Abstract
A gel shift assay that distinguishes the aminoacylated form from the deacylated form of tRNAs was used to study the requirements for aminoacylation of Escherichia coli tRNA(Asn) in vivo. tRNA(Asn) derivatives containing single base changes in their anticodons or discriminator bases were constructed, and the extent of in vivo aminoacylation was determined directly. Substitution of U35 with C35 or U36 with C36 abolished aminoacylation of the tRNA. Substitution of G34 with C34 converted tRNA(Asn) into a lysine acceptor. Thus, each of the anticodon nucleotides are important for aminoacylation of tRNA(Asn). Substitution of discriminator base G73 with A73 affected the extent of aminoacylation in vivo indicating that the discriminator base also contributes to aminoacylation of tRNA(Asn).
Highlights
A gel shift assatyhat distinguishes the aminoacylated using specific 32P-labeledprobes
Since all tRNAs adopt similar three-dimen- type tRNAAsn(GUU) and the mutant anticodon derivatives sional structures[1,2,3], an interesting questionis: how is each tRNA recognized by its cognate synthetase? It is well known that each tRNA hasa unique combination of nucleotides, known as the “identity elements” of the tRNA, which tRNAAs”(GUC),tRNAAsn(GCU)a, nd tRNAAsn(CUU).Oligonucleotides 5, 6, 7, and 8 are complementary, respectively, to nucleotides 60-76 of tRNAA””(GUUw) ith discriminator base G73, A73, C73, or U73
A m - tRNAAsn+ 0 discriminator base are both importantfor aminoacylation of tRNAAs"
Summary
All Three Anticodon Nucleotides in tRNAAS"Are Required reprobed with labeled oligonucleotide complementary to the for Efficient Aminoacylation by AsnRS-To study the contri- wild type tRNAA8".In both cases, the endogenous tRNAA8" bution of anticodon nucleotides 34,35, and 36 to tRNAA8" was found to be fully aminoacylated (data notshown). These derivatives differs from wild type tRNAAs"by a single The tRNAA8"(CUU)mutant was aminoacylated to about nucleotide substitution in the anticodon (Fig. 1).The wild 50%(Fig. 2, lane 4 ) while the endogenous tRNAAa"(GUU)in type and mutant tRNAs were expressed in E. coli, isolated, the same tRNA preparation was found to be essentially fully and analyzed as described [10]. Cylated in vivo as revealed by a mobility shift upon base tRNAA""(CUU) Is Aminoacylated with Lysine- treatment (Fig. 2, compare lanes 1 and 2). In the tRNAA8"(CUU)is partially aminoacylated, it is not clear case of tRNAAa"(GUC)and tRNAAa"(GCU)mutants, similar whether it is aminoacylated with asparagine or other amino shifts of mobility were not observed, indicating that these acid(s). Since tRNAA8"(CUU)is expected to read AAG, a mutant tRNAswere not aminoacylated by AsnRS in vivo or lysine codon, if the tRNA is aminoacylated with asparagine that theaminoacylation level wastoo low to be detected(Fig. or anyamino acid other thanlysine, it would have the poten-.
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