Poster abstracts

Poster number 20 submitted by Daoming Qin

Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

Daoming Qin (Ohio state biochemistry program), Abdi Nimo (Department of Microbiology, The Ohio state University), Kurt Fredrick (Department of Microbiology, The Ohio state University)

Abstract:
During translation initiation in bacteria, the start codon is recognized by the initiator tRNA (fMet-tRNAfMet) in the 30S P site. The initiation factors (IF1, IF2, and IF3) are thought to work in conjunction with the 30S subunit to ensure faithful recognition of the start codon. Mutations in infC, the gene encoding IF3, cause spurious initiation at non-canonical start codons, suggesting an important role for IF3 in initiation fidelity. Here, we characterize two 16S rRNA mutations, G1338A and A790G, which decrease initiation fidelity in vivo. G1338 is a universally conserved nucleotide that interacts with the minor groove of the anticodon stem of P-site tRNA, forming a type II interaction. Previous genetic studies have shown that G1338A can suppress other P-site mutations, suggesting that G1338A may increase the affinity of tRNA for the 30S P site. Indeed, 30S subunits harboring G1338A have increased affinity for tRNAfMet in vitro, suggesting that the fidelity defect may result from a more favorable type II A minor interaction with the initiator tRNA. When G1338A is combined with various infC alleles, effects of the rRNA and protein mutations are consistently additive, suggesting that G1338 and IF3 may affect fidelity independently. Mutation A790G seems to act in a different way to decrease fidelity. A790G decreases the affinity of IF3 for the 30S subunit substantially, and does not confer an increased fidelity defect when combined with infC mutations. These data suggest that A790G may decrease fidelity by inhibiting IF3 interaction with the 30S subunit.

References:
Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli. Abdi NM, Fredrick K RNA 2005 Nov 11 (11) 1624-32.
Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics Carter AP, Clemons WM, Brodersen DE, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V. Nature 2000 Sep 21;407 (6802):340-8
Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo. Sussman JK, Simons EL, Simons RW. Mol Microbiol.1996 Jul; 21(2):347-60.

Keywords: G1338, A790, if3