The Escherichia coli tRNA-Guanine Transglycosylase Can Recognize and Modify DNA

Abstract

tRNAs contain a large number of modified nucleosides (1). One of the more elaborately modified nucleosides is queuine (7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanine). tRNA-guanine transglycosylase (TGT) 1 catalyzes the exchange of queuine (or a precursor) for guanine 34 in the anticodon of certain tRNAs. The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs (tyrosine, aspartate, asparagine, and histidine) (2, 3). The UGU sequence is undeniably the major determinant for tRNA recognition by TGT. However, provided that TGT can position the UGU sequence in the active site in the proper orientation, the UGU sequence need not reside in the anticodon loop to be recognized. Recent studies have shown that TGT can recognize the UGU sequence in at least 2 additional minihelical contexts, at the base of the T⌿C stem in yeast tRNA Phe and in the anticodon position in the absence of U33 (4, 5). Thus, tRNA recognition by TGT is more flexible than previously believed. This observation prompted an examination of the ability of TGT to recognize RNAs containing modifications of the UGU sequence. The initial studies that identified the UGU sequence as the major identity element utilized only canonical base (C, G, A, U) replacements (2, 3). Previous experiments have demonstrated that a DNA analogue of an RNA minihelix corresponding to the anticodon arm of Escherichia coli tRNA Tyr , ECYMH (dEC-YMH) was inactive.2 However, there are two fundamental differences between RNA and DNA, the lack of the 2Ј-hydroxyls and the presence of thymidine rather than uracil in DNA. Therefore, dECYMH has a TGT sequence rather than a UGU sequence.Given the importance of the UGU sequence in TGT recognition, it is possible that the inactivity of dECYMH was due to the presence of thymidine rather than the loss of the 2Ј-hydroxyls. To investigate the role of the 2Ј-hydroxyl in TGT recognition and catalysis, we have studied a deoxyguanosine 34 analogue of ECYMH (Fig. 1, dG 34 ECYMH). This analogue is a substrate for TGT with less than a 10-fold reduction in activity. To further probe the ability of TGT to recognize RNA analogues lacking the 2Ј-hydroxyl, modified DNA analogues of the previously described minihelix ECYMH (2, 6) ( Fig. 1, dUdECYMH) and the alternative TGT minihelical substrates UGU ϩ1 (dUdUGU ϩ1 ) (4) and SCFMH(T⌿C) (dUdSCFMH(T⌿C)) (5) were synthesized and characterized. These analogues (containing deoxyuracil (dU) bases rather than thymidine bases) all serve as substrates for TGT, indicating that the tRNA-guanine transglycosylase from E. coli can recognize and modify DNA.

Novel genomic island modifies DNA with 7-deazaguanine derivatives

“…2B). The key residues that catalyze the G exchange (Asp102 and Asp280 of Zymomonas mobilis bTGT and Asp95 and Asp249 of Pyrococcus horikoshii aTGT) (19), as well as the Zinc binding site (CXCXXCX 22 H motif), are conserved in TgtA5 (Fig. 2B).…”

“…The role of 7-deazaguanosine derivatives as precursors of modified bases in tRNAs and of secondary metabolites is well established (9), and the preQ 0 molecule itself was recently found to have anticancer properties (20). These modified bases can also be detected in rRNA in vivo if labeled preQ 1 is fed to Escherichia coli (21) or inserted in DNA in vitro with the bTGT enzyme (22), but the biological relevance of these last two observations is not clear.…”

Transglycosylation: A mechanism for RNA modification (and editing?)

“…This RNA contains a 2′-deoxyguanosine at the wobble position (position of TGT catalysis) and has been previously reported to be a substrate in the TGT reaction [30]. Interestingly, the K m value for this substrate is approximately 3-to 5-fold lower for the D264E mutant than for wild-type or the D89E mutant, respectively.…”

“…In addition to a thorough understanding of the kinetics of the TGT reaction, studies over the last decade have helped define the recognition of both the tRNA [22,[25][26][27][28][29][30][31][32][33] and heterocyclic base substrates [34][35][36] and have also helped unveil the roles that certain active-site amino acid residues serve in catalysis. Early work in our laboratory determined that TGT is a zinc metalloenzyme.…”

“…It is known from studies in our lab and others' [22,25,[27][28][29][30]110] that the RNA recognition properties of eubacterial tRNA-guanine transglycosylase are limited to a U-G-U sequence in a stem-loop structure. This sort of recognition motif is likely to occur in many RNAs.…”