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

2002; Elsevier BV; Volume: 277; Issue: 9 Linguagem: Inglês

10.1074/jbc.m111077200

1083-351X

Susanne T. Nonekowski, Fan‐Lu Kung, George A. Garcia,

DNA and Nucleic Acid Chemistry

tRNA-guanine transglycosylase (TGT) catalyzes the exchange of queuine (or a precursor) for guanine 34 in tRNA. The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs. Recent studies have shown that the enzyme is capable of recognizing the UGU sequence in alternative contexts (Kung, F. L., Nonekowski, S., and Garcia, G. A. (2000) RNA 6, 233–244) and have investigated the role of the first U of the UGU sequence in tRNA recognition by TGT (Nonekowski, S. T., and Garcia, G. A. (2001) RNA 7, 1432–1441). The TGT reaction involves the breakage and re-formation of a glycosidic bond. To rule out a potential chemical mechanism involving the 2′-hydroxyl at position 34, we synthesized and evaluated an RNA minihelix with 2′-deoxy-G at 34. The high level of activity exhibited by this analogue indicates that the 2′-hydroxyl of G34 is not required for catalysis. Furthermore, we find that TGT can recognize analogues composed entirely of DNA, but only when 2′-deoxyuridines replace the thymidines in the DNA. The requirement for uridine bases for recognition is perhaps not surprising given the UGU recognition motif for TGT. However, it is not clear if the uracil requirement is due to specific recognition by TGT or due to the effect of uracils on the conformation of the oligonucleotide. tRNA-guanine transglycosylase (TGT) catalyzes the exchange of queuine (or a precursor) for guanine 34 in tRNA. The minimal RNA recognition motif for TGT has been found to involve a UGU sequence in the anticodon loop of the queuine-cognate tRNAs. Recent studies have shown that the enzyme is capable of recognizing the UGU sequence in alternative contexts (Kung, F. L., Nonekowski, S., and Garcia, G. A. (2000) RNA 6, 233–244) and have investigated the role of the first U of the UGU sequence in tRNA recognition by TGT (Nonekowski, S. T., and Garcia, G. A. (2001) RNA 7, 1432–1441). The TGT reaction involves the breakage and re-formation of a glycosidic bond. To rule out a potential chemical mechanism involving the 2′-hydroxyl at position 34, we synthesized and evaluated an RNA minihelix with 2′-deoxy-G at 34. The high level of activity exhibited by this analogue indicates that the 2′-hydroxyl of G34 is not required for catalysis. Furthermore, we find that TGT can recognize analogues composed entirely of DNA, but only when 2′-deoxyuridines replace the thymidines in the DNA. The requirement for uridine bases for recognition is perhaps not surprising given the UGU recognition motif for TGT. However, it is not clear if the uracil requirement is due to specific recognition by TGT or due to the effect of uracils on the conformation of the oligonucleotide. tRNAs contain a large number of modified nucleosides (1Grosjean H. Benne R. Modification and Editing of RNA. ASM Press, Washington, D. C.1998Crossref Google Scholar). 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) 1TGTtRNA-guanine transglycosylasequeuine7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosineRUMTm5U54-tRNA methyltransferase 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) (2Curnow A.W. Garcia G.A. J. Biol. Chem. 1995; 270: 17264-17267Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 3Nakanishi S. Ueda T. Hori H. Yamazaki N. Okada N. Watanabe K. J. Biol. Chem. 1994; 269: 32221-32225Abstract Full Text PDF PubMed Google Scholar). 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 TΨC stem in yeast tRNAPhe and in the anticodon position in the absence of U33 (4Nonekowski S.T. Garcia G.A. RNA. 2001; 7: 1432-1441PubMed Google Scholar, 5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar). 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 (2Curnow A.W. Garcia G.A. J. Biol. Chem. 1995; 270: 17264-17267Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar,3Nakanishi S. Ueda T. Hori H. Yamazaki N. Okada N. Watanabe K. J. Biol. Chem. 1994; 269: 32221-32225Abstract Full Text PDF PubMed Google Scholar). Previous experiments have demonstrated that a DNA analogue of an RNA minihelix corresponding to the anticodon arm of Escherichia coli tRNATyr, ECYMH (dECYMH) was inactive. 2A. W. Curnow and G. A. Garcia, unpublished data. However, there are two fundamental differences between RNA and DNA, the lack of the 2′-hydroxyls and the presence of thymidine rather than uracil in DNA. Therefore, dECYMH has a TGT sequence rather than a UGU sequence. tRNA-guanine transglycosylase 7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine m5U54-tRNA methyltransferase 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 2′-hydroxyls. To investigate the role of the 2′-hydroxyl in TGT recognition and catalysis, we have studied a deoxyguanosine 34 analogue of ECYMH (Fig. 1,dG34ECYMH). 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 2′-hydroxyl, modified DNA analogues of the previously described minihelix ECYMH (2Curnow A.W. Garcia G.A. J. Biol. Chem. 1995; 270: 17264-17267Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 6Curnow A.W. Kung F.L. Koch K.A. Garcia G.A. Biochemistry. 1993; 32: 5239-5246Crossref PubMed Scopus (71) Google Scholar) (Fig. 1, dUdECYMH) and the alternative TGT minihelical substrates UGU+1(dUdUGU+1) (4Nonekowski S.T. Garcia G.A. RNA. 2001; 7: 1432-1441PubMed Google Scholar) and SCFMH(TΨC) (dUdSCFMH(TΨC)) (5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar) 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. Reagents were purchased from Sigma, Aldrich, or Invitrogen unless otherwise noted. Buffered phenol, glycerol, and HEPES were from United States Biochemical (USB). Tris/HCl buffer was from Research Organics. 8-[3H]Guanine (10 Ci/mmol) was from Moravek Biochemicals. TGT was isolated as previously described (4Nonekowski S.T. Garcia G.A. RNA. 2001; 7: 1432-1441PubMed Google Scholar,5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar). The RNA minihelix, ECYMH, was chemically synthesized by automated chemical synthesis performed on an Expedite nucleic acid synthesis system (model 8909, PerSeptive Biosystems) using the manufacturer's protocols and reagents. However, the RNA phosphoramidite monomers (G, C, A, and U) and CPG columns were from Glen Research. dG34ECYMH was the generous gift of Dr. Houng-Yau Mei (Bioorganic Chemistry, Pfizer Global Research and Development, Ann Arbor, MI). The dU-DNA analogues, dUdECYMH, dUdUGU+1, and dUdSCFMH(TΨC) were from Invitrogen. The oligos were resuspended in 300–800 μl of HM 7.3 buffer (10 mm HEPES, pH 7.3, 1 mmMgCl2). Concentrations of the analogues were determined spectrophotometrically using the extinction coefficients at 260 nm calculated from the base composition of each oligonucleotide. All native and denaturing PAGE were performed on a Phast System (Amersham Biosciences) as previously reported (2Curnow A.W. Garcia G.A. J. Biol. Chem. 1995; 270: 17264-17267Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7Curnow A.W. Garcia G.A. Biochimie (Paris). 1994; 76: 1183-1191Crossref PubMed Scopus (31) Google Scholar). Typical native band shift assays were performed as follows: TGT (3 μm) was incubated with excess RNA (45–100 μm) at 37 °C for 30 min in a 10-μl reaction mixture containing 10 mm HEPES, pH 7.3, 1 mm MgCl2, 1 mmdithiothreitol, and 1 mm sodium phosphate. The reaction mixtures were then analyzed by native PAGE using 8–25% gradient polyacrylamide gels. Approximately 4 μl were loaded onto each lane. The capability of the RNAs to form a stable complex with TGT was assayed via denaturing PAGE as follows: TGT (7 μm) was incubated with excess RNA (45–100 μm) in the presence of 400 μm 9-methylguanine at 37 °C for 30 min in a 10-μl reaction mixture containing 10 mm HEPES, pH 7.3, 1 mm MgCl2, 1 mm dithiothreitol, and 1 mm sodium phosphate. SDS buffer (10 μl of 60 mm Tris/HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.01% bromphenol blue) was added to the reaction mixtures, and the incubation was continued for an additional hour at 25 °C. Approximately 4 μl were loaded onto each lane. In both native and denaturing PAGE, the gels were stained with Coomassie Blue to visualize the protein, although the gels were scanned in gray scale to generate the figures. A guanine incorporation assay was used to obtain the steady-state kinetic parameters as previously reported (2Curnow A.W. Garcia G.A. J. Biol. Chem. 1995; 270: 17264-17267Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar,5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar, 8Kung F.-L. Garcia G.A. FEBS Lett. 1998; 431: 427-432Crossref PubMed Scopus (13) Google Scholar). A slight modification was made to the precipitation step of the assays: the analogues were precipitated for 1 h at −20 °C rather than at room temperature. This improved the assay by reducing the background radioactivity. The dU-DNA analogues, dUdUGU+1 and dUdSCFMH(TΨC), were assayed at concentrations ranging from 0.2 to 20 μm, whereas the concentration for dUdECYMH ranged from 0.2 to 40 μm and the concentration for dG34ECYMH ranged from 0.1 to 60 μm. The TGT concentration was 180 nm. Aliquots (70 μl) were taken at 5, 10, 20, 40, and 80 min after the reaction had been initiated with enzyme and quenched by precipitation with 2 ml of ethanol and 10 μl of 3 m sodium acetate, pH 5.3. After the addition of the ethanol, the test tubes containing the samples were covered with parafilm and placed in the freezer (−20 °C) to chill for 1 h. The precipitated RNAs were collected on glass fiber filters (GF/C filter, Whatman), washed three times with EtOH, and dried. The amount of radioactivity on each filter was quantified by liquid scintillation. Disintegrations per minute (DPM) were converted into picomoles of radiolabeled guanine using the appropriate specific activities. The initial velocities (vi) obtained from linear regression of guanine incorporation versus time were plotted versussubstrate concentrations. V max andKm were obtained by nonlinear regression of these hyperbolic plots to the Michaelis-Menten equation. Values fork cat were obtained by dividing theV max values by the TGT concentration (180 nm) and the aliquot volume (70 μl). Assays were conducted in triplicate, and the average of the data points (vi) and the error bars generated from the standard deviation within each point were plotted. The initial velocity of ECYMH at 10 μm was determined in all assays to normalize the specific activity of TGT from assay to assay. The observation that TGT recognition of tRNA is flexible enough to accommodate the UGU sequence in alternate contexts (4Nonekowski S.T. Garcia G.A. RNA. 2001; 7: 1432-1441PubMed Google Scholar, 5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar) prompted the investigation of the capability of TGT to recognize a modified RNA minihelix. A 2′-deoxyguanosine 34 analogue of ECYMH (dG34ECYMH) was synthesized and shown to be a substrate for TGT with an 8-fold decrease in k cat and a 3-fold decrease in Km with respect to ECYMH (TableI). The substantial activity of dG34ECYMH rules out any requisite participation of the 2′-hydroxyl in the chemical mechanism of the TGT reaction. This conclusion is consistent with mutagenesis studies that implicate an aspartic acid residue as the enzymic nucleophile (9Romier C. Reuter K. Suck D. Ficner R. Biochemistry. 1996; 35: 15734-15739Crossref PubMed Scopus (46) Google Scholar, 10Kittendorf J.D. Barcomb L.M. Nonekowski S.T. Garcia G.A. Biochemistry. 2001; 40: 14123-14133Crossref PubMed Scopus (24) Google Scholar). Thus, it seems that any potential H-bonding interaction between the 2′-hydroxyl of G34 and TGT is not critical for binding or activity.Table IKinetic parameters for the dU-DNA analoguesAnalogueK m 1-aStandard errors are shown in parentheses. Standard errors fork cat/K m were calculated as in the following equation. S.E.(k cat/K m) = (k cat/K m) × [(S.E.kcat/kcat]2+[(S.E.KM)/Km]21-bKinetic parameters are determined from the average of two (ECYMH & dG34ECYMH) or three (ECY, dUdECYMH, UGU+1, dUdUGU+1, SCFMH(TΨC) & dUdSCFMH(TΨC)) replicate determinations of initial velocity data.k cat1-aStandard errors are shown in parentheses. Standard errors fork cat/K m were calculated as in the following equation. S.E.(k cat/K m) = (k cat/K m) × [(S.E.kcat/kcat]2+[(S.E.KM)/Km]21-bKinetic parameters are determined from the average of two (ECYMH & dG34ECYMH) or three (ECY, dUdECYMH, UGU+1, dUdUGU+1, SCFMH(TΨC) & dUdSCFMH(TΨC)) replicate determinations of initial velocity data.k cat/K m 1-aStandard errors are shown in parentheses. Standard errors fork cat/K m were calculated as in the following equation. S.E.(k cat/K m) = (k cat/K m) × [(S.E.kcat/kcat]2+[(S.E.KM)/Km]21-bKinetic parameters are determined from the average of two (ECYMH & dG34ECYMH) or three (ECY, dUdECYMH, UGU+1, dUdUGU+1, SCFMH(TΨC) & dUdSCFMH(TΨC)) replicate determinations of initial velocity data.Relativek cat/K mμm10 −3 ·s −110−3s−1μm −1ECY1-cKinetic parameters for ECY, ECYMH, & SCFMH(TΨC) are from Kung et al. (5).3.63 (0.44)4.92 (0.19)1.36 (0.17)1ECYMH1-cKinetic parameters for ECY, ECYMH, & SCFMH(TΨC) are from Kung et al. (5).4.68 (1.61)2.32 (0.26)0.50 (0.18)0.37dG34ECYMH1.40 (0.31)0.30 (0.01)0.21 (0.047)0.15dUdECYMH6.01 (0.82)0.46 (0.02)0.076 (0.11)0.056UGU+11-dKinetic parameters for UGU+1 are from Nonekowski and Garcia (4).6.22 (1.19)0.20 (0.01)0.032 (0.015)0.024dUdUGU+16.46 (0.82)0.28 (0.01)0.044 (0.058)0.032SCFMH(TΨC)1-cKinetic parameters for ECY, ECYMH, & SCFMH(TΨC) are from Kung et al. (5).1.57 (0.49)0.19 (0.01)0.12 (0.038)0.088dUdSCFMH(TΨC)4.06 (0.82)0.17 (0.02)0.041 (0.01)0.0301-a Standard errors are shown in parentheses. Standard errors fork cat/K m were calculated as in the following equation.S.E.(k cat/K m) = (k cat/K m) × [(S.E.kcat/kcat]2+[(S.E.KM)/Km]21-b Kinetic parameters are determined from the average of two (ECYMH & dG34ECYMH) or three (ECY, dUdECYMH, UGU+1, dUdUGU+1, SCFMH(TΨC) & dUdSCFMH(TΨC)) replicate determinations of initial velocity data.1-c Kinetic parameters for ECY, ECYMH, & SCFMH(TΨC) are from Kung et al. (5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar).1-d Kinetic parameters for UGU+1 are from Nonekowski and Garcia (4Nonekowski S.T. Garcia G.A. RNA. 2001; 7: 1432-1441PubMed Google Scholar). Open table in a new tab To determine the importance of the remaining 2′-hydroxyls, a DNA analogue of ECYMH (dUdECYMH) was synthesized and characterized. This analogue is comprised entirely of 2′-deoxyribonucleotides. However, because of the importance of the UGU sequence in TGT recognition, it contains deoxyuracil bases in place of thymidine bases. This allows the effect of the ribose backbone to be examined exclusively. In addition to dUdECYMH, the dU-DNA analogues of the alternate substrates UGU+1 (4Nonekowski S.T. Garcia G.A. RNA. 2001; 7: 1432-1441PubMed Google Scholar) (dUdUGU+1) and SCFMH(TΨC) (5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar) (dUdSCFMH(TΨC)) (Fig.1) were also synthesized and evaluated. Native PAGE band shift experiments demonstrate that all of the dU-DNA analogues bind to TGT (Fig.2 A). This indicates that none of the 2′-hydroxyls are critical for binding to TGT. The RNA analogue with the single deoxyribose substitution, dG34ECYMH, qualitatively exhibited the highest ratio of RNA-bound TGT to free TGT (Fig. 2 A, lane 3). It is not clear why the removal of the 2′-hydroxyl of G34 results in apparently tighter binding than the RNA analogue, ECYMH. Although it is unlikely that the overall conformation of dG34ECYMH differs significantly from ECYMH, it is possible that a local change in the sugar pucker of the G34 could be responsible for the apparently tighter binding. 2′-deoxyribonucleotides typically favor a C2′-endo(N) sugar pucker, whereas ribonucleotides are frequently found in a C3′-endo(S) conformation (11Saenger W. Principles of Nucleic Acid Structure. Springer Advanced Texts in Chemistry, Springer-Verlag, New York1984: 55-65Google Scholar). It seems likely that the predominantly RNA nature of dG34ECYMH yields a structure that is virtually identical to that of the native RNA substrate for TGT. The almost certain change in conformation at position 34 because of the deoxyguanosine could result in an orientation in the active site that is suboptimal for catalysis, but does not interfere with binding. dUdECYMH and the other dU-DNA analogues do show qualitatively less band shift than either ECYMH or dG34ECYMH (Fig. 2). Furthermore, the slightly higher Km values of the dU-DNA analogues are consistent with weaker binding. All of these results suggest that the loss of the 2′-hydroxyl at position 34 leads to a reduction in catalysis, possibly because of suboptimal orientation. The loss of the remaining 2′-hydroxyls appears to have a very small effect on binding, which may be because of a conformational effect. All the analogues were able to form a complex that was stable to mild denaturing conditions (∼50 kDa, Fig. 2 B). Previous reports strongly suggest that this complex is a covalent intermediate formed between RNA and aspartate 89 of TGT (9Romier C. Reuter K. Suck D. Ficner R. Biochemistry. 1996; 35: 15734-15739Crossref PubMed Scopus (46) Google Scholar, 10Kittendorf J.D. Barcomb L.M. Nonekowski S.T. Garcia G.A. Biochemistry. 2001; 40: 14123-14133Crossref PubMed Scopus (24) Google Scholar). Consistent with its tighter binding, dG34ECYMH formed a significantly more intense covalent complex (Fig. 2 B, lane 3). In fact, dG34ECYMH produces the largest amount of complex formation of any substrate analogue (RNA or DNA) tested to date, to our knowledge. Although it has not yet been firmly established that the stable complex represents a true mechanistic intermediate (e.g. chemical and kinetic competence of the covalent complex), there is a direct correlation between the formation of this complex and enzymatic activity (5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar). This correlation is consistent with our findings that all the dU-DNA analogues are substrates for TGT (Fig. 3). In general, the activity of the dU-DNA analogues mirrored their RNA analogues (Table I). For example, dUdECYMH, which was the most active DNA analogue, was derived from the "normal" RNA substrate ECYMH. The reduction in thek cat values for dUdECYMH (5-fold) and dG34ECYMH (7-fold) with respect to ECYMH suggests that the orientation of dG34 is not optimal for catalysis. The decreased activities of the alternate RNA substrates UGU+1and SCFMH(TΨC) demonstrate that catalysis for these analogues is also affected by suboptimal orientation of the UGU sequence as compared with the normal RNA substrate. The essentially identicalk cat values of dUdUGU+1 and dUdSCFMH(TΨC), relative to their respective RNA analogues, indicate that the ribose backbone does not significantly aid in any rearrangement that might enhance catalysis. The most prominent result presented in Table I is that removal of all the 2′-hydroxyls has a relatively minor effect (5- to 14-fold reduction) onk cat. Despite the small increases inKm , the overall specificities (k cat/Km) are quite comparable to the RNA analogues, especially for the alternate substrates UGU+1 and SCFMH(TΨC). The activity of the dU-DNA analogues can be explained in one of two ways. The first explanation is that TGT is not sensitive to the differences between RNA and DNA. However, we know that this is not strictly accurate because the analogue containing thymidine (dECYMH) is inactive. Yet, replacement of the thymidine bases with deoxyuracil does restore activity with TGT; thus, TGT recognition is not strictly dependent upon the ribose backbone or any conformational effects of the 2′-hydroxyls. The capability of TGT to recognize the UGU sequence in alternate contexts (4Nonekowski S.T. Garcia G.A. RNA. 2001; 7: 1432-1441PubMed Google Scholar, 5Kung F.L. Nonekowski S. Garcia G.A. RNA. 2000; 6: 233-244Crossref PubMed Scopus (20) Google Scholar) suggests that TGT recognition is fairly indiscriminate. However, recent results from our laboratory (4Nonekowski S.T. Garcia G.A. RNA. 2001; 7: 1432-1441PubMed Google Scholar) show that TGT recognition is blocked when the UGU sequence is locked into certain conformations (e.g. the anticodon loop "U-turn"). An alternative explanation is that the dU-DNA analogues are able to emulate the RNA analogues despite differences in the preferred conformations of deoxyribonucleotides versusribonucleotides. The most prevalent (and most recognizable) form of DNA is the B-form double helix in which the sugar conformation is 2′-endo (11Saenger W. Principles of Nucleic Acid Structure. Springer Advanced Texts in Chemistry, Springer-Verlag, New York1984: 55-65Google Scholar). Because it lacks the 2′-hydroxyls, DNA can also exist in several different forms, including A-DNA and Z-DNA (11Saenger W. Principles of Nucleic Acid Structure. Springer Advanced Texts in Chemistry, Springer-Verlag, New York1984: 55-65Google Scholar). Conversely, RNA exists predominantly in the A-form with a 3′-endo ribose conformation. Given the greater flexibility of DNA, it is possible that DNA analogues can adopt a conformation that is similar to the corresponding RNA in solution. Paquette et al. (12Paquette J. Nicoghosian K., Qi, G.-R. Beauchemin N. Cedergren R. Eur. J. Biochem. 1990; 189: 259-265Crossref PubMed Scopus (34) Google Scholar) compared the conformation of a tRNA with its "tDNA" and "anti-tDNA" (the complement of tDNA) analogues. By examining the mung bean nuclease cleavage patterns of tRNAMet, tDNAMet, and anti-tDNAMetthese authors demonstrated that the global structures of tRNA and tDNA were quite similar, although the core regions of the tDNAs are more exposed than the tRNA. Furthermore, tDNAMet and anti-tDNAMet are both cleaved by the restriction enzymesHhaI and CfoI, which verifies the presence of base-paired stems. Based on these studies, the authors conclude that nucleic acid conformation is largely determined by the interactions of the bases and those elements common to both DNA and RNA. One role of the 2′-hydroxyls (in tRNA at least) is to increase the stability of the molecule (12Paquette J. Nicoghosian K., Qi, G.-R. Beauchemin N. Cedergren R. Eur. J. Biochem. 1990; 189: 259-265Crossref PubMed Scopus (34) Google Scholar). Studies using transition metal complexes have confirmed that the global structure of tDNAPhe resembles that of tRNAPhe but that the base paired stem conformations differ somewhat (13Lim A.C. Barton J.K. Biochemistry. 1993; 32: 11029-11034Crossref PubMed Scopus (27) Google Scholar). Those results are most consistent with a more B-like conformation for the tDNA double helical regions. Additionally, Holmes and Hecht have shown that Fe·bleomycin cleaves tRNAHisand tDNAHis at the same major site, U35 or T35 respectively (14Holmes C.E. Hecht S.M. J. Biol. Chem. 1993; 268: 25909-25913Abstract Full Text PDF PubMed Google Scholar). All of the above studies were conducted using canonical base replacements (A, C, G, T, and deoxyribose for the tDNA analogues) and demonstrate that tDNA and tRNA can have similar conformations in solution. Additional studies have revealed that the presence of modified bases further increases the capability of DNA to mimic RNA (15Guenther R.H. Hardin C.C. Sierzputowska-Gracz H. Dao V. Agris P.F. Biochemistry. 1992; 31: 11004-11011Crossref PubMed Scopus (24) Google Scholar, 16Dao V. Guenther R. Agris P.F. Biochemistry. 1992; 31: 11012-11019Crossref PubMed Scopus (37) Google Scholar, 17Dao V. Guenther R. Malkiewicz A. Nawrot B. Sochacka E. Kraszewski A. Jankowska J. Everett K. Agris P.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2125-2129Crossref PubMed Scopus (55) Google Scholar, 18Basti M.M. Stuart J.W. Lam A.T. Guenther R. Agris P.F. Nat. Struct. Biol. 1996; 3: 38-44Crossref PubMed Scopus (27) Google Scholar). For example, DNA analogues of the yeast tRNAPhe anticodon stem loop containing modifications such as deoxyuracil, 5-methylcytidine (m5C), and 1-methylguanine (m1G) are able to bind Mg+2. This binding is dependent upon the presence of the modified bases (15Guenther R.H. Hardin C.C. Sierzputowska-Gracz H. Dao V. Agris P.F. Biochemistry. 1992; 31: 11004-11011Crossref PubMed Scopus (24) Google Scholar). The Mg+2 is bound in the upper part of the DNA hairpin (16Dao V. Guenther R. Agris P.F. Biochemistry. 1992; 31: 11012-11019Crossref PubMed Scopus (37) Google Scholar) in a position that is analogous to that seen in the x-ray crystal structure of yeast tRNAPhe (19Quigley G. Teeter M. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 64-68Crossref PubMed Scopus (360) Google Scholar). Furthermore, the modified DNA analogues were able to bind to poly(U)-programmed 30 S ribosomal subunits and competitively inhibit the binding of native tRNAPhe (17Dao V. Guenther R. Malkiewicz A. Nawrot B. Sochacka E. Kraszewski A. Jankowska J. Everett K. Agris P.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2125-2129Crossref PubMed Scopus (55) Google Scholar). They were also able to inhibit protein expression in a coupled transcription-translation system (18Basti M.M. Stuart J.W. Lam A.T. Guenther R. Agris P.F. Nat. Struct. Biol. 1996; 3: 38-44Crossref PubMed Scopus (27) Google Scholar). The solution structure of the fully modified DNA analogue demonstrates that the helix is in the B-form. However, the conformation of the anticodon loop includes the formation of the U-turn and is strikingly similar to that of yeast tRNAPhe (18Basti M.M. Stuart J.W. Lam A.T. Guenther R. Agris P.F. Nat. Struct. Biol. 1996; 3: 38-44Crossref PubMed Scopus (27) Google Scholar). This demonstrates that the structure of tRNA is not strictly dependent on the ribose backbone and underscores the importance of modified bases in tRNA structure and function. In light of these experiments, it is perhaps less surprising that tDNA analogues of tRNA can be aminoacylated. E. colitDNAPhe (with a 3′-terminal riboadenosine) and E. coli tDNALys were aminoacylated by their respective aminoacyl-tRNA synthetases (20Khan A.S. Roe B.A. Science. 1988; 241: 74-78Crossref PubMed Scopus (41) Google Scholar). In a similar fashion,E. coli methionyl-tRNA synthetase will aminoacylate a tDNAfMet analogue (21Perreault J.-P. Pon R.T. Jiang M.-Y. Usman N. Pika J. Ogilvie K.K. Cedergren R. Eur. J. Biochem. 1989; 186: 87-93Crossref PubMed Scopus (24) Google Scholar). Giegé et al. (22Aphasizhev R. TheobaldDietrich A. Kostyuk D. Kochetkov S.N. Kisselev L. Giege R. Fasiolo F. RNA. 1997; 3: 893-904PubMed Google Scholar) have utilized a mutant T7 RNA polymerase to selectively incorporate deoxyribose derivatives of each base (dA, dG, dC, or dU) in order to study the effect of replacing a subset of 2′-hydroxyls. Their results indicate that the yeast methionyl-tRNA synthetase will tolerate dA and dU substitutions, but large decreases in charging occur for dG or dC analogues. Similarly, yeast aspartyl-tRNA synthetase will efficiently charge dC and dA analogues but not dG or dU analogues (22Aphasizhev R. TheobaldDietrich A. Kostyuk D. Kochetkov S.N. Kisselev L. Giege R. Fasiolo F. RNA. 1997; 3: 893-904PubMed Google Scholar). The recognition of DNA analogues of RNA substrates also extends to some RNA editing and modifying enzymes. For example, a substrate containing all DNA residues (except for a single ribonucleotide at the cleavage site) can be cleaved by the hammerhead ribozyme, albeit with reduced efficiency (23Dahm S.C. Uhlenbeck O.C. Biochimie (Paris). 1990; 72: 819-823Crossref PubMed Scopus (40) Google Scholar, 24Yang J.-H. Perreault J.-P. Labuda D. Usman N. Cedergren R. Biochemistry. 1990; 29: 11156-11160Crossref PubMed Scopus (66) Google Scholar, 25Pyle A.M. Cech T.R. Nature. 1991; 350: 628-631Crossref PubMed Scopus (177) Google Scholar). From a catalytic perspective, a predominately DNA analogue of the ribozyme domain is capable of cleaving an RNA substrate (26Yang J.-H. Usman N. Chartrand P. Cedergren R. Biochemistry. 1992; 31: 5005-5009Crossref PubMed Scopus (111) Google Scholar). Other examples of RNA catalysis with DNA substrates include Group I introns (27Herschlag D. Cech T.R. Nature. 1990; 344: 405-409Crossref PubMed Scopus (109) Google Scholar) and RNase P (28Perreault J.-P. Altman S. J. Mol. Biol. 1992; 226: 399-409Crossref PubMed Scopus (89) Google Scholar, 29Lee J.-Y. Characterization of Saccharomyces cerevisiae RNase P and Its RNA SubunitDoctoral dissertation. University of Michigan, 1991Google Scholar). There is also some evidence that other tRNA modification enzymes are capable of recognizing DNA analogues. A tDNAfMet analogue is threonylated to a small degree by a crude yeast extract and is able to inhibit the threonylation of tRNAfMet (21Perreault J.-P. Pon R.T. Jiang M.-Y. Usman N. Pika J. Ogilvie K.K. Cedergren R. Eur. J. Biochem. 1989; 186: 87-93Crossref PubMed Scopus (24) Google Scholar); however, these results need to be verified under more stringent conditions. A dU-containing DNA minihelix analogue of the TΨC stem and loop of yeast tRNAPhe was reported to be a substrate for E. coli m5U54-tRNA methyltransferase (RUMT) (30Guenther R.H. Bakal R.S. Forrest B. Chen Y. Sengupta R. Nawrot B. Sochacka E. Jankowska J. Kraszewski A. Malkiewicz A. Agris P.F. Biochimie (Paris). 1994; 76: 1143-1151Crossref PubMed Scopus (14) Google Scholar). This analogue, presumably acting as a weak competitive substrate, was also able to inhibit the methylation of tRNA substrates and reduced the aminoacylation of yeast tRNAPhe. Although some activity was seen, this activity was not linear with respect to time. Furthermore, these authors later reported that RNA mutants with single (dU54 or dU55) or double (dU54dU55) mutations were not substrates forE. coli RUMT (31Sengupta R. Vainauskas S. Yarian C. Sochacka E. Malkiewicz A. Guenther R.H. Koshlap K.M. Agris P.F. Nucleic Acids Res. 2000; 28: 1374-1380Crossref PubMed Google Scholar). No explanation was given in this later report to account for the activity previously seen with the entirely deoxyribose analogue. Therefore, there is some ambiguity as to whether or not RUMT will recognize a DNA analogue. The tRNA editing enzyme that catalyzes the precise addition of the 3′-terminal CCA sequence to tRNAs (the CCA-adding enzyme, ATP(CTP):tRNA nucleotidyl-transferase) will recognize tDNA analogues provided they have a 3′-terminal ribonucleotide (32Shi P.Y. Weiner A.M. Maizels N. RNA. 1998; 4: 276-284PubMed Google Scholar). Both full-length and minihelix DNA analogues of the TΨC stem and loop of tDNAVal and tDNAAlawere substrates for the E. coli CCA-adding enzyme. Interestingly, the minihelix analogues were slightly better substrates than the full-length analogues for the CCA-adding enzyme (32Shi P.Y. Weiner A.M. Maizels N. RNA. 1998; 4: 276-284PubMed Google Scholar). The activity of the deoxyuridine-containing DNA analogues with E. coli TGT clearly demonstrates that TGT recognition is not critically dependent upon the native ribose backbone. However, the 2′-hydroxyl probably does influence binding, either through conformational effects or direct interactions with TGT. These experiments also demonstrate that TGT is capable of recognizing DNA provided that a UGU sequence can be found. This suggests that under certain conditions (e.g. in the E. coli mutant that lacks the enzymes deoxyribouracil-triphosphatase (dUTPase) and uracil N-glycosylase (E. coli dut − ung − strain, Ref. 33Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4905) Google Scholar) it is possible that there may be a physiological role for queuine modification of DNA. We thank members of our laboratory for critical reviews of this manuscript and helpful discussions.