tRNA-guanine transglycosylase from Escherichia coli: gross tRNA structural requirements for recognition.
tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the post-transcriptional modification of specific tRNAs (those for Asn, Asp, His, and Tyr) with the hypermodified base, queuine. In Escherichia coli this enzyme catalyzes the exchange of guanine-34 in the anticodon with preQ1, which is subsequently further modified to queuine. There is evidence that such hypermodified tRNA molecules may play a role in the control of cell proliferation and differentiation. In order to perform detailed, in vitro mechanistic studies and to probe the tRNA-enzyme interaction, we have generated unmodified E. coli tRNA(Tyr) and truncated analogues using an in vitro RNA synthesis system suggested by Milligan and Uhlenbeck [Milligan, J. F., & Uhlenbeck, O. C. (1989) Methods Enzymol. 180, 51-62]. From this system we have generated three tRNA analogues totally devoid of any post-transcriptional modifications. In order to compare the unmodified tRNA with the true physiological substrate for TGT, that is, tRNA that contains all modified bases except queuine, we have isolated E. coli tRNA(Tyr) from an overexpressing clone in a TGT-deficient strain of E. coli. We report here that unmodified, full-length tRNA(Tyr) serves as a substrate for TGT with kinetic parameters that are, within experimental error, the same as those for in vivo isolated tRNA(Tyr). This indicates that other post-transcriptional modifications have negligible effects upon TGT recognition of tRNA. A 17-base oligoribonucleotide, corresponding to the anticodon loop and stem, is also a substrate for TGT with only a 20-fold loss in Vmax/KM, versus the full-length tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Curnow
A W
AW
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065.
Kung
F L
FL
Koch
K A
KA
Garcia
G A
GA
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010430:Pentosyltransferases / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012365:RNA, Transfer, Tyr / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012365:RNA, Transfer, Tyr; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase