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.     

A New Model for the Comparison of the Efficiency and Selectivity of Photoreactive Groups Towards the Nucleic Acid and Protein Functional Groups

Abstract

A new model for the comparison of the efficiency and selectivity of photoreactive groups towards the nucleic acid and protein functional groups is described. It was shown that reagents carrying p-azidoaniline residue are the most efficient and selective for the modiication of the side radicals of amino acids.     

Hybridization analysis of nucleic acids using stained latex]

Method of the latex hybridization analysis has been developed: after hybridization of NA-target with biotinylated probe visualization of hybrids was carried out using latex particles, containing fluorescent dye pyronin G and coated with streptavidin. Due to encapsulation of the fluorescent dye in polymer particles sensitivity of the analysis was increased by several orders of magnitude in comparison with methods, using fluorescently labelled probes. Possessing a number of advantages, the method yields to none of any other methods of NA hybridization analysis in sensitivity.