Protein Synthesis Prof.Dr. Gönül Kanıgür

Protein synthesis Protein synthesis requires more than a hundred macromolecules [mRNA,tRNA,activating enzymes,protein factors,ribosomes] There are three steps in protein synthesis -initiation Elongation termination

Activation of aminoacids +Activated precursors of prot.synthesis are aminoacil-tRNAs The carboxyl group of an aa is joined to the 3-terminus of a tRNA The linking of aa to its coresponding tRNA is catalyzed by an aminoacyl-tRNA synthetase ATP Reaction aa+ATP+tRNA---aa-tRNA+AMP+PPi

İn 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.

İnitiation 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

Formation of initiatiator tRNA Methionyl-tRNAsynthetaze adds methionine to the methionyl tRNAs Second step is the formylation Formylation is catalized by transformylase (N10- formyltetrahydrofolate) This product is called formyl methionyl tRNA

RİBOSOMES Ribosoms 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)

The bacterial ribosome

Protein 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

initiation of protein synthesis: 1) Ternary complex formation (IF2.GTP.initiator tRNA) This complex binds to mRNA to form 30S initiation complex The intereacting components are(mRNA+30S subunit+fMet tRNAf+GTP+Initiation factors) The fmet-tRNAf is located to the AUG (initiator)codon 50S 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.

tRNA-mRNA-rRNA base-pairing interactions determine accuracy of protein synthesis.

İnitiation Formation of a 30S initiation complex 30S ribosomal subunit mRNA Formylmethionyl-tRNA GTP İnitiation factors

[E,P,A] three tRNA binding sites on 30S subunit

ELONGATİON CYCLE IN PROTEIN SYNTHESIS This cycle consist of three steps: binding of aa-tRNA to the A site[codon recognation] Peptid bond formation Translocation

The 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 site The initiator tRNA is located to the P site aa-tRNA is brought to the A site by EF-Tu complexed with GTP Following GTP hydrolysis ,EF-Tu.GDP leaves the ribosome,with aa-tRNA correctly placed at the A site

Peptid bond formation aa-tRNA is located to the A site,initiator tRNA is in P site A peptid bound is formed,resulting in the transfer of formyl- methionine to the aa-tRNA at the A site This reaction is catalyzed by peptidyltransferase,is located in the 50S Dipeptidyl-tRNA occupies the A site,an uncharged tRNA occupies P site

Elongation : Peptide Bond Synthesis [peptide bond formed and growing peptide moves from P-site to A-site]

The next step is translocation Three movements occur The uncharged tRNA leaves the P site The peptidyl-tRNA moves from A site to the P site The ribosome moves three nucleotides along the mRNA This process requires EF-G (translocase) and GTP

50S Peptidyl transferase : A ribozyme activity 23S RNA-catalyzed peptide bond format

Termination Termination codons are UAA;UAG;UGA Aa-tRNA does not bind to the A site if the codons are stops These codons are recognized by release factor RF-1 recognized UAA or UAG RF-2 recognized UAA or UGA

Termination: • Protein release factor(s) recognizes a stop codon Termination: • Protein release factor(s) recognizes a stop codon. • Stimulates release of new protein

Antibiotic inhibitors of protein synthesis

Protein synthesis in eucaryotes İt is very complex process Protein synthesis requires,ATP,GTP,tRNAs,mRNAs,aminoacids,aminoacyltRNA-synthetases,two sets of enzymes One set,is for the process of initiation,the other for elongation and release of the nascent of peptid chain

Protein 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-5 Elongation factors eEF-1alfa,eEF-1Beta,gama,eEF-2 Termination factors eRF-1,eRF-3

Ribosomes 80S ribosome consists of 60S and 40S subunits 60S contains 50 proteins,and 3 rRNA molecules[5s rRNA,28SrRNA,5.8S rRNA] 40S contains 33 proteins and28S rRNA molecule

initiation Eukaryotic Initiation complex EIF-2(GTP) for start AUG only EIF2-GDP + Pi Elongation 1) Ribosome binds to cap 2) Moves to 1st AUG 3) Large + small subunits associate

eIF-3and eIF-1A,eIF-1,eIF-5 bind to the 40S eIF-2 binds initiator tRNA and GTP the 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.

Scanning requires energy [ATP] When AUG is identified eIF-5 triggers the hydrolysis of GTP bound to eIF-2 eIF-2 .GDP and other factors are released 60S ribosomal subunit then joins the 40S complex Elongation and termination steps are similar to procaryotes

Elongation 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 ribosome A peptide bound is then formed

-ribosome moves three nucleotides along the mRNA. This movement translocates the peptidyl-tRNA to the P site The uncharged tRNA to the E site -empty A site ready for adition to the next aa-tRNA Translocation is mediated by eEF-2,coupled to GTP hydrolysis.

termination Eucaryotic 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.

Posttranslational Processing Folding: chaperon protein; inclusion body Secretion: signal sequence (20~25 AA); outer membrane block in E. coli Exocytosis: constitutive vs. regulated Glycosylation: addition of sugar; glycosylation pattern: location, degradation; glycoforms (glycosidase) Phosphorylation