Binding of aminoacyl-tRNA to ribosomes promoted by elongation factor Tu.  Studies on the role of GTP hydrolysis.
The role of guanosine triphosphate (GTP) in the elongation factor Tu(EF-Tu)promoted binding of aminoacyl-tRNA to ribosomes has been investigated.  In the presence of EF-Tu and GTP, phenylalanyl-tRNA (Phe-tRNA) was bound to the A site of the poly(U).ribosome complex having N-acetylphenylalanyl-tRNA on its P site.  EF-Tu could be utilized repeatedly in this binding reaction in the presence of GTP but was used only once and remained bound to ribosomes when GTP was replaced by 5'guanylyl methylenediphosphonate (Gpp(CH2)p), a nonhydrolyzable analog of GTP.  The binylalanyl-tRNA, while little dipeptide was formed in the latter case.  However, when EF-Tu and Gpp(CH2)p bound to the ribosomal complex were released by centrifugation through 10% sucrose containing 0.2 mM GDP, the yield of the dipeptide was correspondingly increased.  It was concluded that the role of GTP in  this reaction is to facilitate the transfer of Phe-tRNA to ribosomes by virtue of the high affinity of EF-Tu.GTP for ribosomes.  The subsequent conversion of EF-Tu.GTP to EF-Tu.GDP, a form of EF-Tu with low affinity for ribosomes as well as for Phe-tRNA, resulted in the detachment of EF-Tu.  Thus, the hydrolysis of GTP seems to be required for the release of EF-Tu from ribosomes, which is necessary for peptidyl transfer and the reutilization of EF-Tu.  A freely reversible interaction between ribosome and Phe-tRNA.EF-Tu.Gpp(CH2)p complex was also demonstrated.
D001665:Binding Sites; D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010649:Phenylalanine; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002250:Carbon Radioisotopes; D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D012343:RNA, Transfer