The influence of tRNA located at the P-site on the turnover of EF-Tu.GTP on ribosomes.
The turnover of EF-Tu.GTP on poly-U programmed ribosomes was measured both in the presence and in the absence of N-acetylated Phe-tRNA(Phe) at the P-site. The reaction was uncoupled from protein synthesis by omitting Phe-tRNA(Phe) at the A-site. In this reaction, the ribosome can be considered as an enzyme catalysing the transition of EF-Tu.GTP to EF-Tu.GTP. A constant EF-Tu.GTP concentration is maintained by regenerating GDP to GTP at the expense of phosphoenolpyruvate by pyruvate kinase. The rate constants are determined using a procedure which corrects for the reduction in specific activity of GTP due to regeneration of the nucleotide. Ribosomes with an occupied P-site are more efficient in stimulating the GTPase of EF-Tu.GTP than ribosomes with an empty P-site. The data suggest that this is mainly caused by an increased affinity of EF-Tu.GTP for ribosomes with a filled P-site rather than by an enhanced reactivity of the GTPase centre.
Abrahams
J P
JP
Department of Biochemistry, Leiden University, The Netherlands.
Acampo
J J
JJ
Kraal
B
B
Bosch
L
L
D001665:Binding Sites; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D012360:RNA, Transfer, Phe; D011072:Poly U; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu