Affinity labeling at the A-site of Escherichia coli ribosomes by a non-hydrolyzable gamma-amide analog of GTP.
gamma-Amides of GTP and affinity and photoaffinity derivatives of gamma-amides of GTP: gamma-anilide of GTP, gamma-(4-azido)anilide of GTP, gamma-[N-(4-azidobenzyl)-N-methyl]amide of GTP, gamma[4-N-(2-chloroethyl)-N-methylaminobenzyl]amide of GTP and gamma-[4-N-(2-oxoethyl)-N-methylaminobenzyl]amide of GTP substituted efficiently for GTP in the EF-Tu-dependent transfer of aminoacyl-tRNA to the ribosome but, in contrast to GTP, they were not hydrolyzed in this process. They represent a new class of non-hydrolyzable GTP analogs with preserved gamma-phosphodiester bond. The radioactive analog of GTP: gamma-[4-N-(2-chloroethyl)-N-methylamino[14C]benzyl]amide of GTP was used as an affinity labeling probe for the identification of components of the GTPase center formed in the EF-Tu-dependent transfer reaction of aminoacyl-tRNA to the ribosomal A-site. Within a six-component complex of poly(U)-programmed E. coli ribosomes with elongation factor Tu, Phe-tRNA(Phe) (at the A-site), tRNA(Phe) (at the P-site) and the [14C]GTP analog, mainly the ribosomal 23S RNA and to a lesser extent the ribosomal proteins L17, L21, S16, S21 and the ribosomal 16S RNA were labeled by the reagent. No significant modification of EF-Tu was detected.
D000345:Affinity Labels / Q000378:metabolism*; D000577:Amides / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006868:Hydrolysis; D010452:Peptide Biosynthesis; D010444:Peptide Elongation Factor Tu / Q000502:physiology; D010455:Peptides; D010777:Photochemistry; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D000345:Affinity Labels; D000577:Amides; D010455:Peptides; D012346:RNA, Transfer, Amino Acyl; C012001:polyphenylalanine; C057026:(4-N-(2-chloroethyl)-N-methylaminobenzyl)amide guanosine-5'-triphosphate; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu