title: An extended Shine-Dalgarno sequence in mRNA functionally bypasses a vital defect in initiator tRNA.
abstract: Initiator tRNAs are special in their direct binding to the ribosomal P-site due to the hallmark occurrence of the three consecutive G-C base pairs (3GC pairs) in their anticodon stems. How the 3GC pairs function in this role, has remained unsolved. We show that mutations in either the mRNA or 16S rRNA leading to extended interaction between the Shine-Dalgarno (SD) and anti-SD sequences compensate for the vital need of the 3GC pairs in tRNA(fMet) for its function in Escherichia coli. In vivo, the 3GC mutant tRNA(fMet) occurred less abundantly in 70S ribosomes but normally on 30S subunits. However, the extended SD:anti-SD interaction increased its occurrence in 70S ribosomes. We propose that the 3GC pairs play a critical role in tRNA(fMet) retention in ribosome during the conformational changes that mark the transition of 30S preinitiation complex into elongation competent 70S complex. Furthermore, treating cells with kasugamycin, decreasing ribosome recycling factor (RRF) activity or increasing initiation factor 2 (IF2) levels enhanced initiation with the 3GC mutant tRNA(fMet), suggesting that the 70S mode of initiation is less dependent on the 3GC pairs in tRNA(fMet).
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and.
authors.forename: Sunil
authors.initials: S
authors.lastname: Shetty
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and.
authors.forename: HimaPriyanka
authors.initials: H
authors.lastname: Nadimpalli
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and.
authors.forename: Riyaz Ahmad
authors.initials: RA
authors.lastname: Shah
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and.
authors.forename: Smriti
authors.initials: S
authors.lastname: Arora
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and.
authors.forename: Gautam
authors.initials: G
authors.lastname: Das
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India; and Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India varshney@mcbl.iisc.ernet.in.
authors.forename: Umesh
authors.initials: U
authors.lastname: Varshney
mesh_terms: D000617:Aminoglycosides / Q000494:pharmacology; D000926:Anticodon / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D039881:Prokaryotic Initiation Factor-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000235:genetics* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D000617:Aminoglycosides; D000926:Anticodon; D039881:Prokaryotic Initiation Factor-2; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012358:RNA, Transfer, Met; D012269:Ribosomal Proteins; C089238:fMet-tRNA(fMet); C087267:ribosome releasing factor; C100305:kasugamycin
keywords: 3GC base pairs; 70S initiation; A1339; G1338; rrsC