1. Initiation of Translation in Bacteria
Chapter 17

2. Initiation of Translation in Bacteria
Two important events- before translation initiation
- Generate a supply of aminoacyl-tRNAs
Amino acids must be covalently bound to tRNAs - tRNA charging
- Dissociation of ribosomes into their two subunits
The cell assembles the initiation complex on the small ribosomal subunit
The two subunits must separate to make assembly possible
Two important events must occur before translation initiation can take place - Process of bonding tRNA to amino acid is called tRNA charging

3. tRNA Charging All tRNAs have same 3 bases at 3’-end (CCA)
Terminal adenosine is the target for charging with amino acid
Amino acid attached by ester bond between
Its carboxyl group
2’- or 3’-hydroxyl group of terminal adenosine of tRNA

4. Aminoacyl-tRNA Synthetase Activity
Aminoacyl-tRNA synthetases join amino acids to their cognate tRNAs - done in a two-step reaction:
Begins with activation of the amino acid with AMP derived from ATP
The energy from the aminoacyl-AMP is used to transfer the amino acid to the tRNA

5. Dissociation of Ribosomes
E. coli ribosomes dissociate into subunits at the end of each round of translation
IF1 actively promotes this dissociation
IF3 binds to free 30S subunit and prevents reassociation with 50S subunit to form a whole ribosome
70S ribosomes of E.coli contain one 30S and one 50S subunit. Each subunit has one or two ribosomal RNAs and large collection of ribosomal proteins. The 30S subunit binds the mRNA and the anticodons ends of tRNAs. It is the decoding agent of ribosome that reads the genetic code in mRNA and allows binding with appropriate aminoacyl-tRNAs. The 50S subunit binds the ends of tRNAs that are charged with amino acids and has peptidyl transferase activity that links amino acids together through peptide bonds.

6. Formation of the 30S Initiation Complex
When ribosomes - dissociated into 50S and 30S subunits - cell builds a complex on the 30S subunit:
mRNA
Aminoacyl-tRNA
Initiation factors
IF3 binds by itself to 30S subunit
IF1 and IF2 stabilize this binding
IF2 can bind alone - but is stabilized with help of IF1 and IF3
When ribosomes have been dissociated into 50S and 30S subunits, cell builds a complex on the 30S subunit. IF1 does not bind alone

7. First Codon and the First Aminoacyl-tRNA
Prokaryotic initiation codon is:
Usually AUG, can be GUG, rarely UUG
Initiating aminoacyl-tRNA is N-formyl-methionyl-tRNA
N-formyl-methionine (fMet) is the first amino acid incorporated into a polypeptide
This amino acid is frequently removed from the protein during maturation
The 30S initiation complex is formed from a free 30S ribosomal subunit plus mRNA and fMet-tRNA

8. Binding mRNA to the 30S Ribosomal Subunit
Binding between the 30S prokaryotic ribosomal subunit and the initiation site of a message depends on base pairing between
Short RNA sequence
Shine-Dalgarno sequence
Upstream of initiation codon
Complementary sequence
3’-end of 16S rRNA

9. Initiation Factors and 30S Subunit
Binding of the Shine-Dalgarno sequence with the complementary sequence of the 16S rRNA is mediated by IF3
- Assisted by IF1 and IF2
All 3 initiation factors have bound to the 30S subunit at this time
IF2 is the major factor promoting binding of fMet-tRNA to the 30S initiation complex
GTP is also required for IF2 binding at physiological IF2 concentrations
The GTP is not hydrolyzed in the process

10. Complete 30S Initiation Complex
The complete 30S initiation complex contains one each:
30S ribosomal subunit
mRNA
fMet-tRNA
GTP
Factors IF1, IF2, IF3

11. Formation of the 70S Initiation Complex
GTP is hydrolyzed after the 50S subunit joins the 30S complex to form the 70S initiation complex
This GTP hydrolysis is carried out by IF2 in conjunction with the 50S ribosomal subunit
Hydrolysis purpose is to release IF2 and GTP from the complex so polypeptide chain elongation can begin

12. Bacterial Translation Initiation
IF1 influences dissociation of 70S ribosome to 50S and 30S
Binding IF3 to 30S, prevents subunit reassociation
IF1, IF2, GTP bind alongside IF3
Binding mRNA to fMet-tRNA forming 30S initiation complex
Can bind in either order
IF2 sponsors fMet-tRNA
IF3 sponsors mRNA
Binding of 50S with loss of IF1 and IF3
IF2 dissociation and GTP hydrolysis

13. Initiation in Eukaryotes
Eukaryotic
Begins with methionine
No Shine-Dalgarno
mRNA have caps at 5’end
Bacterial
N-formyl-methionine
Shine-Dalgarno sequence to show ribosomes where to start

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