title: Interaction of translation initiation factor IF1 with the E. coli ribosomal A site.
abstract: Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.
authors.affiliation: Department of Structural Biology, Stanford University, School of Medicine, CA 94305-5126, USA.
authors.forename: K D
authors.initials: KD
authors.lastname: Dahlquist
authors.forename: J D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D000483:Alleles; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002074:Butanones; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D015977:Eukaryotic Initiation Factor-1 / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D010641:Phenotype; D039861:Prokaryotic Initiation Factor-1; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000447:Aldehydes; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002074:Butanones; D015977:Eukaryotic Initiation Factor-1; D039861:Prokaryotic Initiation Factor-1; C034477:RNA primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D013463:Sulfuric Acid Esters; D012313:RNA; D012343:RNA, Transfer; C005135:kethoxal; C007482:dimethyl sulfate