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

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 tRNAPhe-C-C-A(3'NH) from yeast and to the minor nucleoside x in E. coli tRNAArg, tRNALys, tRNAMet, tRNAIle and tRNAPhe. 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 tRNALys-C-C-A from E. coli can be phenylalanylated by phenylalanyl-tRNA synthetase from yeast, whereas this is not the case for fluorescamine treated tRNALys-C-C-A(XF47). Pre-tRNAPhe-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.

Undecagold cluster modified tRNAPhe from Escherichia coli and its activity in the protein elongation cycle

“…GTP could be visualized by STEM imaging and mass measurement (Blechschmidt et al, 1993). Sprinzl and Faulhammer (1978) demonstrated that the fluorescence of the fluorescamine-modified Phe-tRNAP'" is not affected upon ternary complex formation with EF-Tu .…”

“…This low modification rate could not be explained by sterical hindrance of the gold cluster because the modification with NEM gives the same yield. Sprinzl and Faulhammer (1978) describe a modification of 72% of the tRNAPhe with fluorescamine at acp3U. Asuming that all molecules containing the acp3U base can react with fluorescamine, this would indicate a partial undermodification of tRNAPhc in respect to acp3U47.…”

“…Poly (U)-dependent poly(phe) synthesis was performed in a purified E. coli cell-free system (Sprinzl and Faulhammer, 1978). The reaction mixture (30 p1) contained 15 mM Mops/KOH, pH 7.5, 100 mM NH4CI, 10 mM MgC12, 1 mM dithiothreitol and 0.4 mM GTP.…”

“…Unfortunately, selective labeling of a sulfhydry1 group on 2-thiocytidine enzymically inserted at position 75 of the 3' end of yeast tRNAPhe with the bulky Au,, cluster renders the tRNA inactive in enzymic aminoacylation. However, Sprinzl and Faulhammer (1978) investigated the reaction of fluorescamine with primary amino groups of several tRNAs from Escherichia coli. The reagent was attached to their 3-(3-amino-3-carboxypropyl)uridine nucleoside (acp'U) located in the small, five-membered variable loop of some tRNAs (Steinberg et al, 1993).…”

Structural features in aminoacyl‐tRNAs required for recognition by elongation factor Tu

“…However, when its base )<47 is labeled with fluorescamine, no change in fluorescence is observed upon ternary complex formation [62], suggesting that the variable region although possibly 'covered' to some extent by EF-Tu, is probably not in very close contact with the factor. In any event, the variable region is not believed to be essential for ternary complex formation.…”