Polypeptide chain initiation in E. coli: studies on the function of initiation factor F1.
The requirement of initiation factors F(1) (highly purified) and F(2) (electrophoretically homogeneous) for ribosomal binding of N-formylmethionyl transfer RNA (fMet approximately tRNA) at low Mg(2+) concentration (3.5 mM), with the trinucleoside diphosphate ApUpG as messenger, was studied under various experimental conditions with 30S + 50S ribosomes and with 30S subunits alone. The results were qualitatively the same in both cases but the amount of binding was two to three times higher when both 30S and 50S subunits were present. Although there was a virtually absolute requirement for F(2) in all cases, considerable binding occurred at 0 degrees in the absence of added F(1). F(1) addition stimulated binding up to twofold under these conditions. However, at 25 degrees , the temperature at which the reaction is usually carried out, there was very little binding with F(2) alone and addition of F(1) stimulated the reaction five- to sixfold. Contrary to current belief, the GTP analog 5'-guanylyldiphosphonate (GMP-PCP) cannot replace GTP in the binding reaction. In particular, there was but little stimulation of binding (about 1.5-fold) by addition of F(1) to F(2)-containing samples when GMP-PCP was used. In marked contrast, binding was stimulated up to sevenfold by addition of F(1) when GTP was substituted for the analog. Under these conditions, there was an ApUpG and F(1)-dependent hydrolysis of GTP. This is observable with 30S subunits alone and can hardly be related to the occurrence of translocation. The results may be interpreted to mean that a complex relatively stable at 0 degrees , but less stable at 25 degrees , is formed upon addition of F(2) alone. Conversion of the less stable to the more stable form of complex is made possible by addition of F(1). This is accompanied or mediated by cleavage of GTP.
D001426:Bacterial Proteins / Q000096:biosynthesis*; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000378:metabolism*; D005815:Genetic Code; D006150:Guanine Nucleotides / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008715:Methionine; D010452:Peptide Biosynthesis; D011485:Protein Binding; D012343:RNA, Transfer; D012270:Ribosomes
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D006150:Guanine Nucleotides; D012343:RNA, Transfer; D008715:Methionine; D008274:Magnesium