THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 7, Issue of March 5, pp. 3799-3805,1989 Printed in U.S.A.

Recognition of tRNA by the Enzyme ATP/CTP:tRNA Nucleotidyltransferase INTERFERENCE BY NUCLEOTIDES MODIFIED WITH DIETHYL PYROCARBONATE OR HYDRAZINE*

(Received for publication, February 16, 1988)

Peter Spacciapoli, Loriann Doviken, Julio J. Mulero, and David L. Thurlow From the Department of Chemistry, Clark University, Worcester, Massachusetts 01610

Treatment of tRNA with diethyl pyrocarbonate or hydrazine prior to incubation with the enzyme ATP/ CTP:tRNA nucleotidyltransferase and [(U-~~PIATP re- sults in exclusion of modified bases from labeled mol- ecules. Purines modified with diethyl pyrocarbonate, which interfere with enzyme recognition, cluster at the corner of the tRNA molecule, where the D- and *- loops are juxtaposed in a11 15 tRNAs used in this study. When the enzyme is isolated from Escherichia coli, few other sites of interference are evident near the 3’-end; when the homologous enzyme from yeast is used, more exclusions are apparent near the 3’-end. Modification of uridines with hydrazine has no effect on interaction with the enzyme, except for one uridine near the 3‘- end of tRNAGIy. Interference of enzyme activity by modified bases can be overcome by longer incubation times or increased concentrations of enzyme.

tRNA moIecuIes participate in several highly specific and biologically important interactions with proteins. Restoration of CMP and AMP residues onto the 3’-end of a tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase is one such interaction, which also constitutes an attractive model system for the study of principles that govern protein recognition. Nucleotidyltransferase has been isolated from several orga- nisms, and its biochemical and physical properties have been studied extensively (reviewed in Ref. 1). The ability of nu- cleotidyltransferase, from almost any source, to restore CMP and AMP to all tRNAs without regard to their amino acid specificity (2) and to many plant viral RNAs that have a tRNA-like structure on their 3’-ends (3-6) suggests that structural features of the tRNA substrate required for recog- nition must be common among all tRNAs.

Low levels of enzyme activity have been reported on a variety of other substrates including rRNA (7-9) and tRNA molecules with intact 3’ termini (8), but all such reactions are slow and require high concentrations of enzyme. Further- more, cytidine by itself or the dinucleoside monophosphate CpC has been used as model acceptor substrate for nucleoti- dyltransferase from rabbit liver (10, 11). However, our inter- ests are centered on structural aspects of the RNA that are

* Preliminary experiments were carried out at the University of Massachusetts with the support of Biomedical Research Support Group funds awarded to D. L. T. from National Institutes of Health Grant RR07048-19. The majority of the work was sponsored by funds awarded to D. L. T. at Clark University from National Institutes of Health Academic Research Enhancement Awards Grant R15 GM37445-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

required for observed specific recognition of normal tRNA, and tRNA-like, substrates.

Much of the recent work on nucleotidyltransferase from Escherichia coli (E!), yeast (13, 14), and rabbit liver (15-17) focuses on mechanisms of enzymatic activity and utilizes a kinetic approach. Based on such studies, models of the en- zyme’s active site have been proposed (9, 14) in which the enzyme has separate binding sites for the tRNA (at a region of the molecule distinct from the 3‘-end as well as at the 3’- end itself), CTP (two separate sites), and ATP. As yet, very little is known about the nature of the structural aspects of tRNA which are required for recognition. It has been shown that combinations of complementary tRNA fragments and, in some cases, the half-molecule fragments themselves can act as substrates, although to greatly reduced extents com- pared with intact tRNA (18).

Construction of strains that overproduce the E. coli enzyme (19,20), together with rapid purification schemes (13,ZO-23), should permit characterization of the protein, as well as its interaction with tRNA. For example, physical studies on the protein, possibly including x-ray crystallography, should be possible now that substantial amounts of pure enzyme are available. In addition, protection studies may be performed in which sites on the tRNA molecule that are protected from chemical or enzymatic probes by bound protein are identified.

We adopt an approach, hereafter referred to as damage selection, in which the tRNA is chemically modified prior to incubation with the enzyme. Molecules that are competent to act as substrates are then analyzed for their content of mod- ified bases. Since we include [LY-~’P]ATP in the incubation, such tRNAs are easy to identify and analyze because they become labeled with 32P at their 3”ends. We assume that modified nucleotides, which are excluded from the labeled tRNA, had interfered with enzyme activity either by inhibit- ing the ability of the tRNA to bind to the enzyme or by reducing the rate at which the modified tRNA is used as substrate.

Using several tRNAs as substrates in such experiments, we are able to identify bases that are required intact as a general rule, ones that are required only in individual tRNAs, and ones that do not interfere with enzyme activity when modi- fied. Furthermore, the observation that at least some of the interference with activity can be overcome by increasing the duration of the incubation or concentration of enzyme sug- gests that future substrate titration experiments should allow one to determine whether modified bases affect the K , or V,,, of the enzyme.

MATERIALS AND METHODS

All chemicals used in this study were of reagent grade. Buffers were prepared using deionized distilled water (Barnstead NANOpure

3799

3800 Recognition of tRNA by ATP/CTP:tRNA Nucleotidyltransferae

11) and were stored at 4 "C. Purified isoacceptor species of tRNA were purchased from Boehringer Mannheim or Subriden RNA (Rolling Bay, WA). Some contaminating species were resolved on gels follow- ing labeling with 32P; they were recovered and analyzed separately. [cI-~*P]ATP (>3000 Ci/mmol) was purchased from ICN Radiochem- icals (Irvine, CA). Snake venom phosphodiesterase (29.1 units/mg) was obtained from Worthington. The enzyme ATP/CTPtRNA nu- cleotidyltransferase (EC 2.7.7.25), prepared from E. coli according to Carre et al. (22), was a generous gift of Dr. D. C. Fritzinger (George- town Medical Center, Washington D. C.). A crude preparation of the homologous enzyme from Saccharomyces cereuisiue was kindly pro- vided by Dr. Jacek Wower (University of Massachusetts, Amherst, MA) as the eluant from the DEAE-cellulose column step in the procedure of Sternbach et al. (24).

To remove the 3"terminal A (or CpA) residue(s), aliquots of individual tRNA (50 pg) were treated with 1 p1 of snake venom phosphodiesterase (1 mg/ml) in 50 mM Tris acetate (pH 8.0), 10 mM magnesium acetate for 20 min at 23 "C. A 10-fold concentrated solution of LiCI-SDS' buffer was added to final concentrations of 10 mM Tris acetate (pH 7.6), 100 mM LiC1, 2.5 mM Na2EDTA, 0.5% SDS, and the solution was extracted with an equal volume of phenol saturated in LiC1-SDS buffer. Following precipitation with 4 volumes of ethanol, the tRNA lacking its terminal A (CpA) was washed with 200 pl of 0.3 M sodium acetate, reprecipitated, rinsed with 95% ethanol, dried, and resuspended in water.

Treatment of tRNA prepared in this manner with diethyl pyrocar- bonate and hydrazine was carried out as described by Peattie (251, except that the length of incubation with diethyl pyrocarbonate was shortened to 3 min, and 23 S rRNA (10 pg) was used as carrier.

Prior to restoration of AMP, 5 pg of tRNA substrate was heated to 70 "C in 20 pl of CCA buffer (50 mM Tris glycine (pH 8.5), 5 mM MgCl2, 8 mM dithiothreitol, 25 pM CTP) and cooled slowly to 37 "C. One pl of the enzyme preparation was added to the substrate, with the E. coli enzyme at 2.5 unitsjml and that from yeast at 6 units/ml; 1 unit is defined as the amount of enzyme required to add 1 pmol of [cI-~~PIATP to tRNA in 1 h at 37 "C under the assay conditions described in Ref. 23. Fifty pCi of [cI-~'P]ATP was added, and incu- bation at 37 "C was carried out for 30 min. The samples were dried to 5 pl, an equal volume of formamide, 0.02% xylene cyanol, 0.02% bromphenol blue was added, and the labeled tRNA was purified on a 0.4 mm X 18 cm X 35 cm 10% polyacrylamide "sequencing" gel (acry1amide:N-N'-methylene bisacrylamide, 19:l) in 100 mM Tris borate (pH 8.3), 2.5 mM NazEDTA, 8 M urea. Labeled material was eluted from the gel slice in 0.5 M ammonium acetate, 0.5% SDS, 0.1 mM NasEDTA overnight at 23 "C, precipitated with ethanol, washed with sodium acetate, and rinsed with ethanol.

Control 32P-labeled material that had not previously been chemi- cally modified was treated with diethyl pyrocarbonate or hydrazine as described above and then repurified on a sequencing gel. In some instances, as indicated in the figure legends, control material was used without being repurified. Cleavage of the tRNA at modified bases was achieved by incubation with 1 M aniline acetate (pH 4.5) at 65 "C for 5 min, followed by drying and washing twice with water as described by Peattie (25).

Aniline cleavage products were analyzed on 20% polyacrylamide sequencing gels in 100 mM Tris borate (pH 8.3), 2.5 mM Na'EDTA, 8 M urea. Samples were adjusted such that their Cerenkov counts/

using Kodak X-Omat AR film and were exposed at -70 "C for 12-60 min were approximately equal (t-15%). Autoradiograms were made

h.

RESULTS

In order to characterize regions of tRNA molecules required for recognition by E. coli and yeast ATP/CTP:tRNA nucleo- tidyltransferases, tRNAs from both organisms were treated with diethyl pyrocarbonate or hydrazine and then used as substrates. Unless otherwise designated, all tRNAs are from E. coli. Sites of exclusion of modified bases in labeled tRNA molecules were identified by the absence, or reduction in intensities, of bands on autoradiograms after aniline-induced cleavage products of 32P-labeled tRNAs were resolved on polyacrylamide gels. Exclusions of this sort are evident when bands are compared with those derived from control material,

The abbreviation used is: SDS, sodium dodecyl sulfate.

which had been labeled with 32P prior to modification. The experimental approach is outlined in Fig. 1. Exclusion of modified bases from E. coli tRNAPhe by nucleotidyltransfer- ases from both E. coli and yeast is illustrated in the autora- diogram presented in Fig. 2.

The importance of gel-purifying control material following modification is demonstrated for tRNA'" (NAU), in which case exclusion of modified bases occurs at positions 68-70, in the aminoacyl stem, as a result of reisolation of modified material on a gel (Fig. 3, lanes 2) . This can be seen by comparing the band pattern in lanes 2 (Fig. 3) with that for material that was subjected to treatment with aniline imme- diately following chemical modification, i.e. without gel puri- fication (Fig. 3, lunes 1) . In tRNA modified prior to labeling (Fig. 3, lanes 3 ) , exclusion occurs as a result of gel purification (positions 68-70) and clue to interaction with the enzyme

Q V + u.

8 V V I REMOVE I

MODIFY RESTORE

Q * V

MODIFY

u

SOME tRNAs ARE V

NOT LABELED DUE - TO MODIFICATIONS

MODIFIED BASE WITH ANILINE

RESOLVE FRAGIIENTS ON POLYACRYLAMIDE

MODIFIED LABELED

LABELED MODIFIED THEN THEN I

EXCLUSION OF MODIFIED BASE Y

I J La I I

AUTORADIOGRAM OF GEL FIG. 1. Diagram of damage selection experiment involving

tRNA and ATP/CTP:tRNA nucleotidyltransferase. Sites of modified bases are indicated as solid circles on the cloverleaf diagram oftRNAs. The asterisk indicates location of the 32P label. 32P-Labeled fragments of tRNA, generated by treatment with aniline and resolved according to site on denaturing polyacrylamide gels, are depicted in diagrammatic form on the side of the hypothetical autoradiogram.

Recognition of tRNA by ATP/CTP:tRNA Nucleotidyltransferase 3801 a.

123

"

0 -

b. 1 2 3

D -A38

*a-

- a- -

C. (positions 19, 57, and 58). The same effect is apparent in 1 2 3 tRNAPhe in both the aminoacyl stem (bases 69-71) and the

anticodon loop (positions 27-31), as can be seen by the absence of the corresponding bands in control material that had been gel-purified prior to treatment with aniline (Fig. 2, lanes 2). Such artifactual exclusion a t positions other than 19, 57, and 58 is rare, observed in only three (tRNAPhe, tRNA"', and tRNACIy) of 15 cases, and occasionally variable

*G19 (see Fig. 2a, positions 69-71 at bottom of gel). By gel purifying control material following modification, one can easily distin- guish such exclusions from those due to interaction with the enzyme.

We do not fully understand the physical basis for exclusion due to gel purification but suggest that it may result from alterations in electrophoretic mobility of some tRNAs modi- fied in tightly base-paired stem regions, in which case tRNAs containing modified bases in stem regions would not be found

'A38 in the main band that is recovered from the purification gel and then treated with aniline. Hence tRNAs with modified bases in such positions would be excluded from material analyzed on the final sequencing gels. If this suggestion is correct, running gels a t higher temperatures or in the presence of formamide to eliminate residual secondary structure of stem regions may overcome the problem of exclusion of mod- ified bases due to gel purification. Such experiments are in

1

FIG. 2. Effects of modifying E. coli tRNAPhe with diethyl pyrocarbonate. tRNAPh' was modified with diethyl pyrocarbonate, labeled with '?P using nucleotidyltransferase from E. coli (lanes 3 ) or from yeast (lanes 1 ), gel-purified, and cleaved with aniline. The resulting fragments were resolved on a 20% polyacrylamide gel. Control material was labeled first, then treated with diethyl pyrocar- bonate, gel-purified, and cleaved with aniline (lanes 2) . Gels were run for 2 ( a ) , 4 ( b ) , or 7 (c) h. Exclusion of modified purines by the enzyme a t positions 19, 38, 57, and 58 is indicated by an arrow.

a. b. C.

0 I

FIG. 3. Effects of modifying E. coli tRNA"' (NAU) with diethyl pyrocarbonate. tRNA"' (NAU) was labeled with '*P using nucleotidyltransferase from E. coli, modified with diethyl pyrocarbon- ate, and cleaved with aniline, and the resulting fragments were resolved on a 20% polyacrylamide gel (lanes I ) . Another sample was gel-purified following modification with diethyl pyrocarbonate and prior treatment with aniline (lanes 2 ) . A third aliquot was treated with diethyl pyrocarbonate first; then labeled, gel-purified, and cleaved with aniline (lanes 3 ) . Gels were run for 2 ( a ) , 4 ( b ) , or 7 (c) h. Exclusion of modified purines by the enzyme at positions 19, 57, and 58 is indicated by an arrow, and exclusion as a result of gel purification is indicated at bases 68-70.

progress. To determine the generality of exclusion of modified bases

from positions 19, 57, and 58 due to interaction with nucleo- tidyltransferase, a total of 15 tRNAs were chemically modi- fied, labeled with nucleotidyltransferase from E. coli or yeast, and analyzed for their content of modified bases. In all cases where the tRNA had been treated with diethyl pyrocarbonate, enzymes from both sources were used to label the tRNA, and fragments from different regions of the molecule were resolved on 20% polyacrylamide gels run for different lengths of time (see Fig. 2). Sites of interference by bases modified with diethyl pyrocarbonate are summarized in Table I.

Total exclusion of carbethoxylated purines from positions 19 and 57 by E. coli nucleotidyltransferase is common to all tRNAs tested. Total, or near total, exclusion is apparent at position 58 in all tRNAs. Detectable reduction of band inten- sities is observed in four of the 15 tRNAs near their 3'-ends (i.e. in the region of bases 66-73), of which only two are substantial (initiator tRNAMe' and tRNAAn).

Interference with labeling by nucleotidyltransferase from yeast occurs when a diethyl pyrocarbonate-modified nucleo- tide is located a t position 57 in all tRNAs except initiator tRNAMe'from yeast. Total, or near total, exclusion is observed a t position 58 in only five of the 15 tRNAs. Partial reduction of band intensity at position 58 is also evident in five tRNAs. Only four tRNAs show total (and five partial) exclusion of modified bases by the yeast enzyme at G", in contrast with nucleotidyltransferase from E. coli, in which case exclusion is common to all tRNA9 tested at this site. In the region near the 3'-end, there are 10 tRNAs in which altered bases inter- fere with restoration of A (CpA) by nucleotidyltransferase from yeast, most of which exhibit only partial exclusion.

The importance of structural features of tRNA distant from the 3'-end has been noted previously for nucleotidyltransfer- ase from rabbit liver (11). In this instance it was suggested that the effects of regions distant from the 3'-end vary, depending on whether CMP or AMP is restored. Since tRNA substrates used in this study had been treated with phospho- diesterase, the resulting population of molecules may have contained both tRNAs lacking only the 3'-terminal A and those missing CpA (9). To test whether our results depended

3802 Recognition of tRNA by ATP/CTP:tRNA Nucleotidyltransferase TABLE I

Extent of interference by diethyl pyrocarbonate-modified adenosines and guanosines with E. coli or yeast ATP/CTP:tRNA nucleotidyltransferase activity in 15 tRNAs

Symbols in parentheses refer to results obtained using the yeast enzyme. Unless otherwise specified, tRNAs are obtained from E. coli. Nucleotide positions are numbered according to Sprinzl et al. (31). +++, total (>go%); ++, substantial (50-90%); and +, partial (20-50%) extents of exclusion; inferred from reduction of band intensities on autoradiomams as iudzed bv visual comparison with control bands.

Nucleotide position

19 57 58 Other tRNA

Phe-GAA +++ (+++) +++ (+++) +++ (+++) (73; +)

Phe-GAA +++ (none) +++ (+++) +++ (+++) 73,+; 38,+++

MetrCAU +++ (++) +++ (none) +++ (+)

MetrCAU (yeast)

Met-CAU +++ (none) +++ (+++) ++ (++) (73,+)

Ser-VGA +++ (+++) +++ (+++) +++ (++) (73,+) Arg-ICG +++ (none) +++ (+++) +++ (+) 71,++

Gln-CUG +++ (++) +++ (+++) ++ (none) (72,+) Gln-NUG +++ (+) +++ (+++) ++ (none) (72,+)

Ile-NAU +++ (none) +++ (+++) ++ (+)

(yeast)

(73,+; 38,+++)

+++ (none) +++ (+++) +++ (+) 73,+; 72,+++ (73,+; 72,+++)

Tv-CCA +++ (+++) +++ (+++) +++ (+++) (73,+)

(73,+; 71,++)

Asp-GUC +++ (+) +++ (+++) ++ (none)

Leu-CAG +++ (none) +++ (+++) +++ (+) 71,+ (73,+)

Val-VAC +++ (none) +++ (+++) + (none) Gly-GCC +++ (+) +++ (+++) +++ (none)

on restoration of AMP alone or both CMP and AMP, we examined the interaction of nucleotidyltransferase with tRNA lacking only its terminal AMP residue in the following man- ner.

E. coli tRNAPhe, previously treated with phosphodiesterase, was incubated briefly (5 min) with homologous nucleotidyl- transferase in CCA buffer with no added ATP. This material, in which any missing CMP residue would have been restored, was then extracted with phenol, recovered by precipitation with ethanol, and modified with diethyl pyrocarbonate. Sub- sequent restoration of the 3"terminal AMP in buffer lacking CTP and analysis of the content of modified bases revealed no differences in the pattern of excluded bases from that observed when we used tRNAs to which CMP had not been restored (data not shown). From these data we conclude that the effects we observe are on restoration of AMP alone.

In general, uridine residues modified by treatment with hydrazine appear to have no effect on interaction with the E. coli enzyme (14 of 15 tRNAs), or, where tested (tRNAsPhe from E. coli and yeast, initiator tRNAMet from yeast, tRNAC'" (NUG), and tRNAGly (GCC)), with nucleotidyltransferase from yeast. The only tRNA in which we observe noticeable reduction of a band's intensity when the tRNA had been modified with hydrazine is tRNAcly (GCC) (Fig. 4). In this instance, U73, at the end of the aminoacyl stem, is partially excluded in modified form by the E. coli enzyme (Fig. 4a). U4' is also excluded, but in this instance the exclusion is due to gel purification (Fig. 4, lanes 2). Uridine residues in all other tRNAs tested fail to significantly affect the interaction.

Extending the duration of the labeling reaction or increas- ing enzyme concentration reduces the extent of exclusion in some but not all instances, depending both on the tRNA and the source of enzyme. The band intensity a t position 58, for example, is dramatically increased in two tRNAs when longer times or increased amounts of the yeast enzyme are used (Fig. 5, c and d ) yet is only slightly affected in the case of the E. coli enzyme (Fig. 5a). Similarly, exclusion a t G"' is reduced

FIG. 4. Effects of modifying E. coli tRNAGIy (CCC) with hydrazine. tRNACIY (GCC) was labeled with 32P using nucleotidyl- transferase from E. coli modified with hydrazine and cleaved with aniline, and the resulting fragments were resolved on a 20% poly- acrylamide gel (lanes 1 ). Another sample was gel-purified following modification with hydrazine and prior to treatment with aniline (lanes 2). A third aliquot was treated with hydrazine first; then labeled, gel- purified, and cleaved with aniline (lanes 3). Gels were run for 2 ( a ) , 4 ( b ) , or 7 (c) h. Partial exclusion of modified uridines by the enzyme at position 73 is indicated by an arrow, and exclusion as a result of gel purification is indicated at U".

somewhat with longer incubations or increased concentra- tions of the yeast enzyme but is not detectably affected when nucleotidyltransferase from E. coli is employed (Fig. 5). Ex- clusion a t position 19 by the E. coli enzyme can be partially overcome in tRNAVa' but is not affected in tRNAPh' (Fig. 6).

Recognition of tRNA by A TP/CTP:tRNA Nucleotidyltransferase 3803

e. b. C. d. a.

FIG. 5. Effects of longer incubation times and increased amount of enzyme on exclusion of modified purines at posi- tions 57 and 58. E. coli tRNAPhe (a, b) , tRNATv (c), or tRNAQr ( d ) was treated with diethyl pyrocarbonate and incubated with 1 pl of nucleotidyltransferase from E. coli ( a ) or yeast (b-d) for 10 min (lanes 3), 90 min (lanes 4 ) , or with 3 pl enzyme for 3 min (lanes 5). Control material was labeled prior to modification and was then gel-purified (lanes 2) or not (lanes 1 ) and treated with aniline. The 32P-labeled fragments were resolved on 20% polyacrylamide gels run for 4 h.

a. b.

W Q - " 89-

1 2 3 4 1 2 3 4

G19'

c -0 4

"

FIG. 6. Effects of longer incubation times and increased amount of enzyme on exclusion of modified GIg. E. coli tRNAPh" ( a ) or tRNA""' ( b ) was treated with diethyl pyrocarbonate and incu- bated with 1 pl of nucleotidyltransferase from E. coli for 10 min (lanes 2), 90 min (lanes 3). or with 3 pl of enzyme for 3 min (lanes 4 ) . Control material was labeled prior to modification and was then gel- purified and treated with aniline (lanes I ) . The 3ZP-labeled fragments were resolved on 20% polyacrylamide gels run for 7 h.

Reducing the tRNA concentration by 2 orders of magnitude does not affect the pattern of excluded bases (data not shown). This suggests that the effects we see at 10 p~ tRNA, which is near reported values of the K , for the E. coli enzyme (1, 7, 12), are not likely to reflect subtleties of the interaction, which might be decisive only at very low concentrations of substrate.

DISCUSSION

Recognition of tRNAs by E. coli Nucleotidyltransferase- From x-ray analysis of several tRNA crystals, it is clear that nucleotides at positions 18, 19, 57, and 58 are stacked, as the D- and *-loops are brought together at the comer of the L- shaped three-dimensional structure (26-29). Since positions 19, 57, and 58 are the only sites of interference by modified bases with E. coli nucleotidyltransferase activity that are common to all tRNAs examined in this study, we infer that this enzyme interacts with the region of the tRNA molecule where these bases cluster.

Using tRNA"' (NAU) as a specific example (Fig. 7) and assuming a tertiary structure comparable to that in crystals of yeast tRNAPhe (26), one can see that the tertiary base pair between CS and G" is located at the outermost edge of the long stretch of stacked bases, constituting the top of the L- shaped structure (26). This provides a logical point of contact with the enzyme, since it appears to be a highly conserved structural feature and since nucleotidyltransferase must rec- ognize aspects of tRNA structure that are common to all tRNAs. Exclusion of carbethoxylated guanosines from posi- tion 19 (Fig. 3) would be expected if the enzyme makes contact

A C C A

G C G C C G C G C G C G

A

U G A A C ~ A C C ~ ~ A - S S 0, C U C G

G A G C

A-57 OCUQG C

19' D o A U X

G cuGm7G

C G G C

NAU'

b.

8 FIG. 7. Secondary and tertiary structures of E. coli tRNAm.

a, sites of exclusion of diethyl pyrocarbonate-modified bases; positions 19, 57, and 58 are indicated on the cloverleaf secondary structure of tRNA"" (31); b, artist's drawing of the tertiary structure of tRNA"', assuming it is comparable to that for tRNAPhe from yeast (26). Structures for nucleotides 18, 19, 56,57, and 58 are depicted as they appear in tRNAPh' (28). N-7 atoms at which carbethoxylation inter- feres with labeling by nucleotidyltransferase from E. coli are indicated by solid circles. The *- and D-loops are stippled and cross-hatched, respectively. The minor groove of the *-stem along the top of the molecule is shaded.

with a tRNA starting at the outside comer of the molecule and then extends back toward the 3'-end. Inasmuch as CS is directly above G" at the comer of the molecule, one would predict that it too should be excluded, when chemically mod- ified, from tRNAs that successfully act as substrates for nucleotidyltransferase. Preliminary experiments using di- methyl sulfate and hydrazine to alter C residues indicate that this is the case for tRNAPhe from either E. coli or yeast (data not shown). Further studies are in progress to establish the generality of these observations.

The next stacked base toward the 3'-end is an adenosine, located at position 57 (Fig. 7). Carbethoxylation by diethyl pyrocarbonate at this base also interferes with enzyme rec- ognition (Fig. 3). In most other tRNAs, in which this base is a guanosine, exclusion occurs to the same extent (see Fig. 2). This is understandable, since N-7s of both adenosine and

3804 Recognition of tRNA by ATP/CTP:tRNA Nucleotidyltransferase

guanosine are structurally homologous and are both sites of carbethoxylation by diethyl pyrocarbonate (30). Furthermore, since N-7 is the only site of modification by diethyl pyrocar- bonate in guanosines (30), we infer that it is alteration of N- 7, and not the exocyclic amino group in adenosine 57 of tRNA"', which is responsible for interference with enzyme activity.

Residue 58, an adenosine located one nucleotide away from position 57 in the stacked arrangement, is also excluded in modified form (Fig. 3), as if the protein extends toward the 3'-end along the minor groove of the *-stem (Fig. 7). How- ever, G", stacked between bases 57 and 58, can be fully modified by diethyl pyrocarbonate and has no apparent effect on the interaction (Fig. 3). We note that its N-7 points in the opposite direction from N-7 atoms in residues 57 and 58 (Fig. 7). It is also apparent that N-7 of G" is located far away from any other portion of the molecule, allowing a carbethoxy group ample room to reside and still allow a protein to interact with the corner of the tRNA.

In contrast with N-7 in G", N-7 of both residues 57 and 58 points toward the above mentioned groove. Carbethoxylation of these N-7 atoms would be expected to destabilize the sharp turn made by the *-loop backbone and thereby interfere with protein recognition if the enzyme were interacting with this region. We assume that similar tertiary structural arrange- ments exist in most tRNAs and that the nature of interaction with nucleotidyltransferase is common among them. From the universality of exclusions of modified bases from positions 19, 57, and 58 by the E. coli enzyme, we conclude that the junction of the \k- and D-loops, near the minor groove of the *-stem, is an essential structural feature of the tRNA mole- cule required for proper recognition by E. coli nucleotidyl- transferase.

In two tRNAs, purines near the 3'-end also appear to substantially affect recognition by the E. coli enzyme: initiator tRNAMet and tRNAArg. That residues near the 3'-end would be in a position to interact with the enzyme is not surprising, since this is near the site where the CMP and AMP residues are joined to the tRNA by nucleotidyltransferase. However, it is surprising to note that we observe such effects with modified bases in such few cases, suggesting that nucleotidyl- transferase from E. coli does not, in general, require unmodi- fied bases next to the 3'-terminal CpCpA.

Recognition of tRNAs by Yeast Nucleotidyltransferase-In 14 of 15 tRNAs, the yeast enzyme is inhibited by modified bases located in at least two of the three sites required intact by the homologous enzyme from E. coli, i.e. 19, 57, and 58 (Table I). Based on these observations, it is apparent that the yeast enzyme is similar to its counterpart from E. coli, at least in terms of the general location of its primary site of inter- action.

Of these three sites, position 57 is excluded most often (14 of 15), with nucleotide 58 being at least partially excluded in 10 of 15 tRNAs. The absence of effects due to a modified base at position 19 in eight cases, the absence of exclusion at base 58 in five tRNAs, and the reduced extents of exclusion at nucleotide 58 in 12 cases imply that the detailed nature of interaction with the yeast enzyme differs somewhat from that of the bacterial nucleotidyltransferase.

In addition, we infer from the many instances in which modified bases at positions 19 and 58 fail to affect enzyme recognition by the yeast protein that exclusion at these sites by the E. coli nucleotidyltransferase is not due to some gross alteration of tertiary structure. Rather, our interpretation of these data as identifying sites of interaction with the enzyme is supported.

A second difference between the two enzymes is the fre- quency at which bases near the 3'-end (residues 71-73) affect recognition. In addition to the four tRNAs for which modified bases at these positions interfere with the E. coli interaction, seven other tRNAs behave in a similar fashion with the yeast enzyme (Table I). Thus, nucleotidyltransferase from E. coli appears to interact in a most discriminating manner at the corner of the tRNA molecule and extends toward the 3'-end with less stringent requirements for attachment at the end of the aminoacyl stem. In contrast, the yeast enzyme interacts at the corner of the molecule but has fewer defined points of interaction near the minor groove of the *-stem and extends to the 3'-end with a more rigidly defined binding site for bases located at positions 71-73.

Modification with Hydrazine-Surprisingly, modification of uridines by hydrazine, in general, has no apparent effect on enzyme recognition, as judged by the fact that only one uridine in all of the 15 tRNAs used here is significantly excluded from material labeled by nucleotidyltransferase from either E. coli (all 15 tRNAs tested) or yeast (5 tRNAs tested). It is not surprising that U73 in tRNAGIY, which is the only tRNA used in this study that contains a U at this position, affects interaction with the E. coli enzyme, since it is the base to which the first C would be ligated. However, the absence of any effect on interaction with the yeast enzyme by a modified U73 in tRNAG", and at all other positions in all other tRNAs, is especially noteworthy in view of the fact that hydrazine attacks carbons 4 and 6 in uridine, initially opening the pyrimidine ring and subsequently excising it entirely (25). Consequently, we conclude that uridines do not in general participate in an essential direct contact with nucleotidyl- transferase, nor does excision of the pyrimidine ring from most uridines initiate a conformational change that affects enzyme recognition.

Interference with Enzyme Activity Is Reversible in Some Cases-Where exclusion of modified bases is total, with the E. coli enzyme at positions 19,57, and 58, longer incubations or increased concentration of enzyme have little or no effect (Figs. 5a and 6a). Similarly, with the yeast enzyme at position 57, exclusion is only slightly overcome by longer incubations (Fig. 5, b-d, lanes 4 ) . However, where exclusion is partial, with the yeast enzyme at position 58, interference by modified bases is substantially relieved in tRNATp (Fig. 5c, lane 4 ) and nearly totally relieved in tRNASe' (Fig. 5d, lane 4 ) by longer incubations. In addition, in three tRNAs, when the yeast enzyme is used, increasing the enzyme concentration also substantially overcomes inhibition (Fig. 5, b-d, lanes 5 ) . Fur- thermore, at position 19 in tRNASe', where exclusion is nearly total with the E. coli enzyme, interference with enzyme activ- ity is partially overcome by increased enzyme concentrations (Fig. 6b, lane 4 ) . The observation that inhibition is reversible in some cases suggests that future substrate titration experi- ments may permit determination of whether chemically al- tered bases at these positions affect the K,,, or V,, of the enzyme.

Conclusions-Activity of the enzyme ATP/CTPtRNA nu- cleotidyltransferase, which restores CMP and AMP residues onto the 3'-end of tRNA molecules, is inhibited by diethyl pyrocarbonate-modified purines located at the corner of the tRNA, where the \k- and D-loops are juxtaposed, and, less frequently, by modified purines near the 3'-end. Both E. coli and yeast enzymes require unmodified nucleotides in the same general regions of the tRNA, yet they differ sufficiently to suggest that they do not interact with tRNAs in an identical fashion. The damage selection experimental approach has provided both a detailed map of sites on the tRNA molecule

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