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1. M.Sc. Biotech./Biochem./Microbio. (Sem –I)
Molecular Biology
Translation
By – Dr. Ravi Kant
Assistant Professor (Biotechnology)
Email – ravi.kant@nirmauni.ac.in

2. Translation – protein synthesis
• Nucleotide sequence is translated into an amino acid sequence. The codons on mRNA are
read and corresponding amino acids are added from carboxy to the amino terminus.
• Amino acids arrive at the growing chain in activated form, as aminoacyl tRNAs.
• Aminoacyl tRNAs are the tRNAs that have an amino acid attached to its 3’ OH group.
• Aminoacyl tRNA synthetases are the enzymes that link an amino acid to a particular tRNA.

3. tRNA – structure & function
• tRNAs serve as an adaptor molecule that binds to a specific codon and at the same time
brings a specific amino acid with it for incorporation into the growing polypeptide chain.
• tRNAs structural features: 73 to 93 nucleotides, ~25kDa.
• Clover leaf-shaped or L-shaped molecule.
• Has several modified bases like pseudouridine, ribothymidine,
dihydrouridine, inosine, etc. the modified nucleotides are
formed by enzymatic modification of bases post transcription.
• About half of the bases pairs form a double helix (A form).
• CCA at the 3’ end remains single-stranded, this is where
amino acid attaches, acceptor arm.
• In addition there are DHU, TpsiC loop, and anticodon arm.
• 5’ end is usually G and is phosphorylated.

4. tRNA – structure & function

5. Codon degeneracy
• Some tRNAs can recognize more than one codon.
• E.g. yeast alanyl-tRNA recognizes GCU, GCC, and GCA where the first two base pairs are the
same only last one is different, probably recognition of 3rd is less stringent.
• The recognition of third bp is less stringent, because of the presence of Inosine.
• Inosine base in tRNA is formed by deamination of Adenosine post-transcriptional level.

6. Aminoacyl tRNA synthetases
• Links an amino acid with a particular tRNA, hence establishes genetic code.
• In addition to establishing the genetic code, tRNAs activate the amino acids, as free amino
acids can not form the peptide bond.

7. Aminoacyl tRNA synthetases recognize various features
of tRNA
• How do aminoacyl tRNA synthetases recognizes specific
tRNA? It is an important point at which translation takes
place.
• Aminoacyl tRNA synthetases, the only molecules that
actually know the genetic code, some time referred to as
second code.
• Two classes of amino acyl tRNA synthetases: 1. acylates
2’OH group and 2. acylates 3’OH group.

8. Aminoacyl tRNA synthetases
• Aminoacyl tRNA synthetases are highly specific for a given amino acid.
• Chances of incorporating an incorrect amino acid to a tRNA are less than 104 or 105.
• How is this level of specificity is achieved?
• The binding sites for amino acids in tRNA synthetases are highly
specific.
• What will happen if we incubate threonyl-tRNA synthetase with
threonine-tRNA that has been wrongly charged with serine?
• and if incubated with threonine-tRNA that has been charged with
serine.
• Indicate that aminoacyl tRNA synthetases have proofreading activity
i.e tRNA synthetases have an additional functional site that has
hydrolysis activity, editing site.
• The ‘’editing site’’ responsible for the proofreading activity is 20A
away from the active site.
• “Editing site” and “activation site” acts as a double sieve.

9. Ribosome
• Are the molecular machines that coordinate the interplay of aminoacyl-tRNA, mRNA, and
proteins.
• Is a mass of ribonucleoprotein assembly with a mass of 2500kDa, diameter 250A, and
sedimentation coefficient of 70S.
• 70S = 30S+50S.
• 30S subunit has 16sRNA and 21 proteins named S1-S21.
• 50S subunit has 23S and 5S RNA and 34 proteins (L1-L34).

10. rRNAs
• Three rRNAs i.e. 23S, 16S, and 5S are the
cleavage products of 30S subunit.
• rRNAs are critical for the architecture and
function of the ribosomes.
• rRNAs form extensive secondary structures.
• Initially proteins in the ribosomes were
believed to play a critical role, but now with
the discovery of catalytic RNAs view is
reversed. Something that favors RNA word
hypothesis.

11. Ribosome – A, P, and E sites
• mRNA molecule binds to 30S subunit.
• tRNA interacts with both 30S and 50S subunit.
• At a given moment 2 of three tRNA interacts with mRNA
through codon-anticodon interactions.
• A –site (aminoacylation).
• P-site (peptidyl).
• E-exit (exit).
• A channel connects with P site through which polypeptide is
released.

12. Ribosome binding site: Shine-Dalgarno sequence
• How does protein synthesis begin? In simplest possibility, the first 3 nucleotide act as start
codons. But in bacteria translation always begins 25 nt downstream, the first amino acid is
usually methionine.
• Start codon – AUG (methionine), less common is GUG (leucine).
• In addition to the initiator codon preceding sequence is purine-rich, SD sequence.
• SD sequence pairs with complementary sequences in 16sRNA in 30S subunit.
• Thus 2 things determine initatiation events.

13. Formylmethionine –tRNA (fMet-tRNAf initiates
the translation in prokaryotes

14. Translation – formation of
intiation complex

15. Translation – formation of peptide bond

16. Translation - elongation
• Peptidyl tRNA binds to P site , aminoacyl tRNA binds to A site.
• With both the sites occupied peptide bond is formed.

17. Translation - translocation
• EF-G a GTPase binds to A site on 50S ribosome.

18. Polyribosome
• Transcription and translation goes on simultaneously.
• Many ribosomes may be attached to a particular
mRNA.

19. Translation-termination
• What happens when a stop codon is encountered?
• Stop codons are recognized by release factors, RF1, RF2 and RF3.
• RF1 recognize UAA and UAG.
• RF2 recognize UAA and UGA.
• RF3, a GTPase, helps RF1 and RF2 to be released.
• RF3 interacts with peptidyl transferase centre in 23s RNA.

20. Translation in eukaryotes
• Initiation: pre-initiation complex (eIF2+tRNA+40S ribosome),
eIF4E helps PIC to bind to 5’ cap, with the help of helicases PIC
moves on mRNA till start codon is found. In addition eukaryotic
mRNA (virus) have internal ribosome binding sites.
• Ribosome: 80S (40S and 60S); 40S (18S rRNA); 60S (5S, 28S and 5.8S
rRNA).
• Initiator tRNA (Met-tRNAi).
• Start codon is always AUG, no purine rich stretch (SD sequence). AUG
closest to the 5’ end is selected as the start codon.

21. Translation in eukaryotes
• Elongation: EF1a and EF1bg are the counterparts of EF-Tu and EF-Ts.
• Termination: Unlike RF1,2,3 in prokaryotes in eukaryotes we have only
one release factor in eukaryotes i.e. eRF1 an eIR3 accelerates the
process.

22. Antibiotics/toxins that inhibit
Translation
• Streptomycin (other aminoglycosides) blocks the binding of fMet-tRNA to 30S ribosome.
• Erythromycin binds to 50S subunit and blocks translocation.
• Puromycin, acts an analog of aminoacyl tRNA and terminates polypeptide prematurely.
• DT modifies EF2 and hence blocks translocation.
• Ricin, highly potent (500ug), cleaves 28sRNA.

23. Posttranslational modifications
• What are post-translational modification?
• What role do they play?
• Precursor enzymes.