title: Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study.
abstract: The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed Escherichia coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2, and IF-3) and GTP on this process have been studied by fluorescence stopped-flow kinetic measurements. The formation of the ternary complex was followed by an increase of both intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA. The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+). In the presence of the three initiation factors and GTP the formation of the ternary complex could be resolved into two partial reactions: a fast apparently second-order step (k12 = 5 x 10(6) M-1 s-1, k21 = 1.4 s-1) followed by a slow rearrangement step (k23 less than or equal to 0.1 s-1). The data suggest a mechanism in which the ternary complex is formed by at least two rearrangements of an initially formed preternary complex. The accelerating effects of both IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other two factors.
mesh_terms: D004926:Escherichia coli / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010448:Peptide Initiation Factors / Q000494:pharmacology*; D039665:Prokaryotic Initiation Factor-3; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D012346:RNA, Transfer, Amino Acyl; C006383:tRNA, N-acetylphenylalanine-; D006160:Guanosine Triphosphate; D008274:Magnesium