Random‐splitting of tRNA transcripts as an approach for studying tRNA‐protein interactions

Abstract

Location of phosphodiester bonds essential for ammoacylation of bovine tRNATn' was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21. 22, 36-38. 57-59, 62 and 64 were critical for aminoacylation capacity of tRNA'IP -transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and m the regions presumably outside the contact area as well. These results indicate that for mamtenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non-modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed m this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.

A new assay for tRNA aminoacylation kinetics

“…The difference in gel mobility between aminoacylated and unaminoacylated tRNAs is primarily due to the extra positive charge on the amino acid, suggesting that the acid gel assay will be suitable for all tRNAsynthetase pairs+ Indeed, significant gel shifts have been reported for many different intact aminoacyl-tRNAs (Varshney et al+, 1991)+ Although we used a nicked tRNA substrate to improve resolution, it seems possible that the gel system might be improved sufficiently such that intact tRNAs can be used+ Even if this is not possible, it is likely that a fully active nicked tRNA substrate can be found for any synthetase by varying the position of the nick+ Many sites in yeast tRNA Phe can tolerate a nick without altering its global folding (Pan et al+, 1991), and a useful strategy has been published for finding sites in a tRNA that can be nicked without affecting aminoacylation (Aphasizhev et al+, 1993)+ Although unmodified nicked tRNA was used in these experiments, a nick in the specific position of a modified tRNA may be prepared using deoxynucleotide-directed RNAse H cleavage (Hayase et al+, 1990;Lapham et al+, 1997)+ Additionally, whereas nonradioactive versions of all amino acids are readily available, certain radiolabeled amino acids are either unavailable or are not stable over long periods+ The generality of the gel assay can therefore be considered another advantage over the traditional assay+ Finally, one of the greatest advantages of the gel assay is its much higher sensitivity+ By using a [ 32 P]-labeled RNA substrate instead of an [ 3 H]-labeled amino acid, the gel assay can potentially detect as little as 0+01 fmol of tRNA, making it at least 1,000-fold more sensitive than the traditional assay+ A major consequence is that this assay can be used in pre-steadystate kinetics where the enzyme is in excess of the tRNA+ This permits the kinetic dissection of the tRNA binding step from the aminoacyl tRNA release step in the kinetic reaction mechanism+ In preliminary experiments, we have observed aminoacylation of 40 fmol nicked tRNA substrate by 8 pmol AlaRS, suggesting that such pre-steady-state measurements are feasible+ The ability to accurately measure both the first and second steps of the aminoacylation reaction should permit the efficient determination of all the kinetic rate constants of the aminoacylation reaction by redundant steady-state and pre-steady-state methods+ This information will provide a framework for the subsequent analysis of mutant proteins and tRNAs+…”

Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

“…The conditions for formation of tRKI-protein complexes were as described for tRNA-aminoacyl-tRNA synthetase complexes (Aphasizhev et al, 1993), with minor modifications. Reactions were carried out in a final volume of 20 gI containing S iM radiolabelled tRNA (2-5 X 105 c.p.m., 0.1-0.2 ,ug), 1-5 gl of protein extracts (5-25 tg) and 25 mM HEPES, pH 6.8, 10 mM MgCl2, 5 mM DTT, with or without ATP (1 mM), for 10 min at 20°C.…”

tRNATrp as cofactor of gelonin, a ribosome‐inactivating protein with RNA‐N‐glycosidase activity features required for the cofactor activity

“…Alternatively, since ATP participates in the interaction between tR_NA Trp and tryptophanyltKNA synthetase (19), such complex might constitute the active form of tKNA Trp in geloninpromoting activity. However all attempts to increase the gelonln-promoting activity of tR-NA Trp with purified tryptophanyl-tKNA synthetase have been unsuccessful.…”