2Protein synthesisProtein synthesis requires more than a hundred macromolecules [mRNA,tRNA,activating enzymes,protein factors,ribosomes]There are three steps in protein synthesis-initiationElongationtermination
6Activation of aminoacids +Activated precursors of prot.synthesis are aminoacil-tRNAsThe carboxyl group of an aa is joined to the 3-terminus of a tRNAThe linking of aa to its coresponding tRNA is catalyzed by an aminoacyl-tRNA synthetaseATPReaction aa+ATP+tRNA---aa-tRNA+AMP+PPi
7İn the first step,the aa is joined to AMP,forming an aminoacyl AMP intermediate İn the second step,the aa is transfered to the 3’CCA terminus of the acceptor tRNA and AMP is released.Both step of the reaction are catalyzed by aminoacyl tRNA synthetases.
8İnitiation of protein synthesis İn both eucaryotic and procaryotic cells,translation always initiates with the aa methionine usually encoded by AUG.Alternative initiation codon such as GUG are used in bacteria.İn most bacteria protein synthesis is initiated with a modified methionine(N-formyl methionine),whereas unmodified methionine initiate prot.synt.in eucaryotes
9Formation of initiatiator tRNA Methionyl-tRNAsynthetaze adds methionine to the methionyl tRNAsSecond step is the formylationFormylation is catalized by transformylase (N10- formyltetrahydrofolate)This product is called formyl methionyl tRNA
10RİBOSOMESRibosoms in procaryotes consist of a large and small subunit.30S subunit contains 21 proteins and 16S ribosomal RNA.50S subunit contains 34 proteins and 2 rRNA molecules. (23S and 5S)
12Protein factors:Initiation factor 1 (IF1) prevents the reassociation of diassociated 50S and 30S subunits.IF2 is necessary to the formation of IF2.GTP.fMet tRNAf complex (ternary complex)IF3 is similar to IF1
13initiation of protein synthesis: 1) Ternary complex formation (IF2.GTP.initiator tRNA)This complex binds to mRNA to form 30S initiation complexThe intereacting components are(mRNA+30S subunit+fMet tRNAf+GTP+Initiation factors)The fmet-tRNAf is located to the AUG (initiator)codon50S subunit joins to 30S initiation complex to form a 70S initiation complex. GTP is hydrolysed, all these factors are released.The 70S initiation coplex is ready for elongation step of protein synhesis.
14tRNA-mRNA-rRNA base-pairing interactions determine accuracy of protein synthesis.
15İnitiationFormation of a 30S initiation complex30S ribosomal subunitmRNAFormylmethionyl-tRNAGTPİnitiation factors
17ELONGATİON CYCLE IN PROTEIN SYNTHESIS This cycle consist of three steps:binding of aa-tRNA to the A site[codon recognation]Peptid bond formationTranslocation
18The ribosome has three tRNA binding sites; P [peptidyl],A [aminoacyl],E [exit] The first step starts with the insertion of aa-tRNA into the empty A siteThe initiator tRNA is located to the P siteaa-tRNA is brought to the A site by EF-Tu complexed with GTPFollowing GTP hydrolysis ,EF-Tu.GDP leaves the ribosome,with aa-tRNA correctly placed at the A site
19Peptid bond formationaa-tRNA is located to the A site,initiator tRNA is in P siteA peptid bound is formed,resulting in the transfer of formyl- methionine to the aa-tRNA at the A siteThis reaction is catalyzed by peptidyltransferase,is located in the 50SDipeptidyl-tRNA occupies the A site,an uncharged tRNA occupies P site
20Elongation : Peptide Bond Synthesis [peptide bond formed and growing peptide moves from P-site to A-site]
21The next step is translocation Three movements occurThe uncharged tRNA leaves the P siteThe peptidyl-tRNA moves from A site to the P siteThe ribosome moves three nucleotides along the mRNAThis process requires EF-G (translocase) and GTP
2250S Peptidyl transferase : A ribozyme activity 23S RNA-catalyzed peptide bond format
23Termination Termination codons are UAA;UAG;UGA Aa-tRNA does not bind to the A site if the codons are stopsThese codons are recognized by release factorRF-1 recognized UAA or UAGRF-2 recognized UAA or UGA
24Termination: • Protein release factor(s) recognizes a stop codon Termination: • Protein release factor(s) recognizes a stop codon. • Stimulates release of new protein
26Protein synthesis in eucaryotes İt is very complex processProtein synthesis requires,ATP,GTP,tRNAs,mRNAs,aminoacids,aminoacyltRNA-synthetases,two sets of enzymesOne set,is for the process of initiation,the other for elongation and release of the nascent of peptid chain
27Protein synthesis in Eucaryotes İnitiation factors[10 different factors]eIF-1,eIF-1A,eIF-2,eIF-2B,eIF-3,eIF-4A,eIF-4B,eIF-4E,eIF-4G,eIF-5Elongation factorseEF-1alfa,eEF-1Beta,gama,eEF-2Termination factorseRF-1,eRF-3
29initiation Eukaryotic Initiation complex EIF-2(GTP) for start AUG only EIF2-GDP + PiElongation1) Ribosome binds to cap2) Moves to 1st AUG3) Large + small subunitsassociate
30eIF-3and eIF-1A,eIF-1,eIF-5 bind to the 40S eIF-2 binds initiator tRNA and GTPthe mRNA binds eIF-4E ( binds to the 5 cap)eIF-4G binds to the eIF-4E and PABP (poly A binding protein)eIF-4A and 4B bring mRNA to 40S.Ribosome than scans down the mRNA to identify AUG initiation codon.eIF-5, catalizes the binding of the 60S to the 40Sinitiation complex.
31Scanning requires energy [ATP] When AUG is identified eIF-5 triggers the hydrolysis of GTP bound to eIF-2eIF-2 .GDP and other factors are released60S ribosomal subunit then joins the 40S complexElongation and termination steps are similar to procaryotes
32Elongation The ribosome has three binding sites(P,A,E) İnitiator tRNA is located P site,aa-TRNA is then brought to the A site by eEF-1alfa (complexed with GTP)Following GTP hydrolysis ,EF-1alfa.GDP leaves the ribosomeA peptide bound is then formed
33-ribosome moves three nucleotides along the mRNA. This movement translocates the peptidyl-tRNA to the P siteThe uncharged tRNA to the E site-empty A site ready for adition to the next aa-tRNATranslocation is mediated by eEF-2,coupled to GTP hydrolysis.
34terminationEucaryotic cells contain only one relase factor (eRF) which recognize all three termination codons.Release factor binds termination codon at the A site.İt stimulates hydrolysis of the bond between the tRNA and the polypeptide chain at the P site.The tRNa released,and the ribosomal subunits and the mRNA dissociate.
35Posttranslational Processing Folding: chaperon protein; inclusion bodySecretion: signal sequence (20~25 AA); outer membrane block in E. coliExocytosis: constitutive vs. regulatedGlycosylation: addition of sugar; glycosylation pattern: location, degradation; glycoforms (glycosidase)Phosphorylation