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.

• 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 Initiationcomplex EIF-2(GTP) for startAUG onlyEIF2-GDP + PiElongation1) Ribosome binds to cap2) Moves to 1st AUG3) Large + small subunitsassociate

• 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