Activity of the 30-S CsCl core in elongation-factor-dependent GTP hydrolysis.
The activity of a 30-S CsCl core lacking proteins S1, S2, S3, S5, S9, S10, S14, S20 and S21 has been studied in the ribosome-dependent FTPase reactions in the presence of the 50-S subunit with and without methanol. Without methanol, the 30-S CsCl core was unable to sustain GTPase activity dependent on elongation factor G (EF-G), while it was only slightly active in the presence of elongation factor T (EF-T). With EF-T, addition of methanol induced in the presence of either 30-S subunits or 30-S CsCl cores an activity which was more than 10-fold higher than that observed with the 30-S subunit in the absence of methanol. Methanol lowered the Mg2+ optimum of the EF-T-dependent GTPase reaction from approximately 20 mM to approximately 10 mM. In the absence of methanol the EF-G-dependent (GTPase reaction at low concentration of monovalent cations depends on the 50-S subunit alone (30-S-uncoupled EF-G GTPase). Addition of the intact 30-S subunit but not of its CsCl core abolished inhibition of the 30-S-uncoupled EF-G-GTPase by NH4+. The 30-S CsCl core caused the same effect as the 30-S subunit when methanol was present. 30-S-uncoupled EF-G GTPase activity was lower than the GTPase activity dependent on 30-S plus 50-S subunits at [EF-G]/[50-S] below 5 but was considerably higher in the presence of a large excess of EF-G. In the presence of methanol the 30-S CsCl core behaved similarly to the 30-S subunit. Our results indicate that the action of the 30-S subunit in elongation-factor-dependent GTPases is supported by structural features that are preserved in the 30-S CsCl core. The 30-S split proteins are therefore not essential for EF-G and EF-T activities in the hydrolysis of GTP. With EF-T, in all conditions tested association of the ribosomal subunits appeared to accompany GTPase activity. Association seems also to be a prerequisite of the EF-G GTPase activity that depends on both ribosomal subunits.
D000641:Ammonia / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010445:Peptide Elongation Factors; D010649:Phenylalanine; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism
D010445:Peptide Elongation Factors; D012269:Ribosomal Proteins; D010649:Phenylalanine; D000641:Ammonia; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010744:Phosphoric Monoester Hydrolases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D008274:Magnesium