©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
tRNA-guanine Transglycosylase from Escherichia coli
MINIMAL tRNA STRUCTURE AND SEQUENCE REQUIREMENTS FOR RECOGNITION (*)

(Received for publication, March 1, 1995; and in revised form, May 19, 1995)

Alan W. Curnow , George A. Garcia (§)

From the Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previously, we have demonstrated that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is capable of utilizing an in vitro generated minihelix consisting of the anticodon stem and loop sequence of E. coli tRNA (Curnow, A. W., Kung, F. L., Koch, K. A., and Garcia, G. A.(1993) Biochemistry 32, 5239-5246). This suggests that the tRNA structural motifs necessary for recognition comprise a loop at the end of a short helix. To gain further insight into the structural requirements for TGT recognition, we have investigated the conformation of this minimal substrate. Thermal denaturation studies and kinetic analyses at 20 and 37 °C indicate that this minihelix is predominantly melted at 37 °C and that the melted conformation is not a substrate for TGT. This is confirmed by the determination that a non-helical analogue of the minihelix is not a substrate for TGT at either temperature. Two additional minihelices designed to be stable at 37 °C, ECYMH (a 4-base pair extension of the previous minihelix) and SCDMH (a yeast tRNA analogue of ECYMH), were generated and characterized. Finally, several sequence mutants of SCDMH, focusing on the GU base pair and UGU loop sequence, have been produced, and kinetic parameter determinations have been performed at 37 °C. Our results are consistent with a recent report (Nakanishi, S., Ueda, T., Hori, H., Yamazaki, N., Okada, N., and Watanabe, K. (1994) J. Biol. Chem. 269, 32221-32225) indicating that a UGU sequence in a 7-base loop is the minimal requirement for TGT recognition.


INTRODUCTION

A key enzyme involved in the post-transcriptional modification of tRNA with queuine (7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine) is tRNA-guanine transglycosylase (TGT,()EC 2.4.2.29)(1) . In Escherichia coli, TGT catalyzes the replacement of guanine 34 in the tRNA anticodon with a queuine precursor, preQ (7-aminomethyl-7-deazaguanine). preQ is, in turn, further modified to queuine. The overall process results in the substitution of guanine 34 with queuine and maturation of the tRNA molecule. The tRNAs (tRNA, tRNA, tRNA and tRNA), which act as substrates for this enzyme, contain the anticodon sequence, GUN, where N may be any base(2, 3) . Our laboratory has initiated the evaluation of the molecular recognition of tRNA by TGT from E. coli using in vitro generated tRNA analogues based upon E. coli tRNA (Fig. 1).


Figure 1: The primary nucleotide sequences and possible secondary structures of the E. coli tRNA structural analogues. Guanine 34 is underlined in each analogue.



Comparison of the activity of totally unmodified tRNA (Fig. 1, ECY2) to that for Q-deficient but otherwise modified tRNA isolated from a tgt-deficient clone of E. coli revealed that the other modified bases do not appear to play a significant role in the recognition of tRNA by TGT (4) . We subsequently found that a dimeric conformer of tRNA has a slightly higher K than monomeric tRNA (ECY2) but has an equal maximum velocity (5) . Generation and evaluation of truncated analogues of tRNA indicated that ECY2-A1, an analogue corresponding to the 17-base anticodon arm of tRNA, was found to have a K value only 6-fold higher than full-length tRNA and V/K only 40-fold lower (4) . This suggests that, other than the anticodon arm, the majority of the tRNA structure has very little effect upon recognition by TGT.

Thermal denaturation curves can be utilized to elucidate the solution conformation adopted by nucleic acids(6) . Such studies in our system of tRNA analogues indicate that ECY2-A1 has a T33 °C(7) . Thus, at 37 °C ECY2-A1 is mostly denatured, suggesting that the specificity constant (V/K) calculated for ECY2-A1 at this temperature may be artificially low since the analogue will exist in a predominantly melted conformation. A recent report by Nakanishi et al.(8) suggests that a YUGU sequence in the anticodon loop of cognate tRNAs is the minimal requirement for recognition by TGT. These analyses were carried out at 37 °C using 5-base pair stem minihelices similar to ECY2-A1. Presumably, the minihelices studied will also be predominantly melted under the conditions of the assay, leaving the results subject to question.

We have pursued several routes to a stable framework upon which to base our sequence mutation studies. Kinetic parameters were determined for several analogues at 20 °C, a temperature at which ECY2-A1 will adopt a stable minihelix conformation. A non-helical analogue of ECY2-A1 was also generated and evaluated to investigate the possibility that the enzyme required sequence but not conformational information for recognition. The anticodon arm of ECY2-A1 was extended by 4 base pairs to provide a minihelix with enhanced stability. A yeast tRNA analogue of this minihelix was used as a framework for several sequence mutations (Fig. 2) and kinetic evaluation at 37 °C.


Figure 2: The primary nucleotide sequences and probable secondary structures of minihelices derived from yeast tRNA. A, SCDMH includes the yeast tRNA anticodon domain with a 3-base pair extension of the helical stem. There are seven sequence mutants whose names are derived by adding the site of the mutation, using the standard numbering system for tRNAs, to SCDMH. Guanine 34 has been underlined, and the site of mutation is indicated with an arrow pointing to the mutation. The mutant indicated by the parentheses is a double mutant. B, there are two loop conformation analogues. In SCDMH6BL cytosine 38 has been deleted to form a 6-base loop, and in SCDMH8BL a cytosine residue has been added between positions 38 and 39 to form an 8-base loop.




EXPERIMENTAL PROCEDURES

Materials

Chemical reagents were purchased from Sigma, Boehringer Mannheim, and Life Technologies, Inc. Nucleoside triphosphates and RNasin were from Boehringer Mannheim. BstNI restriction endonuclease was from Stratagene. [8-C]Guanine was from Moravek Biochemicals. TGT was isolated from an overexpressing clone as described previously(9, 10) . T7 RNA polymerase was isolated from E. coli BL21/pAR1219 following the procedure of Grodberg and Dunn(11) .

Preparation and Purification of the tRNA Transcript

Unmodified E. coli tRNA and minihelix RNA analogues were produced via the transcription of a double-stranded DNA template in an in vitro system and purified as described previously(4) .

Thermal Denaturation Curves

Thermal denaturation profiles were determined on a Cary Cotinua Varian spectrophotometer following the method suggested by Groebe and Uhlenbeck(12, 13) . The absorbance was monitored at 260 nm for 2 µM RNA solutions in 10 mM sodium phosphate buffer, pH 7, 0.1 mM EDTA, and 150 mM NaCl. The temperature was increased at the rate of 1 °C/min with absorbance readings taken through a range from 10 to 90 °C. The denaturation curves for ECY1 and ECY2 (the full-length modified and unmodified analogues of E. coli tRNA), ECY2-A1 (the anticodon microhelix), ECY2-A1NH (the nonhelical analogue of ECY2-A1), and ECYMH and SCDMH (the extended minihelix analogues) are presented in Fig. 3. The total change in absorbance for SCDMH is less than that for the other analogues, most likely due to the fact that SCDMH contains no adenosine, which contributes largely to the UV absorbance change.


Figure 3: Thermal denaturation curves for several tRNA analogues. The relative absorbances at 260 nm for ECY2-A1 (1), ECY2-A1NH (2), ECY2 (3), ECY1 (4), ECYMH (5), and SCDMH (6) are plotted versus temperature. The profiles were measured in 10 mM sodium phosphate buffer, pH 7, 0.1 mM EDTA, and 150 mM NaCl. The temperature was increased at the rate of 1 °C/min with absorbance readings taken through a range from 10 to 90 °C.



Kinetic Assays

The kinetic parameter determinations were performed in a buffer consisting of 100 mM HEPES at pH 7.5, 10 mM MgCl, and 10 mM dithiothreitol. The concentration of [8-C]guanine was maintained at 10 µM (about 10 K(4) ) while the concentration of the tRNA substrates was varied from 0.5 to 100 µM. The general procedure included incubating a 300-µl mixture of buffer, substrate, and 12.6 µg/ml TGT at either 20 or 37 °C. At 3-min intervals 70-µl aliquots were removed from the reaction mixture. For the longer tRNA sequences, >30 nucleotides, the aliquots were quenched in 5% trichloroacetic acid and filtered onto glass fiber filters (Whatman, GF/C). The filters were then washed 3 times with 5% trichloroacetic acid and once with ethanol, dried, and quantitated via liquid scintillation counting. For the shorter sequences, <30 nucleotides, 10 µl of 3 M NaOAc, pH 5.3, was added to the aliquots, which were then precipitated with 1.5 ml of ethanol and collected on glass fiber filters (Whatman, GF/C). The filters were then washed 3 times with ethanol, dried, and quantitated via liquid scintillation counting. Initial velocities were determined by linear regression, and from that data Michaelis-Menten parameters were determined by nonlinear regression and are displayed in Fig. 4and 5. The kinetic parameters (K, V, and V/K) along with the standard errors of the fits for the structural analogues at 37 and 20 °C are shown in Table 1and for the sequence mutants are shown in Table 2. Analogues for which the guanine incorporation was below the limit of detection (about 0.1% of full-length tRNA activity) at 100 µM analogue are denoted as having no detectable activity.


Figure 4: Michaelis-Menten plots of E. coli tRNA structural analogues. A, at 37 °C: ECY2 (solidtriangles), ECYMH (solidcircles), and ECY2-A1 (soliddiamonds). B, at 20 °C: ECY2 (opentriangles), ECYMH (opencircles), and ECY2-A1 (opendiamonds). Solidcurves are calculated fits of the data by nonlinear regression.








RESULTS AND DISCUSSION

In previous studies we have demonstrated that the major elements utilized by TGT in the recognition of tRNA substrates are found within the anticodon arm of E. coli tRNA(4, 5) . This was demonstrated by employing RNA substrates varying widely in gross structure while maintaining a common motif, the anticodon arm. Such structural analogues have included full-length E. coli tRNA, a dimeric form of E. coli tRNA, and the anticodon arm itself. For example, at 37 °C the anticodon arm minihelix has a V/K that is 40-fold lower and a K that is only 6-fold higher than that for the full-length tRNA, suggesting that the predominate effect of the loss of tRNA structure is upon catalysis. This is similar to the conclusion drawn by Gu and Santi(14) , who reported that an 11-base oligoribonucleotide corresponding to the TC arm of tRNA has a k/K that is 300-fold lower and a K that is 5-fold higher than that for the full-length tRNA for the tRNA (mUS4) methyltransferase and concluded that the effect was primarily upon catalysis. A recent report from Nakanishi et al. (8) has probed further into tRNA substrate recognition by the E. coli TGT. In their study, the kinetic parameters for several sequence mutants of an RNA minihelix, similar to ECY-A1, derived from the anticodon arm of E. coli tRNA have been determined. The conclusion drawn by the authors was that the YUGU sequence in the anticodon loop is the minimum requirement for recognition.

The UV thermal denaturation curves in Fig. 3indicate that the modified full-length tRNA (ECY1) has a higher T than the unmodified tRNA (ECY2) (61 versus 56 °C). These results are consistent with earlier findings from studies with tRNA and tRNA, which indicate that base modifications enhance the stability of the tRNA native conformation (15, 16) . In addition, the curves for the full-length analogues have gradual transitions possibly due to the melting of the tertiary base interactions followed by melting of the base pairs of the four helices, which make up the secondary structure. The transitions for the smaller analogues, ECYMH, SCDMH, and ECY2-A1, which presumably have no tertiary interactions, are much sharper and most probably simply reflect the melting of the base pairing in the helices. Perhaps the most important aspect of the thermal denaturation profiles for the minihelices is that the T for ECY2-A1 is 33 °C and the T for ECYMH 75 °C. If TGT requires an intact helix leading into the anticodon loop for recognition, then the kinetic parameters that were calculated for ECY2-A1 at 37 °C must be artificially low since at this temperature this analogue will be predominantly denatured.

Our earlier results indicated that the anticodon arm alone is sufficient for binding and catalysis to occur but did not eliminate the possibility that TGT recognizes oligoribonucleotides containing the UGU sequence in the absence of a defined tertiary structure. The fact that there is no energy coupling for this enzymic reaction suggests that binding energy is used to catalyze this base exchange. If so, it is reasonable to assume that a significant amount of tRNA tertiary structure may be required to provide this binding energy. To address this question we generated ECY2-A1NH. In this analogue, we have replaced the 3` side of the helix (5 bases) with the reverse of the 5` sequence yielding an analogue incapable of helix formation (Fig. 1). We attempted to determine the kinetic parameters for this analogue at both 20 and 37 °C and found that in concentrations up to 100 µM the analogue had no detectable activity at either temperature. The analogue also does not seem to interact with TGT via a previously published bandshift assay (5) (data not shown). Finally the analogue is incapable of inhibiting the incorporation of guanine into the full-length cognate tRNA (data not shown). These results suggest that the non-helical analogue does not, to any significant extent, interact with TGT and that some degree of loop/helix structure is required for TGT catalysis to occur.

The relative activities of the tRNA analogues with TGT observed at 20 °C are very much like the trend we previously reported at 37 °C (Table 1). It is important to note that each analogue displays a V approximately 5-fold lower at 20 than at 37 °C, reflecting an overall lower activity for TGT at 20 °C. At the lower temperature ECYMH has a larger K than at 37 °C, while ECY2 has an identical K at both temperatures. The change in K for ECYMH can be partially explained by the larger error for the values at 20 °C where the enzyme is less active. The relative V/Kratio for ECYMH is similar at both temperatures, suggesting that the analogue is stable at both temperatures. The relative V/K ratio for ECY2-A1 decreases from a factor of 40 at 37 °C to a factor of 10 at 20 °C, suggesting that at 37 °C only 25% of the analogue is in the stable helical conformation required for TGT recognition. This is consistent with the thermal denaturation curve for ECY2-A1 shown in Fig. 3. We conclude that an RNA minihelix with an extended stem, stable at 37 °C, is the minimum stable framework suitable for sequence specificity studies.

We have selected a yeast tRNA minihelix framework, SCDMH, in which to carry out site-specific sequence mutations to investigate the specific bases involved in substrate recognition by TGT. The x-ray crystal structure for the full-length yeast tRNA has been determined(17, 18, 19) . Future modeling studies will use this information to generate structures for our minihelices, which will be used as a framework in which to interpret our kinetic results. Prior to performing sequence mutations, it is necessary to determine the kinetic parameters for SCDMH with TGT. Comparing the results for ECYMH (Table 1) to the results for SCDMH (Table 2) we find that the analogues have similar kinetic parameters.

The sequence mutation studies began in the anticodon loop focusing on the UGU sequence, which was inferred to be the major determinant for TGT recognition in earlier sequence homology studies(3) . The only loop sequence mutant that has any detectable activity is SCDMHU33C, which has an 80-fold loss in substrate specificity relative to SCDMH. The other mutants showed no detectable activity in concentrations up to 100 µM. The inactivity of the one-base insertion, SCDMH8BL, and one-base deletion, SCDMH6BL, mutants (Table 2) suggests that the size of the loop, and presumably its conformation, is also vital for recognition of tRNA substrates by TGT.

G is conserved among the TGT cognate tRNAs, being base paired either with C or U. The establishment of a canonical base pair, GC in SCDMH, results in a modest increase in substrate specificity while mutation to AU results in a 2-fold loss in substrate specificity. Mutation of the GU pair to give CG yields a 7-fold loss in substrate specificity relative to SCDMH. These results suggest that the enzyme does not have a specific amino acid-base interaction at position 30; however, it may be that the conformation imparted in the loop by this base pair plays a role in the recognition of tRNA by TGT.

We have reached a very similar conclusion to Nakanishi et al.(8) that the UGU sequence found in tRNAs, which are active as substrates for TGT, is the major recognition determinant within the anticodon domain. However, we conclude that the 5 base pairs (three G-C and two A-U) in the anticodon stem investigated by Nakanishi et al. are inadequate to maintain the stable helical structure necessary for TGT catalysis to occur optimally at 37 °C. Thus, additional structural stabilization is necessary in order to form a true minimal substrate. This stabilization may be provided by: 1) lowering the assay temperature, 2) additional canonical base pairs and the tertiary interactions of distant bases, in the instance of the full-length tRNA, or 3) by extending the anticodon stem by 3-4 base pairs, in the instance of a minihelix analogue.

It should be noted that these studies are designed to investigate positive recognition elements only and do not address the possibility that negative recognition elements for TGT exist elsewhere in the non-cognate tRNA structure.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM45968 and GM07767 and the University of Michigan, College of Pharmacy. A preliminary report of this work has been presented at the ASBMB Annual Meeting, Washington, D. C., May 21-25, 1994 (7). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 313-764-2202; Fax: 313-763-5633.

The abbreviation used is: TGT, tRNA-guanine transglycosylase.


ACKNOWLEDGEMENTS

We thank Dr. David Giegel (Warner-Lambert/Parke-Davis Pharmaceutical Research Division, Ann Arbor, MI) for assistance with the UV thermal denaturation experiments.

Note Added in Proof-Further support for our conclusions comes from recent footprinting experiments by Mueller and Slany (Mueller, S. O., and Slany, R. K.(1995) FEBS Lett.361, 259-264) that indicate the E. coli TGT protects only the anticodon stem-loop of tRNA. Our results are also consistent with those of Carbon et al. (Carbon, P., Haumont, E., Fournier, M., de Henau, S., and Grosjean, H.(1983) EMBO J.2, 1093) where they found that the UGU sequence was the main determinant for queuine modification in Xenopus oocytes.


REFERENCES
  1. Okada, N., and Nishimura, S.(1979)J. Biol. Chem. 254, 3061-3066 [Abstract]
  2. Harada, F., and Nishimura, S.(1972)Biochemistry 11, 301-308 [Medline] [Order article via Infotrieve]
  3. Tsang, T. H., Buck, M., and Ames, B. N.(1983)Biochim. Biophys. Acta 741, 180-196 [Medline] [Order article via Infotrieve]
  4. Curnow, A. W., Kung, F. L., Koch, K. A., and Garcia, G. A.(1993)Biochemistry 32, 5239-5246 [Medline] [Order article via Infotrieve]
  5. Curnow, A. W., and Garcia, G. A.(1994)Biochimie (Paris)76,1183-1191 [Medline] [Order article via Infotrieve]
  6. Puglisi, J. D., and Tinoco, I.(1989)Methods Enzymol. 180, 304-325 [Medline] [Order article via Infotrieve]
  7. Curnow, A. W., and Garcia, G. A.(1994)FASEB J.8,1348 (Abstr. 519)
  8. Nakanishi, S., Ueda, T., Hori, H., Yamazaki, N., Okada, N., and Watanabe, K.(1994) J. Biol. Chem. 269, 32221-32225 [Abstract/Free Full Text]
  9. Chong, S., and Garcia, G. A.(1994)BioTechniques 17, 686-691 [Medline] [Order article via Infotrieve]
  10. Garcia, G. A., Koch, K. A., and Chong, S.(1993)J. Mol. Biol. 231, 489-497 [CrossRef][Medline] [Order article via Infotrieve]
  11. Grodberg, J., and Dunn, J. J.(1988)J. Bacteriol. 170, 1245-1253 [Medline] [Order article via Infotrieve]
  12. Groebe, D. R., and Uhlenbeck, O. C.(1988)Nucleic Acids Res. 16, 11725-11735 [Abstract]
  13. Groebe, D. R., and Uhlenbeck, O. C.(1989)Biochemistry 28, 742-747 [Medline] [Order article via Infotrieve]
  14. Gu, X., and Santi, D. V. (1991)Biochemistry 30, 2999-3002 [Medline] [Order article via Infotrieve]
  15. Sampson, J. R., and Uhlenbeck, O. C.(1988)Proc. Natl. Acad. Sci. U. S. A. 85, 1033-1037 [Abstract]
  16. Derrick, W. B., and Horowitz, J.(1993)Nucleic Acids Res. 21, 4948-4953 [Abstract]
  17. Westhof, E., Dumas, P., and Moras, D.(1985)J. Mol. Biol. 184, 119-145 [Medline] [Order article via Infotrieve]
  18. Dumas, P., Ebel, J. P., Giege, R., Moras, D., Thierry, J. C., and Westhof, E.(1985) Biochimie (Paris)67,597-606 [Medline] [Order article via Infotrieve]
  19. Westhof, E., Dumas, P., and Moras, D.(1988)Acta Crystallogr. Sect. A 44, 112-123 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.