title: Mechanism of ribosomal translocation. Translocation limits the rate of Escherichia coli elongation factor G-promoted GTP hydrolysis.
abstract: The pre-steady-state kinetics of GTP hydrolysis catalysed by elongation factor G and ribosomes from Escherichia coli has been investigated by the method of quenched-flow. The GTPase activities either uncoupled from or coupled to the ribosomal translocation process were characterized under various experimental conditions. A burst of GTP hydrolysis, with a kapp value greater than 30 s-1 (20 degrees C) was observed with poly(U)-programmed vacant ribosomes, either in the presence or absence of fusidic acid. The burst was followed by a slow GTP turnover reaction, which disappears in the presence of fusidic acid. E. coli tRNAPhe, but not N-acetylphenylalanyl-tRNAPhe (N-AcPhe-tRNAPhe), stimulates the GTPase when bound in the P site. If the A site of poly(U)-programmed ribosomes, carrying tRNAPhe in the P site, is occupied by N-AcPhe-tRNAPhe, the burst of Pi discharge is replaced by a slow GTP hydrolysis. Since, under these conditions, N-AcPhe-tRNAPhe is translocated from the A to the P site, this GTP hydrolysis very probably represents a GTPase coupled to the translocation reaction.
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013997:Time Factors
chemical_list: D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D020963:GTP Phosphohydrolase-Linked Elongation Factors