Protein Synthesis Lecture 12b PDF

Lecture 12b 12b/ Protein Synthesis Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 27: p1015-1041 PROTEIN SYNTHESIS INVOLVES FIVE STAGES 1) Activation of amino acids – tRNA is aminoacylated 2) Initiation of translation – mRNA and aminoacylated tRNA...

Lecture 12b 12b/ Protein Synthesis Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 27: p1015-1041 PROTEIN SYNTHESIS INVOLVES FIVE STAGES 1) Activation of amino acids – tRNA is aminoacylated 2) Initiation of translation – mRNA and aminoacylated tRNA bind to ribosome 3) Elongation – Cycles of aminoacyl-tRNA binding and peptide bond formation…until a STOP codon is reached 4) Termination and ribosome recycling – mRNA and protein dissociate, ribosome recycled 5) Folding and post-translational processing – Catalyzed by a variety of enzymes and helped by chaperones OVERVIEW OF PROTEIN SYNTHESIS The five stages of protein synthesis. 1 The tRNAs are aminoacylated. 2 Translation initiation occurs when an mRNA and an aminoacylated tRNA are bound to the ribosome. 3 In elongation, the ribosome moves along the mRNA, matching tRNAs to each codon and catalyzing peptide bond formation. 4 Translation is terminated at a stop codon, and the ribosomal subunits are released and recycled for another round of protein synthesis. 5 Following synthesis, the protein must fold into its active conformation and ribosome components are recycled. Posttranslational modifications may occur for certain proteins at this stage. COMPONENTS REQUIRED FOR PROTEIN SYNTHESIS IN E. COLI Table 27-5 Components Required for the Five Major Stages of Protein Synthesis in E.coli Stage Essential components Essential components 1. Activation of amino acids 20 amino acids 20 aminoacyl-tRNA synthases 32 or more tRNAs ATP Mg2+ 2. Initiation mRNA N-Formylmethionyl-tRNAfMet Initiation codon in mRNA (AUG) 30S ribosomal subunit 50S ribosomal subunit Initiation factors (IF1, IF2, IF3) GTP Mg2+ 3. Elongation Functional 70S ribosomes (initiation complex) Aminoacyl-tRNAs specified by codons Elongation factors (EF-Tu, EFTs, EF-G) GTP Mg2+ 4. Termination and ribosome recycling Termination codon in mRNA Release factors (RF1, RF2, RF3, RRF) EF-G IF3 5. Folding and posttranslational processing Chaperones and folding enzymes (PPI, PDI); specific enzymes, cofactors, and other components for removal of initiating residues and signal sequences, additional proteolytic processing, modification of terminal residues, and attachment of acetyl, phosphoryl, methyl, carboxyl, carbohydrate, or prosthetic groups THE RIBOSOME IS A KEY PLAYER IN PROTEIN SYNTHESIS Make up ~25% of dry weight of bacteria ~65% rRNA, 35% protein – rRNA forms the core – rRNA does the catalysis of peptide bond formation (the ribosome is a ribozyme) Made of two subunits bound together, (30S and 50S) in bacteria, (40S and 60S) in eukaryotes with mRNA running through them Chloroplasts and mitochondria have ribosomes simpler than those in bacteria COMPOSITION OF RIBOSOMES IN BACTERIA VS. EUKARYOTES In eukaryotes ribosomes: small subunit is 40S, large subunit is 60S. § In 40S: 18S and 33 proteins § In 60S: 5S, 28S and 5.8S rRNA and 47 proteins RIBOSOMES IN PROKARYOTES VS. EUKARYOTES Composition and mass of ribosomes in bacteria and eukaryotes. Ribosomal subunits are identified by their S (Svedberg unit) values, sedimentation coefficients that refer to their rate of sedimentation in a centrifuge. The S values are not additive when subunits are combined, because S values are approximately proportional to the 2/ power of molecular weight and 3 are also slightly affected by shape. THE RIBOSOME ATOMIC STRUCTURE Structure of the 50S subunit At different resolutions STRUCTURE OF RIBOSOMES IN BACTERIA AND EUKARYOTES (a): The bacterial ribosome. The 50S and 30S subunits come together to form the 70S ribosome. A cleft between them is where protein synthesis occurs. (b): The yeast ribosome has a similar structure with somewhat increased complexity. The 60S and 40S subunits come together to form the 80S ribosome rRNAS HAVE COMPLEX SECONDARY STRUCTURES The two ss rRNAs of E.coli (5S, 16S) have specific 2-D structure with extensive intrachain base pairs Diagrams of the secondary structure of E. coli 16S and 5S rRNAs. The first (5′ end) and final (3′ end) ribonucleotide residues of the 16S rRNA are numbered. CONSERVATION OF SECONDARY STRUCTURE IN SMALL SUBUNIT rRNAs FROM BACTERIA, ARCHAEA, AND EUKARYOTES 16 S 16 S/18 S 18 S 2D structure of rRNAs is highly conserved from bacteria to eukarya The red, yellow, and purple indicate areas where the structures of the rRNAs from bacteria, archaea, and eukaryotes have diverged. Conserved regions are shown in green. tRNA MOLECULES HAVE CHARACTERISTIC FEATURES ssRNA of 73–93 nucleotides in both bacteria and eukaryotes Cloverleaf structure in 2-D “Twisted L” shape in 3-D Most have G at 5’-end; all have CCA at 3’-end Have modified bases – Pseudouridine, Dihydrouridine, Methylated bases, etc. tRNA MOLECULES HAVE CHARACTERISTIC FEATURES Amino acid arm – Has amino acid esterified via carboxyl group to the ribose 2’-OH or 3’-OH of the A of the terminal CCA codon Anticodon arm D arm – Contains dihydrouridine (D) – Contributes to folding TyC arm – Contains pseudouridine (y)―has bonding between base and ribose – Helps in folding GENERAL 2-D CLOVERLEAF STRUCTURE OF tRNAS § The large dots on the backbone represent nucleotide residues; the blue lines represent base pairs. Characteristic and/or invariant residues common to all tRNAs are shaded in light red. § Transfer RNAs vary in length from 73 to 93 nucleotides. Extra nucleotides occur in the extra arm or in the D arm. § At the end of the anticodon arm is the anticodon loop, which always contains seven unpaired nucleotides. The D arm contains two or three D (5,6-dihydrouridine) residues, depending on the tRNA. § In some tRNAs, the D arm has only three hydrogen-bonded base pairs. Symbols are: Pu, purine nucleotide; Py, pyrimidine nucleotide; ψ, pseudouridylate; G*, either guanylate or 2’-Omethylguanylate. GENERAL 3-D STRUCTURE OF tRNAs (YEAST tRNAPhe) amino acid anticodon The shape resembles a twisted L. (a) Schematic diagram with the various arms shaded in different colors. (b) A space-filling model, with the same color coding. The CCA sequence at the 3’ end (purple) is the attachment point for the amino acid. AMINOACYL-tRNA SYNTHETASES Amino acid + tRNA + ATP à Aminoacyl-tRNA + AMP + PPi Mg++ Each enzyme binds a specific amino acid and the matching tRNA Most cells contain 20 different aminoacyltRNA synthetases, one for each amino acid Some cells contain less than 20 synthetases; in this case one amino acid is converted to another after charging the tRNA, by a different enzyme (see also the case of selenocysteine and pyrrolysine) SYNTHESIS OF AMINOACYLATED tRNAS: 1) CREATION OF AMINOACYL INTERMEDIATE COO– of amino acid attacks a-phosphate of ATP à creates aminoacyladenylate intermediate Pyrophosphate (PPi) is formed then cleaved, So, the overall reaction is driven forward by two phosphoanhydride bond cleavages. Fate of the aminoacyladenylate varies (two classes of synthetases will be involved). SYNTHESIS OF AMINOACYLATED tRNAS: 2) TRANSFER OF AMINOACYL TO tRNA Aminoacyl-tRNA synthetases (two classes) transfer aminoacyl group from enzyme to the 3’A of tRNA Aminoacyl-tRNA synthetases – 1/ Ribose 2’-OH (class 1 synthetase) or 3’-OH (class II) of 3’A of tRNA attacks the carbonyl of aminoacyl-AMP, creating an ester bond between amino acid and tRNA, and releasing AMP – 2/ In the final product, the tRNA is bound to its cognate (appropriate) aminoacid through the 3’OH of A ACTIVATION OF AMINO ACIDS – AMINOACYL-tRNA SYNTHETASES ATTACH THE CORRECT AMINO ACID TO THEIR tRNAS Mg2+ Amino acid + tRNA + ATP à aminoacyl-tRNA + AMP + PPi 1/ Formation the aminoacyl General structure of aminoacyl-tRNAs adenylate (2 ATP equivalents used: 1 ATP and 1 PPi) 2/ Transfer of the aminoacyl group to the tRNA by 2 classes of synthetases Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step 1 is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases (see Table 27–7). For class I enzymes, the aminoacyl group is transferred initially to the 2’-hydroxyl group of the 3’-terminal A residue, then to the 3’-hydroxyl group by a transesterification reaction. For class II enzymes, the aminoacyl group is transferred directly to the 3’-hydroxyl group of the terminal adenylate. The aminoacyl group is esterified to the 3’ position of the terminal A residue. The ester linkage that both activates the amino acid and joins it to the tRNA is shaded light red. PROOFREADING AND DISCRIMINATION BY AMINOACYL-tRNA SYNTHETASES Aminoacyl-tRNA synthetases must be specific for both amino acid and tRNA: § At the amino acid level: proofreading by tRNA synthetases of the amino acid bound to the cognate (appropriate) RNA § At the tRNA level: Only a few nucleotides in tRNA confer the binding specificity – Matching each amino acid with correct tRNA can be viewed as the “second genetic code” – The “code” is in molecular recognition of a specific tRNA molecule by a specific synthetase on the basis of anticodon region and other regions (example: primary determinant in Ala-tRNA is a single G=U in amino acid arm, see next slide) PROOFREADING AND DISCRIMINATION BY AMINOACYL-tRNA SYNTHETASES (uses one ATP equivalent) Proofreading at the amino acid level: (Double sieve mechanism) Discrimination at the tRNA level (the second genetic code): Discrimination at the tRNA level (Example: tRNAAla): - Correct Amino acid (Ile) binding to site 1 (active site) to form AA-AMP (larger AAs are eliminated, smaller can be accomodated) - If incorrect, the incorrect AA-AMP binds to site 2 (proofreading site) to be hydrolyzed (Val-AMP à Val + AMP). (This step uses 1 ATP if incorrect AA is hydrolyzed). Ex: Ile-tRNA synthetase Nucleotide positions in tRNAs recognized by aminoacyl-tRNA Synthetases (orange and blue points) (a) The GU pair of tRNAAla required for Recognition by Ala-tRNA synthetase, (b) The synthetic helix (made in the lab) can also be aminoacetylated specifically with Ala STRUCTURE OF AMINOACYL-tRNA SYNTHETASES Structure of Class I synthetases (monomeric or dimeric) Gln-tRNA synthetase complex from E. coli Structure of Class II synthetases (dimeric or tetrameric) Asp-tRNA synthetase complex from E. coli fMet OR Met IS THE FIRST AMINO ACID IN A PEPTIDE First codon is AUG (Met) All organisms have two tRNAs for Met – In bacteria, chloroplasts and mitochondria initiation tRNA inserts N-formylmethionine (uses tRNAfMet) – Interior Met is inserted with normal tRNAMet Eukaryote protein begins with Met, not fMet, but still a special tRNA is used ADDITIONAL GENETICALLY CODED AMINO ACIDS 20 genetically encoded amino acids are common in all organisms But 2 additional amino acids are also found in a few proteins and are genetically coded – Selenocysteine (Sec) is introduced in proteins after: Charging a specific type of tRNA for serine (tRNASer*) that recognizes UGA (stop codon) with serine in both bacteria and eukaryotes, using Ser-tRNA synthetase (making Ser-tRNASer*). Then the OH of Ser is modified to SeH by a separate enzyme, thus giving Sec-tRNASer*. UGA-Stop codon is interpreted differently by the association of a specialized EF during elongation introducing Sec in a protein. – Pyrrolysine (Prl) is directly attached to its own tRNA (tRNAprl) by a specific AA-tRNA synthetase. This Prl-tRNAprl recognizes UAG (stop) codon by some archae bacteria INITIATION IN PROTEIN SYNTHESIS The Dintzis experiment -Protein synthesis begins with the N-terminal AA - and then proceeds by the stepwise addition of amino acids to the C-terminal end of the growing polypeptide - The AUG initiation codon specifies an amino-terminal methionine residue. However, formyl methionine (fmet) is introduced instead of met. For initiation codon: first a tRNAfmet is charged with met by met-tRNA synthetase, then the met is transformylated to fmet 1/ Met-tRNA synthetase: methionine + tRNAfmet + ATP à met-tRNAfmet + AMP + PPi 2/ Transformylase: (only tRNAfmet can be formylated, not tRNAmet ) N10-Formyltetrahydrofolate + met-tRNAfmet à TetrahydroFolate + fmet-tRNAfmet 3/ During translation, fMet-tRNAfmet is bound to IF2, whereas met-tRNAmet is bound to EFTu. Reticulocytes (immature erythrocytes) actively synthesizing hemoglobin were incubated with radioactive leucine (selected because it occurs frequently in both the α- and β-globin chains). Samples of completed α chains were isolated from the reticulocytes at various times afterward, and the distribution of radioactivity was determined. The dark red zones show the portions of completed α-globin chains containing radioactive Leu residues. - At 4 min, only a few residues at the carboxyl end of α-globin were labeled, because the only complete globin chains with incorporated label after 4 min were those that had nearly completed synthesis at the time the label was added. - With longer incubation times, successively longer segments of the polypeptide contained labeled residues, always in a block at the carboxyl end of the chain. The unlabeled end of the polypeptide (the amino terminus) was thus defined as the initiating end, which means that polypeptides grow by successive addition of amino acids to the carboxyl end. For an internal met codon: a tRNAmet is charged with met by met-tRNA synthetase. Therfore: Distinction between a 5’ AUG initiation codon and an internal one during translation on the ribosome: - using 2 types of tRNAs: tRNAfmet and tRNAmet: - Using IF2 (initiation) and EFTu (elongation) INITIATION (3 STEPS) REQUIRES A LARGE MOLECULAR ASSEMBLY In bacteria, initiation requires: – 30S ribosomal subunit – mRNA – fMet-tRNA – Initiation Factors IF-1, IF-2, and IF-3 – GTP – 50S ribosomal subunit – Mg2+ STEP 1 OF INITIATION The 30S ribosomal subunit binds IF-1, IF-3 and mRNA - Initiation factor IF-3 keeps 30S and 50S subunits apart The initiating (5’)-AUG codon of mRNA is guided to its correct position on the ribosome 30S by the Shine-Dalgarno sequence (region in mRNA that is complementary to a sequence in 16S ribosomal RNA). STEPS 2 AND 3 OF INITIATION (uses one ATP equivalent) fmet tRNA, recruited by IF-2, binds to the peptidyl (P) site along with initiating(5’)AUG. Large 50S subunit combines with the 30S subunit forming the initiation complex – IF-2-GTP recruits fmet-tRNAfmet then hydrolyzes GTP (thus one ATP equivalent is used) – A functional ribosome is formed, that will proceed in protein synthesis FORMATION OF THE INITIATION COMPLEX IN BACTERIA IN THREE STEPS (one ATP equivalent is used) Initiation Step 1: - 30S ribosomal subunits binds IF-1 and IF-3 and mRNA - 5’ AUG initiating codon is guided to its correct position by the Shine-Dalgarno sequence on the mRNA that serves as signal for Initiation of protein synthesis Initiation Step 2: - the complex formed in step 1 is joined by IF-2-GTP and the initiator fMet-tRNAfmet - the anticodon of fMet-tRNAfmet pairs with the mRNA initiation codon at the P site Initiation Step 3: - the complex formed in step 2 combines with the 50Sribosomal subunit IF-2-GTP is hydrolyzed to GDP and IF-1, IF-2, IF-3 are released. One ATP equivalent is used. - A functional 70S ribosome (initiation complex) is formed Ribosomes have 3 sites for tRNA binding; - A (amino acyl) and - P (peptidyl ) sites bind to aminoacyl-tRNAs - E (exit) site binds to uncharged tNRA ELONGATION: Step 1/ AN AMINOACYL-tRNA BINDS (one ATP equivalent is used) Aminoacyl tRNA (for a second AA) binds to Elongation Factor Tu and GTP (EF-Tu-GTP) The aminoacyl tRNA-EF-Tu-GTP complex binds to the aminoacyl (A) site of the 70S initiation complex After GTP hydrolysis, EF-Tu-GDP leaves the ribosome (one GTP is used). EF-Tu GTP is regenerated using EF-Ts and GTP ELONGATION: Step 2/ PEPTIDE BOND FORMS There are now two amino acids bound to tRNAs positioned for joining – One in the P site (fmet-tRNAfmet) and one in the A site (AA2-tRNA-AA2) fmet group is transferred from its tRNA in the P site to the amino acid in the A site (AA2) – Reaction catalyzed by the 23S rRNA (ribozyme) “Uncharged” (deacetylated) tRNAfMet is now in the P site, and a dipeptidyl-tRNA is now in the A site ELONGATION Step 3/ TRANSLOCATION OF THE RIBOSOME (one ATP equivalent is used) Ribosome moves one codon toward the 3’-end of the mRNA – Uses EF-G (translocase) – Uses energy from GTP hydrolysis (part of EF-G) – The dipeptidyl-tRNA is now in the P site – tRNAfMet ,which is now in E site, leaves the ribosome – The A site is now free and open for new aminoacyltRNA to bind THE 3 STEPS OF ELONGATION IN BACTERIA (2 ATP equivalent are used during elongation) Elongation step 1: binding of the second aminoacyl-tRNA Elongation step 2: formation of the first peptide bond Elongation step 3: translocation Uses one ATP equivalent Uses one ATP equivalent TERMINATION IS SIGNALED BY A STOP CODON UAA, UAG, or UGA in the A site will trigger the binding of termination factors (Release Factors) RF-1, RF-2, RF-3 (not tRNAs) These help to: – Hydrolyze terminal peptide-tRNA bond – Release peptide and tRNA from ribosome – Cause subunits of ribosome to dissociate so that initiation can begin again – Aided by EF-G GTP hydrolysis Ribosome Recycling Factor (RRF) helps in the dissociation of the two subunits of the ribosome TERMINATION OF PROTEIN SYNTHESIS IN BACTERIA Uses one ATP equivalent Model of a nascent polypeptide chain in the tunnel on the large (50s) subumit of an “open” ribosome with the peptidyl-transferase site (PT) and the exit site. Termination occurs in response to a termination codon in the A site. 1/ A release factor, RF (RF-1 or RF-2, depending on which termination codon is present), binds to the A site (RF3 function not yet elucidated). 2/ this leads to hydrolysis of the ester linkage between the nascent polypeptide and the tRNA in the P site and release of the completed polypeptide. 3/ the mRNA, deacylated tRNA, and release factor leave the ribosome, which dissociates into its 30S and 50S subunits, aided by ribosome recycling factor (RRF), IF-3, and energy provided by EF-G-mediated GTP hydrolysis. The 30S subunit complex with IF-3 is ready to begin another cycle of translation FEATURES OF PROTEIN SYNTHESIS Large energy cost (see next slide) Can be rapid when accomplished on clusters of ribosomes called polysomes In bacteria, tightly coupled to transcription – Translation can begin before transcription is finished No coupling in eukarya, where the RNA needs to be completed in the nucleus, then exported to the cytosol where it can be circularized before translation. COUPLING OF TRANSCRIPTION AND TRANSLATION IN BACTERIA vs EUKARYA E. Coli (coupled) (a) Electron micrograph of polysomes forming during the transcription of a segment of DNA from E. coli. Each mRNA is being translated by many ribosomes simultaneously. The nascent polypeptide chains forming on the ribosomes are difficult to see under these conditions. The arrow marks the approximate beginning of the gene that is being transcribed. (b) Each mRNA is translated by ribosomes while it is still being transcribed from DNA by RNA polymerase. This is possible because the mRNA in bacteria does not have to be transported from a nucleus to the cytoplasm before encountering ribosomes as in Eukaryotes. In this schematic diagram the ribosomes are depicted as smaller than the RNA polymerase. In reality the ribosomes (Mr 2.7 x 106) are an order of magnitude larger than the RNA polymerase (Mr 3.9 x 105). Eukaryotes (uncoupled) RNA is transcribed and processed in the nucleus, then needs to exit the nucleus before translation Electron micrograph and schematic drawing of polysomes in Eukaryotes ENERGY COST OF PROTEIN SYNTHESIS Large energy cost: – For each peptide bond made, 2 ATP equivalents are used in the activation step (aminoacetylation), 1 GTP in elongation step, 1 GTP in translocation step: Total of 4 ATP equivalents/peptide bond formation. – Also, 1 GTP is used only once in the initiation step and 1 GTP is used only once in the termination step:Total of 2 ATP equivalents/protein synthesis cycle. In case proofreading is needed, 1 ATP equivalent is used (in proofreading by AA-tRNA synthetases). INITIATION IN EUKARYOTES Uses more initiation factors – Over 12 IFs, including eIFIA and eIF3 (functional homologs of IF-1 and IF-3) – 5’ cap of the mRNA binds an initiation factor. Has a step that circularizes the mRNA ELONGATION AND TERMINATION IN EUKARYOTES Elongation uses eukaryotic elongation factors eEF-1 (similar to prokaryotic EF-Tu and EF-Ts) and eEF-2 (similar to prokaryotic EF-G). The mechanism is very similar to that of prokaryotes. Termination uses eukaryotic termination factors (release factors) and the mechanism is similar to that of prokaryotes, except that one universal release factor (eRF) is used that recognize all 3 stop codons. MANY ANTIBIOTICS TARGET PROTEIN SYNTHESIS IN BACTERIA Puromycin – Similar structure to 3’-end of aminoacyl-tRNA – So it binds to the A site of ribosomes, forms bond with growing peptide – But cannot participate in translocation and dissociation. Thus, puromycin terminates protein synthesis Tetracyclines – block the A site on the ribosome Chloramphenicol and Cycloheximide – Block peptidyl transfer – Chloramphenicol inhibits mitochondrial and chloroplast ribosomes as well as bacterial Streptomycin – Causes code to be misread, inhibits initiation at high concentrations ACTION OF PUROMYCIN ON PEPTIDE SYNTHESIS Disruption of peptide bond formation by puromycin Binding location of other antibiotics to the 30S subunit The antibiotic puromycin resembles the aminoacyl end of a charged tRNA, and it can bind to the ribosomal A site and participate in peptide bond formation. The product of this reaction, peptidyl puromycin, is not translocated to the P site. Instead, it dissociates from the ribosome, causing premature chain termination. MANY INHIBITORS OF PROTEIN SYNTHESIS From Garrett and Grisham, 4th Ed. SUMMARY In this lecture, we learned: Protein synthesis occurs on the ribosomes, which consist of protein and RNA. Ribosomes of bacteria and eukaryotes are different in composition. Transfer RNA has remarkable features with different arms having different functions (amino acid arm , anticodon arm…). The growth of polypeptides on ribosomes begins with the Nterminal amino acid and proceeds by addition of residues towards the C-terminal end. Protein synthesis occurs in five stages (activation of amino acid, initiation, elongation, termination and folding). Many antibiotics and toxins inhibit some aspects of protein synthesis.