Poster abstracts

Poster number 35 submitted by Tek Lamichhane

Analysis of modification at 966 and 967 positions in Escherichia coli 16 S ribosomal RNA

Tek N. Lamichhane (Department of Chemistry/Biological Science, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University), Philip R. Cunningham (Department of Biological Science, Wayne State University)

Abstract:

The eight-nucleotide sequence of the 970 loop of helix 31 is conserved within the Bacteria, Archaea and Eukarya, but only A964, A969, and C970 are conserved among all three domains. The 970 loop also contains two of the eleven modified nucleosides in E. coli 16S rRNA, m2G966 and m5C967. Biochemical and structure studies have placed this loop near the P-site and have shown that it is involved in the decoding process and in binding the antibiotic tetracycline.
To identify functionally important nucleotides, sequence motifs and structural motifs in the 970 loop, all eight of the conserved loop nucleotides (964-971) were randomly mutated, selected, sequenced and assayed the viable mutants in our lab previously. Interestingly, single mutations at positions 966 and 967 produced hyperactive ribosomes. Biochemical and modeling studies suggest that initiation factor 3 (IF3) interacts with the stacked residues 966:967:968. Over-expression of IF3 specifically restores wild-type levels of protein synthesis to the 966 and 967 mutants. The 966 and 967 mutants, however, are not able to initiate translation from mRNAs containing CUG, AAG and ACG as the start codon. These data suggest that m2G966 and m5C967 are key components of IF3 binding to the 30 S subunit, but are not involved in discrimination of the initiator codon. To further determine the effect of those nucleotides in translational fidelity such as involvement in stop codons read-through, we tested all mutants of 966 and 967 with stop codons UAG and UGA in luciferase gene and it is shown that m5C967A and m5C967G showed the higher level of UAG stop codon read-through as compared to the wild type 16 S rRNA while as m2G966C showed higher level read-through in both UAG and UGA stop codons.

Keywords: Hyperactive mutants, Initiation factors, Translational fidelity