Protein Biosynthesis Translation

Protein Biosynthesis translation

The Major Participants in Translation w mRNA w tRNA w Aminoacyl-tRNA synthetases w Ribosomes

mRNA The template for translation. In prokaryotes, the mRNA is polycistronic A polycistronic mRNA 5’pppA/G 5’ UTR

AUG UAA Cistron1

AUG

UAA

Cistron2

AUG

UAA

Cistron3 3’UTR

Monocistronic mRNAs in eukaryotes Processing (CAPing, polyadenylation and splicing ) of the primary transcripts usually results in a a monocistronic mRNA.

A monocistronic mRNA Me7

Gppp 5’ UTR

AUG

UAA

Open reading frame (ORF)

(A)n=200 3’UTR

tRNA The genetic code is read during translation via adaptor molecules, tRNAs.

Aminoacyl-tRNA Synthetases •Amino acids activation is to form aminoacyl-tRNAs catalyzed by Aminoacyl-tRNA Synthetases •This reaction activates the amino acid for protein synthesis

Ribosomes w Ribonucleoprotein particles w Found in the cytosol, mitochondria and

chloroplasts w Move along mRNAs, bind aminoacyl-tRNAs and synthesize proteins w Ribosomes are composed of 2 major units

Ribosome composition

Mitochondrial and chloroplast ribosomes differ from both examples shown.

Ribosome Structure w The two subunits assemble into complete

functional ribosome in the presence of mRNA. w A tunnel runs through the large subunit. w Growing peptide chain is thought to thread through the tunnel during protein synthesis

A 3D model for the E.coli ribosome 30S

70S

50S

Comparison of ribosomes and tRNAs E.coli ribosome

tRNA binding sites: the P site (peptidyl) the A site (aminoacyl) the E site (exit)

What are A, P and E sites on ribosome • Sites in the interface between large and small ribosomal

subunits where tRNAs reside during protein synthesis  A-site is the position where the aminoacyl-tRNA binds P-site is the position where peptide-tRNA binds E-site is the spot where the uncharged tRNA is placed • There are A, P and E sites on both subunits  movement of mRNA and tRNAs seems to be related to a movement of the two subunits • A- and P-sites have been known for long time, the E-site was only discovered in the past decade after ribosome structures have been solved

Process of protein synthesis w Direction of peptide chain growth

N w

C

polypeptides grow by addition of new amino acid residues to the carboxyl end

Process of Prokaryotic Protein Synthesis w initiation w elongation w termination

Initiation w Initiation involves binding of mRNA and

initiator aminoacyl-tRNA to a small subunit, followed by binding of a large subunit w Correct binding of mRNA on ribosome requires alignment of a pyrimidine-rich sequence on 3'end of 16S RNA of 30S small subunit with a purine-rich part of 5'-end of mRNA w The purine-rich segment - the ribosome-binding site - is known as the Shine-Dalgarno (S-D) sequence

Shine-Dalgarno sequences recognized by E.coli ribosomes

Initiation w The initiator tRNA is one with a formylated

methionine: f-Met-tRNAfMet w A formyl transferase adds the formyl group from formyl-FH4. w It is only used for initiation, and regular Met-tRNAMet is used instead for Met addition

Events of Initiation w 30S subunit with initiation factor proteins (IF-1 and

IF-3) binds mRNA w IF-2 delivers the initiator tRNA in a GTPdependent process to the initiation codon. w IFs, GTP, fMet- tRNAf, mRNA and 30S ribosome form the 30S initiation complex w Release of the IFs accompanying GTP hydrolysis leads to binding of 50S subunit forming 70S initiation complex.

 When the two ribosomal subunits join, the

AUG initiator codon with its bound fMettRNA aligns with the P site.  The P site is now occupied by initiator fMet-tRNA, A site is empty. w

initiation 30S subunit (IF-3:IF1) binds mRNA, IF-2 delivers the initiator f-Met-tRNA to the initiation codon

30S initiation complex GTP hydrolysis is accompanied by IFs release and binding of the 50S subunit

70S initiation complex

The Elongation Cycle w Elongation: synthesis of peptide bonds -

with tRNAs bound to aminoacyl (A) and peptidyl (P) sites. w The elongation factors are vital to cell function

Elongation factors w EF-Tu

Binds GTP,delivers aminoacyltRNA to A site w EF-Ts Displaces GDP from EF-Tu, recycles EF-Tu-GTP w EF-G Binds GTP, promotes translocation of ribosome

Elongation step 1: Binding of an aminoacyl-tRNA to A site w Aminoacyl-tRNA binds to A site of ribosome

as a complex with EF-Tu and GTP w GTP is then hydrolyzed and EF-Tu:GDP complexes dissociate w EF-Ts recycles EF-Tu by exchanging GTP for GDP

Elongation second step:peptide bond formation w The amino N of the aa - tRNA in the A site

reacts with the carbonyl C of the aa (with nascent chain) linked to tRNA in the P site. tRNA

P site

O

O O

P O

A site

tRNA

O

CH2

−

H H O

O

R

O

CH2

O−

H H

O

C HC

R

NH3+

Adenine H H OH

O R

:NH2

C HC

P

O

C NH

O

H

O

H OH

O

HC

Adenine

tRNA

P site

O

O O

The nascent polypeptide, one residue longer, is now linked to the tRNA in the A site.

P O

A site

tRNA

O CH2 −

H H

OH

O

Adenine H

O

P O

O CH2 −

H H

H OH O

O

Adenine H H OH

O C

HC

R

NH O

C HC

R

NH

O

C HC

R

NH3+

The tRNA in the P site is now unloaded (It will dissociate during translocation.)

Peptidyl Transferase w This is the central reaction of protein

synthesis w 23S rRNA is the peptidyl transferase!

Ribosome is a ribozyme (catalytic rRNA) w The "reaction center" of 23S rRNA is located in the 50S particle - the catalytic

bases are among the most highly conserved in all of biology.

Elongation step 3:translocation w Ribosome moves one codon toward the 3’

end of the mRNA. w Peptidyl-tRNA is translocated to P site from A site. w Uncharged tRNA is translocated to E (exit) site, then dissociates from ribosome. w Movements of the ribosome along mRNA requires EF-G(translocase) and GTP.

Peptide Chain Termination w Termination occurs when "stop codon"

reached w Proteins known as "release factors" recognize the stop codon at the A site w Presence of release factors with a stop codon at A site transforms the peptidyl transferase into a hydrolase, which cleaves the peptidyl chain from the tRNA carrier

Termination of protein synthesis

Players of translation w Ribosome (RNA, proteins) w mRNA w tRNA w Aminoacyl-tRNA synthetases w Translation factors (initiation,

elongation,termination)

Events of Translation w Initiation (different between prokaryotes and eukaryotes) • Dissociation of ribosome • Binding of initiation factor (IF1/eIF1A) to small particle • Recruitment of f-Met-tRNAMet to small particle • Recruitment of mRNA to small particle • Binding of large particle - dissociation of initiation factors w Elongation (similar between pro- and eukaryotes)

Entry of aa-tRNA to A-site • Peptide-bond formation • Translocation of mRNA and tRNAs to P and E sites • Entry of next aa-tRNA to A-site etc. •

w Termination (similar between pro- and eukaryotes) • Entry of release factor to recognize stop codon • Exit of polypeptide and release factors