tRNA‐guanine transglycosylase from Escherichia coli: recognition of full‐length ‘queuine‐cognate' tRNAs

Abstract

A key enzyme involved in the incorporation of the modified base queuine into tRNA (position 34) is tRNA-guanine transglycosylase (TGT). Studies of the recognition of truncated tRNAs by the Escherichia coli TGT have established a minimal recognition motif involving a minihelix with a 7 base loop containing a U-G-U sequence (where G is replaced with queuine) [Curnow, A.W. and Garcia, G.A. (1995) J. Biol. Chem. 270, 17264^17267;Nakanishi, S. et al. (1994) J. Biol. Chem. 269, 32221^32225]. Still, a clearer understanding of the recognition of full-length`queuine-cognate' tRNAs by TGT remains lacking. In this paper, we report the in vitro transcription and enzymological characterization (K m and k t ) of all four`queuine-cognate' tRNAs from E. coli and from Saccharomyces cerevisiae with the TGT from E. coli. No primary or secondary structures emerge as important recognition elements from this study. The modest differences in substrate specificity (relative k t /K m values vary from 0.5 to 8.4) seen among these`queuine-cognate' tRNAs most likely result from the accumulated effects of many subtle factors. Interestingly, the yeast tRNAs are essentially equivalent to the E. coli tRNAs as substrates for TGT, indicating that there is nothing intrinsic to the yeast tRNAs that accounts for the absence of queuine in yeast.z 1998 Federation of European Biochemical Societies.

The RNA-binding PUA Domain of Archaeal tRNA-Guanine Transglycosylase Is Not Required for Archaeosine Formation

“…The moderate decrease in the k cat value of the ⌬PUA mutant results in a catalytic rate similar to that of E. coli TGT as measured here (2.5 ϫ 10 Ϫ3 s Ϫ1 ) and by others (51). It is also comparable with that reported for pseudouridine synthetases from yeast (47) and E. coli (52,53), which also catalyze a transglycosylation step during their reaction.…”

The RNA-binding PUA Domain of Archaeal tRNA-Guanine Transglycosylase Is Not Required for Archaeosine Formation

“…The moderate decrease in the k cat value of the ⌬PUA mutant results in a catalytic rate similar to that of E. coli TGT as measured here (2.5 ϫ 10 Ϫ3 s Ϫ1 ) and by others (51). It is also comparable with that reported for pseudouridine synthetases from yeast (47) and E. coli (52,53), which also catalyze a transglycosylation step during their reaction.…”

tRNA Recognition of tRNA-guanine Transglycosylase from a Hyperthermophilic Archaeon, Pyrococcus horikoshii

“…The low turnover rate of TGT of Ͻ1/min was in accordance with the multistep mechanism of the base replacement reaction, as proposed for bacterial TGT (20). A low K cat was also reported for E. coli TGT (22,30).…”

tRNA–guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis

“…(It should be noted that these K M values are ca. 10-fold lower than K M values that we have previously published [7,9,18]. The current studies utilize a higher specific activity guanine substrate, allowing for a more sensitive assay.…”

The RNA-binding PUA Domain of Archaeal tRNA-Guanine Transglycosylase Is Not Required for Archaeosine Formation

“…The moderate decrease in the k cat value of the ⌬PUA mutant results in a catalytic rate similar to that of E. coli TGT as measured here (2.5 ϫ 10 Ϫ3 s Ϫ1 ) and by others (51). It is also comparable with that reported for pseudouridine synthetases from yeast (47) and E. coli (52,53), which also catalyze a transglycosylation step during their reaction.…”

tRNA Recognition of tRNA-guanine Transglycosylase from a Hyperthermophilic Archaeon, Pyrococcus horikoshii

“…The low turnover rate of TGT of Ͻ1/min was in accordance with the multistep mechanism of the base replacement reaction, as proposed for bacterial TGT (20). A low K cat was also reported for E. coli TGT (22,30).…”

tRNA–guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis

“…(It should be noted that these K M values are ca. 10-fold lower than K M values that we have previously published [7,9,18]. The current studies utilize a higher specific activity guanine substrate, allowing for a more sensitive assay.…”

The RNA-binding PUA Domain of Archaeal tRNA-Guanine Transglycosylase Is Not Required for Archaeosine Formation

“…The moderate decrease in the k cat value of the ⌬PUA mutant results in a catalytic rate similar to that of E. coli TGT as measured here (2.5 ϫ 10 Ϫ3 s Ϫ1 ) and by others (51). It is also comparable with that reported for pseudouridine synthetases from yeast (47) and E. coli (52,53), which also catalyze a transglycosylation step during their reaction.…”

tRNA Recognition of tRNA-guanine Transglycosylase from a Hyperthermophilic Archaeon, Pyrococcus horikoshii

“…The low turnover rate of TGT of Ͻ1/min was in accordance with the multistep mechanism of the base replacement reaction, as proposed for bacterial TGT (20). A low K cat was also reported for E. coli TGT (22,30).…”

tRNA–guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis

“…(It should be noted that these K M values are ca. 10-fold lower than K M values that we have previously published [7,9,18]. The current studies utilize a higher specific activity guanine substrate, allowing for a more sensitive assay.…”