Transcript Protein synthesis

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 3terminus 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.
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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,a
minoacyltRNA-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,eIF4B,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
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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 peptidyltRNA to the P site
• The uncharged tRNA to the E site
• -empty A site ready for adition to the next aatRNA
• 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