A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase.
Early investigations into the interaction between Escherichia coli glutamyl-tRNA synthetase (GluRS) and tRNAGlu have implicated the modified nucleoside 5-[(methylamino)methyl]-2-thiouridine in the first position of the anticodon as an important contact for efficient aminoacylation. However, the experimental methods employed were not sufficient to determine whether the interaction was dependent on the presence of the modification or simply involved other anticodon loop-nucleotides, now occluded from interaction with the synthetase. Unmodified E. coli tRNA(Glu), derived by in vitro transcription of the corresponding gene, is a poor substrate for GluRS, exhibiting a 100-fold reduction in its specificity constant (kcat/KM) compared to that of tRNA(Glu) prepared from an overproducing strain. Through the use of recombinant RNA technology, we created several hybrid tRNAs which combined sequences from the in vitro transcript with that of the native tRNA, resulting in tRNA molecules differing in modified base content. By in vitro aminoacylation of these hybrid tRNA molecules and of tRNAs with base substitutions at positions of nucleotide modification, we show conclusively that the modified uridine at position 34 in tRNA(Glu) is required for efficient aminoacylation by E. coli GluRS. This is only the second example of a tRNA modification acting as a positive determinant for interaction with its cognate aminoacyl-tRNA synthetase.
Sylvers
L A
LA
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Rogers
K C
KC
Shimizu
M
M
Ohtsuka
E
E
Söll
D
D
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005975:Glutamate-tRNA Ligase / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D012352:RNA, Transfer, Glu / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism; D013379:Substrate Specificity; D013891:Thiouridine / Q000031:analogs & derivatives*; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D012352:RNA, Transfer, Glu; D013891:Thiouridine; C011916:5-methylaminomethyl-2-thiouridine; D016914:Ribonuclease H; D005975:Glutamate-tRNA Ligase