tRNA IDENTITY

Determination of 2′-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: A nucleotide analogue interference study

“…Nucleotide bases have been recognized as identity elements in interaction of tRNAs with synthetases (Normanly & Abelson, 1989;Söll & RajBhandary, 1995)+ 29-Hydroxyl groups have also been shown to play a role in this process+ These experiments were based on a complete exchange of all 29-deoxy groups in the molecule (Khan & Roe, 1988) or just one of the four nucleotides (Aphasizhev et al+, 1997)+ Additionally, kinetic analysis of single 29-deoxy-modified positions in minihelices or tRNA fragments of tRNA Ala or tRNA Pro showed small but distinct effects in aminoacylation efficiency (Musier-Forsyth & Schimmel, 1992;Yap & Musier-Forsyth, 1995)+ We were interested in an in vitro scanning procedure which should rapidly identify such 29-deoxy-sensitive sites in a complete tRNA rather than testing individual positions in a stepwise fashion+ The E. coli tRNA Asp system was chosen because it offered the opportunity to compare the results with an X-ray structural model still under final refinement (L+ Moulinier & D+ Moras, pers+ comm+)+ Although the simplest method for scanning a tRNA for the importance of 29-hydroxyl groups for aminoacylation should be the random incorporation of 29-deoxynucleotides, the identification of the interfering positions by limited alkaline hydrolysis proved impractical in our system because of high background of cleavage, even though this procedure had been applied to a 15-nt-long tRNA anticodon stem-loop fragment to test ribosomal A site binding (von Ahsen et al+, 1997)+ We therefore chose to couple the 29-deoxy modification with a phosphorothioate modification that generates a signal only at the deoxy positions upon iodine cleavage (Gish & Eckstein, 1988) and thus facilitates the 29-deoxy identification+ As any interference observed with this double modification could result from the deoxy and/or the phosphorothioate substitutions, the effect of the phosphorothioate modification was determined separately by transcribing the E. coli tRNA Asp with dNTPaS or NTPaS+ Both pools were then charged with the cognate synthetase and a tRNAs were ligated using one unmodified half and one half containing the deoxynucleotide at the identified position as described in Materials and Methods+ Each measurement was repeated at least twice+ Error Յ 20%+ (Gish & Eckstein, 1988;Schatz et al+, 1991;Verma & Eckstein, 1998)+ Because the incorporation of the analogues is random, this approach does not rely on any assumption about which positions might be important and thus differs from the previous studies+ In the scanning experiments positions U11, A24, U25, C28, U29, C36, C48, G57, A58, and C67 showed only a deoxy effect+ All others were accompanied by considerable phosphorothioate interference+ At G34, C74, and C75 the deoxy effect on top of the phosphorothioate interference was considered strong enough to be indicative of an additional effect+ The only position where the phosphorothioate effect was stronger than the deoxy effect was U35, the central position of the anticodon+ However, in general, comparison between these two effects is made difficult by the fact that the amount of dNTPaS and NTPaS incorporation is not necessarily the same+ The assignment of deoxy effects was therefore conservative+…”

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

“…Analysis of tRNA identity elements has shown that often several sequences are required to specify correct acylation of a given tRNA, and that strong cooperative interactions can occur between these elements to ultimately determine the identity of a tRNA (Normanly and Abelson 1989;Putz et al 1993;Rogers et al 1993). Therefore, although replacement of the other candidate import determinants of the imported glutamine tRNA with nonimported glutamine tRNA sequence had little or no effect on mitochondrial import, the possibility that these sequences acted synergistically to direct import was a viable hypothesis.…”

Identity elements in bovine tRNATrp required for the specific stimulation of gelonin, a plant ribosome-inactivating protein

“…The specificity among mammalian tRNAs of tRNA Trp as a cofactor of gelonin and the observation that yeast tRNA Trp is completely devoid of stimulating activity (Brigotti et al+, 1996) led us to study the elements relevant to the gelonin-promoting activity of mammalian tRNA Trp + The method involving in vitro transcription by T7 RNA polymerase (Sampson & Uhlenbeck, 1988) was used to construct tRNA Trp molecules with substitutions in their nucleotide sequence+ The extensive structure-function analysis was made possible by the observation (Brigotti et al+, 1998b) that the wild-type bovine tRNA Trp transcript, lacking the modified bases normally found in mammalian mature tRNA Trp , is fully active as a stimulator of gelonin+ This situation is akin to that in which synthetic tRNAs, in spite of their somewhat lower stability in tertiary folding (Maglott et al+, 1998), are nonetheless good substrates for cognate aminoacyl-tRNA synthetases (Normanly & Abelson, 1989)+…”