine can specify both the initiating residue and interior Met residues of polypeptides synthesized by E coli Explain exactly how this is possible Are the following statements concerning aminoacyl tRNA synthetase true or false a Two distinct classes of the enzymes have been defined that are not very related to each other b The enzymes scan previously synthesized aminoacyl tRNAs and cleave off any amino acids that are linked to the incorrect tRNA c Proofreading can occur at the formation of either the aminoacyl adenylate intermediate in some synthetases or at the aminoacyl tRNA in other synthetases to insure that the correct amino acid is attached to a given tRNA d The product of the reaction has a hi gh energy ester bond between the carboxyl of an amino acid and a hydroxyl on the terminal ribose of the tRNA A preparation of ribosomes in the process of synthesizing the polypeptide insulin was incubated in the presence of all 20 radiolabeled amino acids tRNA s aminoacyl tRNA synthetases and other components required for protein synthesis All the amino acids have the same specific radioactivity counts per minute per nanomole of amino acid It takes ten minutes to synthesize a complete insulin chain from initiation to termination in this system After incubation for 1 minute the completed insulin chains were cleaved with trypsin and the radioactivity of the fragments determined a Which tryptic fragment has the highest specific activity b In the same system described above the insulin polypeptide chains still attached to the ribosomes after ten minutes were isolated cleaved with trypsin and the specific activity of each tryptic peptide determined Which peptide has the highest specific activity Which component of the protein synthesis machinery of E coli carries out the function listed for each statement a Translocation of the peptidyl tRNA from the A site to the P site of the ribosome b Binding of f Met tRNA to the mRNA on the small ribosomal subunit c Recognition of the termination codons UAG and UAA d Holds the initiator AUG in register for formation of the initiation complex via base pairing a In the initiation of translation in E coli which ribosomal subunit does the mRNA initially bind to b What nucleotide sequences in the mRNA are required to direct the mRNA to the initial binding site on the ribosome c What other factors are required to form an initiation complex BMB 400 146 147 148 149 PART THREE V 2 Chapter 14 Translation What steps in the activation of amino acids and elongation of a polypeptide chain require hydrolysis of high energy phosphate bonds What enzymes catalyze these steps or which protein factors are required POB Maintaining the Fidelity of Protein Synthesis The chemical mechanisms used to avoid errors in protein synthesis are different from those used during DNA replication DNA polymerases utilize a 339 gt 5 exonuclease proofreading activity to remove mispaired nucleotides incorrectly inserted into a growing DNA strand There is no analogous proofreading function on ribosomes and in fact the identity of amino acids attached to incoming tRNAs and added to the growing polypeptide is never checked A proofreading step that hydrolyzed the last peptide bond formed when an incorrect amino acid was inserted into a growing polypeptide analogous to the proofreading step of DNA polymerases would actually be chemically impractical Why Hint Consider how the link between the growing polypeptide and the mRNA is maintained during the elongation phase of protein synthesis POB Expressing a Cloned Gene You have isolated a plant gene that encodes a protein in which you are interested What are the sequences or sites that you will need to get this gene transcribed translated and regulated in E cali The three codons AUU AUC and AUA encode isoleucine They correspond to quothybridquot between a codon family 4 codons and a codon pair 2 codons The single codon AUG encodes methionine Given the prevalence of codon pairs and families for other amino acids what are hypotheses for how this situation for isoleucine and methionine could have evolved 1410 Use the following processes to answer parts a c 1 synthesis of aminoacyl tRNA from an amino acid and tRNA 2 binding of aminoacyl tRNA to the ribosome for elongation 3 formation of the peptide bond between peptidyl tRNA and aminoacyl tRNA on the ribosome 4 translocation of peptidyl tRNA from the A site to the P site on the ribosome 5 assembly of a spliceosome for removal of introns from nuclear pre mRNA 6 removal of introns from nuclear pre mRNA after assembly of a spliceosome 7 synthesis of a 5 cap on eukaryotic mRNA a Which of the above processes require ATP b Which of the above processes require GTP c For which of the above processes is there evidence that RNA is used as a catalyst
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'