Translation

Nucleotide sequence i.e. transcript of mRNA is translated into transcript of polypeptide amino acid sequence
in the polypeptide
 Machinery
• Site: Cytoplasm at ribosome
• The RNA Players –
• ribosomal RNA (rRNA) -the site of polypeptide assembly.
rRNA recognizes and binds to start sequence.
 It moves three nucleotides at a time tRNA
 disengages at stop signal
• Messenger RNA (mRNA) attaches to the ribosome. It directs
which amino acids are assembled into polypeptides.
• Transfer RNA (tRNA) attaches to free amino acids in the
cytoplasmic "pool" of amino acids. It transports specific
amino acids based on complementary pairing of a triplet code
(anticodon) with the triplet code (codon) of the mRNA.
Enzyme "hooks" the amino acid to the last one in the chain forming
a peptide bond.
Protein chain continues to grow as each tRNA brings in its amino
acid and adds it to the chain. - This is translation!!
 Transfer RNA
• Cloverleaf structure
• anticodon- 3’ to 5’ sequence that matches the complementary 5’ to
3’sequence (codon) on the mRNA
• Acceptor arm - Amino acid code on 3’ end (binding site of aa)
• T and D loops – provide structure for interface with aminoacyl-tRNA
synthetase

?
 Wobble Hypothesis
Genetic code, codons • There are 61 different codons but only 40
different tRNAs.
• Triplets of nucleotide bases in reading frame called
codons→each codes for one amino acid • Explained by “wobble hypothesis”:
– The anticodon of certain tRNAs can bind
How many triplet codes?
successfully to a codon whose third position
20 common amino acids in a protein requires nonstandard base pairing.
4 diff. bases on DNA A,T,C, & G – Allows one tRNA to be able to base pair
| | | | with more than one type of codon.
4 diff. bases on RNA U,A,G, & C
4 things put together in combinations of 3 = 4 3= 64
Therefore - 64 different DNA triplet codes or RNA codons
• "start” start signal coded by AUG codon
• stop signal coded by one of three nonsense codons:
• UAA, UAG , UGA
• 60 for 20 amino acids
• Degenerate Code- more than one triplet code for
some amino acids
•A site:binding site for first aminocylated
tRNA

•P site – binding site for the peptidyl tRNA

(the growing peptide chain is kept and
new aa are attached)
)

•1) E site – binding site for the uncharged

tRNA(“naked” t RNA exit the ribosome)

•These sites are present at the interface

between the small and the large subunit of
ribosome.

Large subunit does???? Small subunit does ?????

 Charging of tRNA

• Amino acid covalently binds to its specific aminoacyl tRNA sythnetase in presence of Mg++, which
then covelantly attaches the amino acid onto a specific tRNA. Aminoacyl tRNA sythnetase catalyze
the addition of amino acids to tRNAs
• Aminoacyl tRNA = “CHARGED” tRNA
• Is ATP expended in this process? Yes!
• Each tRNA has a distinct anticodon and attached amino acid.
 Initiation:Protein made in 5’ to 3’ direction, with N-terminal
end made first
•Initiation factor-3 (IF-3) binds to 30S ribosomal unit.
• Ribosome binding site on mRNA binds to •Then mRNA binds to 30S ribosomal subunit in such a way that AUG codon lie
Targeting of complementary sequence on small subunit of on the peptidyl (P) site and the second codon lies on aminoacyl (A) site
mRNA ribosome, with the help of proteins called •Shine dalgrno* sequence in the bacterial mRNA guide correct positioning of
initiation factors. AUG codon at P-site of 30S ribosome.

Translation • Initiator aminoacyl tRNA, bringing in •The tRNA carrying formylated methionine for bacteria ie. FMet–tRNAFMet
begins at the the first amino acid, binds to the whereas methionine for eukaryotes is palced at P-site. This specificity is induced
AUG start mRNA’s start codon, AUG by IF-2 with utilization of GTP. The IF-1 prevent binding of FMet–tRNAFMet in
codon A-site.

LARGE subunit of ribosome •After binding of FMet–tRNAFMet on P-site, IF-3, IF-2 and IF-1 are released so
binds, placing initiator that 50S ribosomal unit bind with 30S forming 70S sibosome. The exit site is
aminoacyl tRNA in the P-site. located in 50S.

Shine-Dalgarno sequence: Polypurine sequence AGGAGG centred at 10 bp upstream of AUG initiation codon of bacterial
mRNA complementary to 3’ end of 16sRNA at
This is the only tRNA that can
bind to the small ribosomal
subunit by itself

This is called as 70S Initiation

Complex for prokaryotes
 Elongation: 1st step:Binding of AA-tRNA at A-site
(A) EF-Tu:GTP:aa-tRNA (ternary complex) and the ribosome with mRNA.
(B) Recognition of anticodon at A site by ternary complex to the ribosome
with mRNA.
(C) Codon–anticodon recognition and complementary base pairing between
codon-anticodon of ternary complex on the ribosome with mRNA.
(D) Hydrolysis of GTP to GDP on the ribosome with mRNA.
(E) EF-Tu:GDP dssociation and aa-tRNA accommodation on the ribosome
with mRNA. EF-TU:GDP then and enter into EF-TS cycle
The small circles represent amino acids, the yellow triangle represents GTP,
the yellow lightning bolt represents a chemical transformation (i.e., GTP
hydrolysis to GDP), and the yellow cross represents EF-Tu:GDP
 Elongation: 2nd step:Peptide bond formation
•The aminoacid present in t-RNA of P-site ie Fmet is
transferred to t-RNA of A-site forming peptide bond. This
reaction is catalyzed by peptidyl-transferase activity of 50S
subunit (ribozyme).
•Now, the t-RNA at P-site become uncharged

Enter into next cycle

Elongation: 3rd step:Ribosome translocation
Elongation factor G (EF-G) then binds near the A site,
forcing the tRNAs containing the new amino acid and the
growing chain into the next (P and E) sites on the ribosome,
due to the 3D structural changes of ribosome
EF-G splits GTP to catalyze 5’-3’ movement, changes
conformation and falls off, thus increasing the speed of
translation.
EF-G-GTP is the (translocase enzyme)
This process continues giving long polypeptide chain of
amino acids until stop codon appear on A-site.
 Termination
Stop Codons = UAA, UAG, UGA
No tRNA binds to this set of codons as no anticodon
on tRNA
RF3
•One of these codons at the A site attracts release
factor(RF-1, RF-2 and RF-3) RF3
•RF-1 recognises UAA and UAG
RF-2 recognises UAA and UGA RF3
RF-3 GTPase, dissociate 30S and 50S subunits.
•In case of eukaryotes only one release actor eRF
causes dissociation.
.Ribosome adds a water to the last peptide, creating
the carboxyl end

GDP

Pi
 Post translational modification:polypeptide to biologically functional protein

• Cleavage: N-formylmethionine in case of bacteria is removed from polypeptide chain and some carboxyl terminal are also removed by enzymatic
action to make functional protein. In case of eukaryotic protein, amino terminal is N- acetylated.
• Many proteins fold with the help of molecular chaperones
 Inhibitor
Cell type Inhibitor
Prokaryotes Dauromycin and interfering with the passage of both DNA and RNA polymerase.
Adriamycin
Rifamycine Binds to β subunit of RNA Pol, blocks primer synthesis for replication as well as
initiation of RNA synthesis
Prokaryotes Quinolones inhibiting DNA gyrase
and
eukaryotes
Eukaryotes Aphidicolin inhibits DNA pol α (leading strand synthesis),DNA pol δ and DNA pol Ɛ (lagging
strand synthesis) of eukaryotes
 Inhibitor
Cell type Inhibitor
Prokaryotes Tetracycline binds to the ribosomal small subunit (30S) of prokaryotes and blocking the
aminoacyl-tRNA binding to site A
Streptomycine
chloramphenicol Block the accommodation of aa-tRNA into the A site of the large bacterial
ribosomal subunit
Erythromycine prevents elongation of the polypeptide chain and also inhibit the formation of
the large ribosomal subunit.
Rifamycine Binds to RNA Pol, blocks initiation of RNA synthesis
Prokaryotes Puromycine aa-tRNA analogue, causes premature chain termination
and
eukaryotes Actinomycine D binding to DNA duplexes at the transcription initiation complex and preventing
RNA polymerase movement
Eukaryotes Cycloheximide It binds the ribosome and inhibits eEF2-mediated translocation in eukaryotes
i.e peptidyl transferase activity of large subunits
Aminitin Binds to RNA pol II
 Mutations

Permanent alteration in the nucleotide sequence of the genome of an

organism resulting in:
– Change in sequence of amino acids of the translated protein.

Individuals showing these changes are known as mutants

Factor or agents causing mutation are known as mutagens
 Deletion

Duplication

Chromosomal Insertion

Inversion

Translocation

Silent

Point Missence
Change of a single nucleotide (deletion,
insertion, or substitution of ONE nucleotide
Gene Nonsense
mutation in a gene)
Insertion
Frameshift Inserting or deleting one or more nucleotides •
Deletion Changes the “reading frame” like changing a
sentence
Types of Mutations

• Silence mutation: Does not change amino acid sequence.

• Nonsense mutation: Results in an early stop codon:
shortened protein.
• Missense mutation: results in a single amino acid change
in the protein encoded by that gene
• frameshift mutation: Addition or deletion of a nucleotide
causes entire reading frame to be shifted.