Participation of X47-fluorescamine modified E. coli tRNAs in in vitro protein biosynthesis

Publisher Website
Google Scholar
Abstract
The reaction of fluorescamine with primary amino groups of tRNAs was investigated. The reagent was attached under mild conditions to the 3′-end of tRNA Phe -C-C-A(3′NH 2 ) from yeast and to the minor nucleoside X in E.coli tRNA Arg , tRNA Lys , tRNA Met , tRNA Ile and tRNA Phe . The primary aliphatic amino groups of these tRNAs react specifically so that the fluorescamine dye is not attached to the amino groups of the nucleobases. E.coli tRNA species modified on the minor nucleoside X47 can all be aminoacylated. An involvement of the minor modified nucleoside X47 in the tRNA : synthetase interaction is detected. Native tRNA Lys -C-C-A from E.coli can be phenylalanylated by phenylalanyl-tRNA synthetase from yeast, whereas this is not the case for fluorescamine treated tRNA Lys -C-C-A(XF47). Phe-tRNA Phe -C-C-A(XF47) forms a ternary complex with the elongation factor Tu:GTP from E.coli, binds enzymatically to the ribosomal A-site and is active in poly U dependent poly Phe synthesis. Fluorescamine-labelled E.coli tRNAs provide new substrates for the study of protein biosynthesis by spectroscopic methods.