1. Birth of proteins by translation
Reading:
Any of the biochemistry or Molecular biology texts
Mol. Bio. of the Cell by Alberts et al (4 e) – Chapter 6
Molecular Cell Biol. by Lodish et al (5 e) – Chapter 4.3 – 4.5
Biochemistry by Voet and Voet (2 e) – Chapter 30

2. Key concepts in translation
Genetic information transcribed from DNA to mRNA as a nonoverlapping, degenerate triplet code
1 codon = 1 amino acid but 1 amino acid > 1 codon
2 key molecules responsible for decoding nucleotide sequence into amino acid sequence are tRNAs and aminoacyl-tRNA synthetases
3 base anticodon in tRNA allows base-pairing with corresponding sequence in mRNA
20 specific aminoacyl-tRNA synthetases present
Both pro and eukaryotic ribosomes have a large and small subunit

3. What is translation? mRNA directed synthesis of polypeptides
Translates DNA sequence information into proteins
Genetic code dictates translation of specific RNA triplet codons to amino acids
Occurs in the cytosol

4. Genetic code Triplet code
Degenerate – more than 1 triplet may encode same amino acid
Non overlapping
E.g AUGCGTACT
Start codon mainly AUG (rarely GUG)
Stop codons are UAG, UGA, UAA

5. Exceptions! CODON UNIV UNUSUAL ORGANISM CODE CODE
UGA Stop Trp mycoplasma,
mitochondria (some spp)
CUG Leu Thr Yeast mitochondria
UAA, UAG Stop Gln Paramoecium,
Tetrahymena etc

6. Open Reading frames (ORF)
Uninterrupted sequence of codons in mRNA (from start to stop codon) that is translated into amino acids in a polypeptide chain

7. Mutations
MAN CAN FLY- correct sequence DAN CAN FLY – substitution DAC ANF LY - frameshift mutation

8. Main classes of mutations
Deletions or Insertions: 1bp to several Mbp Single base substitutions Missense mutations: replace one amino acid codon with another Nonsense mutations: replace amino acid codon with stop codon Splice site mutations: create or remove exon-intron boundaries Frameshift mutations: alter the ORF due to base substitutions Dynamic mutations: changes in the length of tandem repeat elements

9. Translation requires…..
1) mRNA
2) Aminoacyl- transfer RNA (aatRNA)
3) Ribosomes

10. 1) Messenger RNA (mRNA)
This class of RNAs are the genetic coding templates used by the translational machinery to determine the order of amino acids incorporated into an elongating polypeptide in the process of translation.

11. 2) Transfer RNA (tRNA)
class of small RNAs which form covalent bonds to amino acids
allows correct insertion of amino acids into the elongating polypeptide chain.

12. Adaptor hypothesis
tRNA acts as a ‘shuttle’ linking amino acid to nucleic aid
Aligns correct amino acids to form a polypeptide
One tRNA per amino acid

13. Translation has 2 important recognition steps1
Correct aminoacylation (‘charging’): Covalently attach the correct amino acid to tRNA (specified by anticodon)
Select the correct charged tRNA as specified by mRNA 2

14. How does the aaRSs select the right tRNA to be acylated especially since most tRNAs are structurally similar?
By recognising specific tRNA identifiers present on the acceptor step & anticodon loop
e.g. AlaRSs recognise G3.U70 bp on the acceptor stem

15. 2 Select the correct charged tRNA as specified by mRNA
Less than 61 tRNAs found in cells
Ribosomes select aa-tRNA based only on their codon –anticodon interactions
This pairing is antiparallel and the base in the third position forms non standard base pairing (Wobble hypothesis)
tRNA anticodon 3’-A A G-5’ or 3’-A A G-5’
mRNA codon 5’-U U C-3’ ’-U U U-3’
Question?
When does translation of the genetic code actually occur, during the charging of the tRNA synthetases or during the binding of tRNAs to the ribosome?

16. 3) Ribosomes
Ribosomal RNA (rRNA) assembled, together with numerous ribosomal proteins, to form the ribosomes.
Ribosomes engage the mRNAs and form a catalytic domain into which the tRNAs enter with their attached amino acids. The proteins of the ribosomes catalyze all of the functions of polypeptide synthesis

17. Ribosomes

18. Ribosomes Made of rRNA & ribosomal proteins 2 subunits – large - small
Subunits are self assembling
combine only in the presence of mRNA and a charged (aminoacylated) tRNA

19. What is the structure of the ribosome?
A, P & E sites

20. Polypeptide synthesis (overview)
3 distinct steps
Chain initiation
Chain elongation
Chain termination

21. Initiation in eukaryotes
Step 1 : Formation of pre-initiation complex
40S-eIF3 bound by eIF1A to a ternary complex of tRNAimet, eIF2 and GTP
Fig 4-26 MCB by Lodish et al)

22. Initiation
Step 2: Formation of initiation complex (cap binding of mRNA to 40S)

23. Initiation Step 3: positioning at start codon –
initiation complex unwinds mRNA using eIF4 helicase
Initiation complex stops at the start site AUG
This recognition allows an irreversible GTP hydrolysis of eIF2 preventing any further unwinding
Kozak sequence
ACCAUGG

24. Initiation Step 4: Association of large subunit (60S)
Irreversible GTP hydrolysis mediates the association of 60S-eIF6 (large subunit ) to the small subunit by the action of eIF5
This becomes the P site

25. Initiation in eukaryotes
Fig 4-25 MCB by Lodish et al)

26. Chain elongation 4 stage reaction cycle 1) aatRNA binding
aatRNA binds to A site on ribosome by base pairing with codon
2) Conformation change in ribosome: induced by GTP hydrolysis of EF1a
3) Transpeptidation
C terminal of polypeptide uncoupled from P site tRNA and peptide bond transferred to amino acid on A site tRNA
catalysed by peptidyltransferase
4) Translocation
GTP hydrolysis of EF2 causes 2nd conformational change P site tRNA is transferred to E site
Simultaneous transfer of A site tRNA moved to P site

27. How does elongation proceed?
Which end of the protein is produced first?

28. Chain elongation 4 Steps

29. Step 1 : aatRNA binding

30. Step 2: conformational change

31. Step 3 : Transpeptidation

32. Step 4 : Translocation

33. termination Release factors (eRFs) recognise and bind to stop codons
This induces peptidyl transferase to transfer peptidyl group to water instead of aatRNA
Uncharged tRNA released from ribosome
Inactive ribosome then release mRNA
Typically the entire process takes 30-60sec!!

34. Antibiotics that inhibit translation
Only prokaryotes
Streptomycin prevents initiation-elongation
Chloramphenicol blocks peptidyltransferase
Only eukaryotes
Cycloheximide blocks translocation
Both
Puromycin causes premature release of polypeptide

35. During the process of translation:
A. the peptide is ‘passed’ from the tRNA in the P- site to the tRNA in the A-site.
B. incoming tRNAs must first bind to the E- site.
C. initiation begins with the binding of the ribosomal SSU to the poly-A tail of the mRNA.
D. the mRNA is translated by one ribosome at a time.

36. Translation begins: A. at the “start codon” of the mRNA.
B. at the 3’ end of the mRNA.
C. at the promoter site on the mRNA.
D. in the nucleolus of the nucleus.

37. During the ‘elongation’ stage of translation, after the arrival of each new tRNA
A. the amino acid is ‘passed’ from the tRNA in the A-site to the tRNA in the P-site.
B. newly arriving tRNAs must first bind to the E-site.
C. the peptide is ‘passed’ from the tRNA in the P-site to the tRNA in the A-site.
D. the new tRNA must first bind to the P-site of the ribosome.

38. Initiation of translation begins when
A. the large and small subunits link together, then bind to the mRNA.
B. the ribosomal small subunit binds to the 5’ end of the mRNA.
C. after ribosome binds to the start codon and an initiator tRNA enters the ribosome.
D. the initiator tRNA binds to the start codon, followed by binding of the ribosomal small subunit.