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
AUG UAA AUG UAA AUG UAA
5’pppA/G
5’ UTR Cistron1 Cistron2 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
AUG UAA
Me7
Gppp (A)n=200
5’ UTR Open reading frame (ORF) 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 C
w 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 GTP-
dependent 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 fMet-
tRNA 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:IF-

1) 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 aminoacyl-
tRNA 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 tRNA A site
O O
Adenine O P O CH2 Adenine
O P O CH2 O
O
O − H H O− H H
H H H H
O OH O OH

O C O C

HC R HC R

NH :NH2

O C
HC R
NH3+
 tRNA P site tRNA A site
O O
O P O CH2 Adenine O P O CH2 Adenine
O O
The nascent O − H H O − H H
H H H H
polypeptide, one OH OH O OH
O C
residue longer, is
HC R
now linked to the NH
tRNA in the A O C
site. 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