Unusual anticodon loop structure found inE.colilysine tRNA

Abstract

Although both tRNA(Lys) and tRNA(Glu) of E. coli possess similar anticodon loop sequences, with the same hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) at the first position of their anticodons, the anticodon loop structures of these two tRNAs containing the modified nucleoside appear to be quite different as judged from the following observations. (1) The CD band derived from the mnm5s2U residue is negative for tRNA(Glu), but positive for tRNA(Lys). (2) The mnm5s2U monomer itself and the mnm5s2U-containing anticodon loop fragment of tRNA(Lys) show the same negative CD bands as that of tRNA(Glu). (3) The positive CD band of tRNA(Lys) changes to negative when the temperature is raised. (4) The reactivity of the mnm5s2U residue toward H2O2 is much lower for tRNA(Lys) than for tRNA(Glu). These features suggest that tRNA(Lys) has an unusual anticodon loop structure, in which the mnm5s2U residue takes a different conformation from that of tRNA(Glu); whereas the mnm5s2U base of tRNA(Glu) has no direct bonding with other bases and is accessible to a solvent, that of tRNA(Lys) exists as if in some way buried in its anticodon loop. The limited hydrolysis of both tRNAs by various RNases suggests that some differences exist in the higher order structures of tRNA(Lys) and tRNA(Glu). The influence of the unusual anticodon loop structure observed for tRNA(Lys) on its function in the translational process is also discussed.

The crystal structure of HIV reverse-transcription primer tRNA(Lys,3) shows a canonical anticodon loop

“…Chemical modifications at 34 and 37 appear clearly and do not present, as previously proposed by others, either mutual interactions (Agris et al+, 1997) or any other unusual feature that could explain the different behavior of tRNA(Lys) versus tRNA(Glu) from Escherichia coli (Watanabe et al+, 1993)+ What is seen for S34 is the perfect stacking of its O2-replacing sulfur onto the aromatic ring of U35 (Fig+ 6)+ This is not unexpected when considering the well-known stabilizing effect of 2-thio modification (Watanabe et al+, 1979;Yokoyama et al+, 1979Yokoyama et al+, , 1985Houssier et al+, 1988;Kumar & Davis, 1997;Grosjean et al+, 1998)+ Less expected, however, is the pronounced stacking of the threonylcarbamoyl moiety of R37 onto A38, and not onto U36 as previously thought (Parthasarathy et al+, 1974(Parthasarathy et al+, , 1977)+ Interestingly, the sulfur of the thiomethyl group on the same R37 is stacked onto C75 of the symmetry-related molecule within a dimer+ That this crystal packing feature can be viewed, as explained above, as a part of a codon-anticodon interaction gives a reasonable view of the positioning of both A37-modifying chemical groups during codon reading by tRNA(Lys,3)+ This will be discussed elsewhere in view of tRNA(Lys) and other tRNAs frameshifting abilities+…”

Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency

“…acryloylaminophenylmercuric acetate) gels+ Thirty-nine percent of the wild-type mitochondrial tRNA Trp showed a slower migration in the APM gel, suggestive of thiolation+ The observed shift in migration of the tRNA Trp was eliminated by oxidation of the sulfur group with hydrogen peroxide, as previously described for another sulfur-containing tRNA (Watanabe et al+, 1994; Fig+ 8, lanes 1 and 2)+ In addition, the shift in migration observed is consistent with that of an in vitro-synthesized tRNA containing a single s 2 U modification (data not shown)+ Similar extents of thiolation were observed for all five tRNA Trp mutants in Figure 7 (Fig+ 8, lanes 3-7), indicating that editing is not required for thiolation+…”

“…Total mitochondrial RNA was electrophoretically separated on 7 M urea/8% acrylamide gels cast with 50 mM APM+ The samples were denatured by heating at 95 8C for 5 min under denaturing conditions (7 M urea)+ Equal amounts of RNA were loaded per lane (2+5 mg)+ After electrophoresis, the gels were soaked in TBE buffer containing 200 mM b-mercaptoethanol, stained with ethidium bromide for visualization under UV light, and blotted onto a Zetaprobe membrane (BioRad) for northern analysis+ Northern analysis was carried out as recommended (BioRad)+ Control northern hybridizations were carried out with each 32 P-labeled oligomer to show that under the hybridization conditions used, the oligomers would only recognize their intended target, thus ruling out the possibility of spurious hybridization between mutant-specific oligomers and the endogenous wild-type tRNA Trp (data not shown)+ The hydrogen peroxide treatment was carried out as previously described (Watanabe et al+, 1994), as a control to show that the observed shift is due to thiolation of the tRNA in question+…”

The tRNA Thiolation Pathway Modulates the Intracellular Redox State in Escherichia coli

“…S5 in the supplemental material). The mnm 5 s 2 U base is used in tRNA lys and tRNA Glu for AAA (AAG) and GAA (GAG) codons, respectively (44). If indirect effects of translation cause reduction of RNR expression, these codon usages of the genes involved in RNR pro-…”