©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence and Structure Requirements for Drosophila tRNA 5`- and 3`-End Processing (*)

(Received for publication, February 9, 1995; and in revised form, May 25, 1995)

Louis Levinger (§) Vikram Vasisht (¶) Vilma Greene (**) Rae Bourne (§§) Alex Birk (¶¶) Srinivas Kolla

From theDepartment of Natural Sciences/Biology, York College of the City University of New York, Jamaica, New York 11451

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Eukaryotic tRNAs are processed at their 5`- and 3`-ends by the endonucleases RNase P and 3`-tRNase, respectively. We have prepared substrates for both enzymes, separated the activities from a Drosophila extract, and designed variant tRNAs to assess the effects of sequence and structure on processing. Mutations affect these reactions in similar ways; thus, RNase P and 3`-tRNase probably require similar substrate structures to maintain the catalytic fit. RNase P is more sensitive to substrate substitutions than 3`-tRNase. In three of the four stems, one substitution prevents both processing reactions while the opposite one has less effect; anticodon stem substitutions hardly affect processing, and double substitutions intended to restore base pairing also restore processing to the wild type rate.

Structure probing suggests that tRNA misfolding sometimes coincides with reduced processing. In other cases, processing inhibition probably results from specific unfavorable stem appositions leading to local helix deformation. A single T loop substitution disrupts the tertiary D-T loop interaction and reduces processing. We have thus begun mapping tRNA processing determinants on the global, local, and tertiary structure levels.


INTRODUCTION

Eukaryotic RNAs are processed before utilization. tRNA is endonucleolytically processed at its 5`-end by RNase P (Altman et al., 1995) and at its 3`-end by 3`-tRNase (Deutscher, 1995); eukaryotic tRNA has a G added to the mature 5`-end after RNase P processing (Cooley et al., 1982). Some tRNAs have intervening sequences removed by splicing (Westaway and Abelson, 1995). In prokaryotes, 3`-CCA is transcriptionally encoded, while in eukaryotes, it is added by tRNA nucleotidyl transferase after the 3`-tRNase reaction (Deutscher, 1995). Many nucleotides in tRNA are post-transcriptionally modified (Björk, 1995).

tRNAs must possess a generalized processing identity, analogous to their identities which are specialized for efficient and accurate aminoacylation (McClain, 1995). Generalized tRNA identity was determined for RNase P using unmodified RNA polymerase T7 transcripts with domain deletions (McClain et al., 1987). Substrate identity for 3`-tRNase has been less thoroughly analyzed (Deutscher, 1995).

RNase P has an RNA component (Guerrier-Takada et al., 1983; Lee et al., 1989, 1990; Baer et al., 1990; Doria et al., 1991), which is catalytic in prokaryotes. Prokaryotic RNase P requires a substrate with a coaxially stacked acceptor and T stem; the D and anticodon arms are dispensable for 5`-end processing (McClain et al., 1987), and 3`-CCA stimulates RNase P (Guerrier-Takada et al., 1984). Eukaryotic RNase P substrate requirements may be more extensive (Hardt et al., 1993) and depend on length of the acceptor and T stems (Carrara et al., 1989). tRNA precursors with one base pair added to the acceptor stem are cleaved at -1 by Escherichia coli RNase P but are cleaved normally by the Schizosaccharomyces pombe enzyme (Krupp et al., 1991).

Eukaryotic 3`-tRNase, which has been less thoroughly investigated than RNase P, appears in at least one case to be a protein enzyme (Castaño et al., 1985). 5`-End processing precedes 3`-end maturation in both the Xenopus (Castaño et al., 1985) and Drosophila (Frendewey et al., 1985) systems. Since 3`-CCA addition can only follow the 3`-tRNase reaction, 3`-CCA is probably not biologically important for eukaryotic RNase P activity. Yeast nuclei do not ordinarily require a tRNA 3`-endonuclease, but they have such activities (Furter et al., 1992). Interestingly, one yeast nuclear 3`-tRNase can process tRNA with a 5`-end leader and requires an AU-rich sequence just downstream from its processing site for activity. Defective 3`-end processing was lethal in the case of an essential yeast mitochondrial tRNA (Zennaro et al., 1989); a tRNA with a 3`-end trailer in place of 3`-CCA would not function properly in translation.

Working with culture cell extracts and synthesized tRNAs, we have separated Drosophila RNase P from an enzyme (3`-tRNase) that is clearly capable of endonucleolytically removing the tRNA 3`-end trailer. We cannot be certain that this is the enzyme that processes tRNA 3`-ends in vivo; its products do appear to be the same as those previously observed using a cloned tRNA gene transcribed and processed with a Drosophila extract (Frendewey et al., 1985). Even though the RNase P and 3`-tRNase enzymes and processing sites differ, our investigation shows the generalized tRNA substrate identity to be similar for five mutant sets in both reactions.

RNase P substrate structure probing with Pb and RNase T1 described herein suggests that in one case, defective RNA folding coincides with processing inhibition. Additionally, disrupting a D-T interaction with a T loop single substitution increases D loop sensitivity to T1 and severely inhibits processing. In two cases where processing is prevented by single substitutions, the RNAs appear to be correctly folded, suggesting that in these instances, the processing defects arise from local helix deformation.


MATERIALS AND METHODS

Cell Culture and Extract Preparation

Drosophila Kc(0) cells were propagated, and extracts were prepared as described previously (Preiser and Levinger, 1991; Price et al., 1987; Dingermann et al., 1981).

tRNA Gene Construction, Site-specific Mutagenesis, and in Vitro Transcription

Processing was unlinked from transcription using a T7 promoter/tRNA gene construct, freeing us to analyze any tRNA variants for their processing effects, even those that have poor RNA polymerase III internal promoters. The Drosophila tRNA gene (Frendewey et al., 1985) linked to a T7 RNA polymerase promoter was constructed using overlapping synthetic oligonucleotides as described previously for the 5 S RNA gene (Preiser and Levinger, 1991). tRNA was analyzed because the stabilized secondary structure at its 3`-end (Fig.1A) helped to establish that tRNA 3`-end processing is endonucleolytic.


Figure 1: Substrates for Drosophila tRNA structure and processing analysis. A, sequence and secondary structure of Drosophila tRNA (redrawn from Frendewey et al.(1985)). Arrows, RNase P and 3`-tRNase processing sites. B, RNase P substrate with a mature 3`-end. C, 3`-tRNase substrate with a mature 5`-end. D, transversions were introduced in sets of three (nucleotides enclosed in rectangles).



An RNase P substrate (Fig.1B) was prepared by introducing an EcoT22I runoff site at A using the Kunkel procedure(1985). A 3`-tRNase substrate (Fig.1C) was obtained by deleting the 16-nt (^1)5`-end leader, initiating transcription at +1, and using a constructed DraI runoff site at +108. 3`-tRNase cuts tRNA after A (Fig.1C, arrow); 3`-CCA is not transcriptionally encoded and must be added after the 3`-tRNase reaction by tRNA nucleotidyl transferase. 3`-tRNase templates produce very poor labeled RNA yields, perhaps owing to the sequence of the first 10 nucleotides (Milligan et al., 1987); to improve yield, these T7 reactions were primed with pGpC (Sigma; as suggested by Jerry Jendrisak). Wizard (Promega) miniprep DNA runoff templates were transcribed, internally labeled with [P]UTP, and gel-purified as described previously for 5 S RNA (Preiser and Levinger, 1991).

The tRNA genes for both the RNase P and 3`-tRNase substrates were cloned between the EcoRI and BamHI sites of Stratagene SK to prepare U-substituted templates for Kunkel mutagenesis(1985). Mismatched oligonucleotides were synthesized to produce the acceptor, D, T and anticodon stem substitutions, and the D/T loop substitutions illustrated in Fig.1D. Double substitutions were made by simultaneously annealing two mismatched oligonucleotides or, in the case of the anticodon stem, by using one longer oligonucleotide. Mutations were confirmed by supercoil DNA sequencing using Sequenase 2.0 (U.S. Biochemical Corp.). The yeast tRNA plasmid p67YF0 (a gift of L. Behlen) with an upstream SP6 promoter and BstNI runoff site at +75 was used to transcribe tRNA with a 37-nt 5`-end leader and a mature 3`-CCA end for control RNase P and structure mapping experiments (data not shown).

Fractionation of 3`-tRNase from RNase P

3`-tRNase was separated from RNase P by chromatography on S-Sepharose or DEAE-Sepharose Fast-Flow (Pharmacia Biotech Inc.; Fig.2). 2.5 ml of S100 extract was equilibrated with 25 mM KCl in column buffer (CB, consisting of 15 mM potassium-HEPES, pH 8, 3 mM MgCl(2), 1 mM dithiothreitol, 0.1% Tween 20, 0.1 mM phenylmethylsulfonyl fluoride and 10% glycerol) using a Sephadex G25 PD-10 column (Pharmacia Biotech Inc.) and loaded on a 3-ml ion exchange column. After collecting the flow-through and washing with 25 mM KCl-CB, the column was eluted with 20 ml each of 0.1, 0.2, and 0.4 M KCl-CB. A peaks were pooled, stored in aliquots at -70 °C, and equilibrated with assay buffer using a G25 spun column or by dilution.


Figure 2: Gel assay of the chromatographic separation of RNase P from 3`-tRNase. A and B, fractionation with S-Sepharose. A, 3`-tRNase reactions. B, RNase P reactions. Lanes1-4, assays of the 25 mM KCl column flow-through and the fractions eluted from the column with 0.1 M, 0.2 M, and 0.4 M KCl, respectively. C and D, same as A and B except chromatography with DEAE-Sepharose. Designations at sides are as follows: pre-3`, precursor for 3`-tRNase processing (108 nt); mature, mature tRNA (72 nt); 3` end, small fragment released from the 3`-end by 3`-tRNase (36 nt; see Fig.1C); Pre-P, RNase P precursor (88 nt); 5` end, small fragment released from the tRNA 5`-end (16 nt; see Fig.1B). Mlanes, a marker mixture of 146-, 135-, 110-, and 105-nt RNAs produced by T7 transcription. E, RNase P reactions performed with 5`-end-labeled RNase P substrate. Lanes1-5, processing reactions with unmodified tRNA T7 transcript substrates were performed for 0, 15, 30, 60 and 120 min, respectively.



Processing Reactions

Processing analysis was performed as described previously for 5 S RNA (Preiser and Levinger, 1991) using the optimal KCl concentration in CB for each enzyme (175 mM for RNase P and 25 mM for 3`-tRNase; data not shown). 2 µl of the S-Sepharose flow-through or 0.2 M KCl pool was used in each 20-µl 3`-tRNase or RNase P reaction, respectively, with a 60-min incubation at 28 °C (Fig.2, A-D) or with sampling after 0, 15, 30, 60, and 120 min (Fig. 2E and 3). Samples were deproteinized, recovered, electrophoresed on 6% sequencing gels until bromphenol blue migrated to 4 cm above the bottom of the gel (to retain the small fragments in Fig.2) or until the xylene cyanol was 4 cm above the bottom of the gel (to optimize resolution of mature tRNA; Fig.3), and autoradiograms were taken for 6-48 h depending on the amount of [P]tRNA loaded. Processing activities were determined as described previously (Preiser and Levinger, 1991) and are qualitatively presented as 100% (+), 25-75% (±), 10-25% (-), or <10% (0) of wild type processing activity.


Figure 3: Processing of wild type and variant tRNAs with sequence transversions. Panels1-5, RNase P reactions. Panels 6-10, 3`-tRNase P reactions. Panels1 and 6: A, wild type; B, G4C; C, C68G; D, 4/68 double transversion. A-D are the same as in panel1. Lanes1-5, processing for 0, 15, 30, 60, and 120 min, respectively. Panels2 and 7, same as panels1 and 6, except analysis of the 11/24 variant set. Panels3 and 8, the 29/41 set; panels4 and 9, the 50/62 set; panels5 and 10, the 18/55 set. Designations below the panels are as follows: +, processing similar to wild type; ±, processing at 25-75% of wild type; -, processing at 10-25% of wild type; 0, no processing.



5`-End Labeling and Structure Probing

To investigate RNA folding, unlabeled RNase P substrate tRNAs (Fig.1B) with the substitutions indicated in Fig. 1D were transcribed, deproteinized, and recovered. 5`-triphosphates were removed with shrimp alkaline phosphatase (U.S. Biochemical Corp.), which was subsequently heat-inactivated. 5`-Ends were labeled with [-P]ATP (DuPont NEN) using T4 polynucleotide kinase (Maniatis et al., 1982) and RNAs were purified on denaturing gels.

Recovered 5`-end-labeled RNAs were redissolved in water and refolded by heating to 70 °C for 10 min and cooling to room temperature in a buffer containing 15 mM potassium-MOPS, pH 7, 3 mM MgCl(2), 175 mM KCl. Results were independent of a wide range of MgCl(2) and KCl concentrations. Cleavages were performed in 5 µl with 1.25 µg of added unlabeled yeast tRNA (Boehringer Mannheim) for 5 min at room temperature using lead acetate at 0.5, 1, or 5 mM or using RNase T1 at 0.5, 1, or 2 units/ml. After incubation, cleavage was terminated by dilution to 400 µl with 0.15 M sodium acetate, pH 5, 10 mM EDTA, 0.5% SDS; RNAs were recovered by ethanol precipitation, including phenol/chloroform deproteinization for the RNase T1 samples, and electrophoresed on a 10% sequencing gel until the bromphenol blue marker dye migrated to 4 cm above the bottom. Autoradiograms were taken for 6-48 h depending on the amount of labeled tRNA loaded.


RESULTS

Separation of tRNA Substrates

Both 5`- and 3`-end-processing reactions have been performed using a single Drosophila tRNA precursor (Fig.1A) and S100 (data not shown; Frendewey et al., 1985). Under these conditions, an intermediate was observed that has a processed 5`-end and a 3`-end trailer (an RNase P product; Fig.1C), but no intermediate was observed that has a 5`-end leader and a processed 3`-end (a 3`-tRNase product; Fig.1B). The absence of the intermediate that is processed first by 3`-tRNase suggested that this enzyme cannot process a substrate with a 5`-end leader and that the RNase P reaction must therefore precede the 3`-tRNase reaction (Frendewey et al., 1985; Castaño et al., 1985). Alternatively, under the conditions of extraction and assay, RNase P may simply be more active than 3`-tRNase. Secondary structure of the 3`-end trailer (Fig.1, A and C) stabilizes it, allowing detection of the small 3`-end fragment (Fig.2A, lane1, band labeled 3` end) and demonstrating that this 3`-tRNase is an endonuclease (Frendewey et al., 1985).

To investigate substrate effects on 5`- and 3`-end processing reactions, we uncoupled the substrate for 5`-end processing from that for 3`-end processing (Fig.1, compare B and C with A). An unprocessed 5`-end could possibly interfere with 3`-tRNase activity (see above). The RNase P substrate with a truncated 3`-end (Fig.1B) also gave more reproducible processing than the natural precursor (Fig.1A). Truncated substrates are useful for in vitro processing experiments because the extra sequence, which does not improve processing, might cause steric hindrance, interfere with RNA refolding, or bind inhibitory proteins.

Another reason for separating the two processing substrates is that RNase P and 3`-tRNase processing determinants could differ. For example, a tRNA variant that is a good 3`-tRNase substrate might be a poor RNase P substrate. If the suggested obligatory reaction order (first RNase P, and then 3`-tRNase; Frendewey et al., 1985; Castaño et al., 1985) is correct, then the relative processing efficiencies for both enzymes could not be established with such a variant using a single substrate (Fig.1A).

Separation of RNase P from 3`-tRNase by Column Chromatography

S100 processing reactions require an ATP regeneration system (data not shown), which was included in an earlier transcription/processing system (Frendewey et al., 1985). An ATP requirement has never been established for RNA processing endonucleases; the ATP dependence of these reactions disappears and the RNase P and 3`-tRNase become more active when they are separated by ion exchange chromatography (data not shown). We have not further investigated the ATP requirement for tRNA processing using the S100; we chose instead to use column fractions for RNase P and 3`-tRNase reactions.

Drosophila RNase P binds to both cation and anion exchange resins more tightly than 3`-tRNase (Fig.2), as previously demonstrated in Xenopus (Castaño et al., 1985). We used the S-Sepharose flow-through and 0.2 M KCl fractions for Drosophila 3`-tRNase and RNase P reactions, respectively. Enzymes were cleaner and more active when separated with S-Sepharose than with DEAE-Sepharose.

The small fragments observed with both 3`-tRNase and RNase P (Fig.2, lanesA1 and B3, designated 3`-end and 5`-end, respectively) demonstrate the occurrence of in vitro 3`- and 5`-endonucleolytic processing. These small fragments were identified on the basis of size: 36 nt for the 3`-end trailer and 16 nt for the 5`-end leader. RNase P reactions performed with 5`-end-labeled RNA (Fig.2E) also illustrate efficient and stable production of the 5`-end fragment. Multiple species observed in the 36-nt 3`-tRNase 3`-end trailer fragment (Fig.2, lanesA1 and C2) and in mature tRNAs produced by RNase P (Fig.2, lanesB3 and D3) could arise from 3`-end heterogeneity of T7 transcripts (Milligan et al., 1987).

Variant Effects on RNase P and 3`-tRNase Reactions

In RNase P (Fig.3, panels 1-5) and 3`-tRNase (Fig.3, panels6-10) reactions with wild type substrates, 72-nt products that migrate identically (designated M on the right of each panel) accumulate with incubation time (0 to 120 min; lanes1-5 in part A of each panel) at the expense of the precursors (88 nt in the case of RNase P substrates and 108 nt in the case of 3`-tRNase substrates; compare Fig.1, B and C). The variant tRNAs are arranged in sets of three, with two single substitutions, which presumably disrupt a base pair (Fig.3, parts B and C of each panel) and a double substitution, which is expected to re-establish base pairing with a sequence transversion (Fig.3, partsD; see Fig. 1D for the tRNA substitutions that were made in the context of RNase P and 3`-tRNase substrates; Fig.1, B and C). These sets survey each of the four stems: acceptor stem (Fig.3, panels1 and 6), D stem (panels2 and 7), anticodon stem (panels3 and 8), T stem (panels4 and 9), and a D/T loop tertiary contact (Fig. 3, panels5 and 10). In every case, the double substitutions that restore base pairing also restore wild type processing activity (Fig.3, partD of each panel).

Singly substituted tRNAs are processed by RNase P and 3`-tRNase with similar time courses (Fig.3, compare partsB and C of panels1-5 with panels6-10). G4C (the substitution of a G at nt 4 in the wild type with a C in the variant; Fig.1D) prevents processing, while C68G does not inhibit processing by either enzyme (Fig.3, partsB and C of panels1 and 6; the 0, -, ±, and + designations below the panels signify broad processing ranges relative to wild type). Thus a CC apposition at 4/68 has a pronounced inhibitory effect, while a GG apposition does not. The CC effect must be position- and not sequence-specific because the double substitutions restore wild type processing (panels1D and 6D). This argument also applies to the other four variant sets.

Like the 4/68 variant sets (Fig.3, panels1 and 6), the 11/24 D stem substitutions (Fig. 1D and Fig. 3, panels2 and 7) affect processing asymmetrically. U11A prevents both RNase P and 3`-tRNase processing (Fig.3, panels2B and 7B), while A24U and the 11/24 double substitutions give wild type processing (Fig.3, panels2C and 7C).

None of the anticodon stem substitutions (Fig.1D) significantly affected processing (Fig.3, panels3 and 8), consistent with observations from other systems that RNase P neither contacts nor requires the anticodon stem for activity (McClain et al., 1987; Hardt et al., 1993). The anticodon stem is also not required for the tertiary D/T fold (Dichtl et al., 1993).

Asymmetric effects like those in the acceptor and D stems were also observed with T stem substitutions. U62A prevents processing (Fig.3, panels4C and 9C); the opposite substitution, A50U, significantly inhibits both RNase P and 3`-tRNase processing (Fig.3, panels4B and 9B). The 50/62 double substitution restores the variant's ability to be processed like the wild type (Fig.3, panels4D and 9D), showing that the inhibitory effects are due to the 50/62 AA apposition, not due to a sequence-specific contact.

Interference with Tertiary Folding Inhibits Processing

The D/T tertiary fold was investigated with transversions at 18/55 (Fig.1D and Fig. 3, panels5, 10). C55G prevented processing by RNase P (Fig.3, panel5C) and reduced processing by 3`-tRNase (Fig.3, panel10C). The D loop substitution G18C inhibited processing less than C55G (Fig.3, panels5C and 10C; compare with partsB). Double substitutions apparently restore the tertiary fold (see below; Fig.4, panel 5) and also restore processing to the wild type rate (Fig.3, panels5D and 10D; compare partsA).


Figure 4: Structure and folding of wild type and variant tRNAs. Structure was analyzed with Pb and RNase T1. Panel1, analysis of the 4/68 transversions. A, wild type; B, G4C; C, C68G; D, 4/68 transversions. Lane1, no cleavage control; lanes2-4, lead acetate at 0.5, 1, and 5 mM. Lanes5-7, RNase T1 at 0.5, 1, and 2 units/ml. Incubations were for 5 min at room temperature. Panels2-5, the 11/24, 29/41, 50/62, and 18/55 transversion sets were analyzed as in panel1. Numbers on the right signify the nucleotide positions of prominent T1 cleavages. Lines on the left signify enhanced lead cleavages in variant tRNAs. Arrows signify enhanced T1 cleavages.



Effects of Variant tRNAs on Structure and Folding

We investigated tRNA structure and folding by 5`-end-labeling RNase P substrates (Fig.1B), which have wild type and variant sequences (illustrated in Fig.1D), and cleaving them with structure probes. The RNase P substrates were chosen because the 5`-end leader has a strong T7 promoter, can be efficiently 5`-end-labeled, and allows structure mapping of the entire tRNA sequence from +1 through +72.

Wild type Drosophila tRNA is more accessible to Pb and T1 cleavage (Fig.4A and 5A) than yeast tRNA (data not shown). In yeast tRNA, Pb binds a specific site in the T loop and cleaves a precise target (nt 17) in the D loop (Behlen et al., 1990); Pb cleaves wild type Drosophila tRNA (Fig.5A, lines) most prominently at nt 19-21, in the anticodon loop at nt 32, and weakly elsewhere in the D loop (nt 13 and 17), the anticodon loop (nt 38), the variable loop (nt 43-48), and the T loop (nt 53-59). The strongest T1 cleavage sites (Fig.5A, arrows) are at nt 34 and 36 in the anticodon loop (as in yeast tRNA; data not shown), with weaker T1 sites in the D loop (nt 15 and 17-18), the T loop (nt 56), elsewhere in the anticodon loop (nt 31 and 37), and at several stem Gs (nt 1, 4, 6, 10, 23, 24, 42, and 43).


Figure 5: Effect of substitutions on tRNA folding. A, wild type tRNA structure map. Lines indicate lead cleavage sites, with the linethickness signifying cleavage frequency. Arrows indicate T1 sites, with arrowdarkness indicating cleavage frequency. B, differences in the cleavage pattern between G4C and wild type tRNA. C, differences in the cleavage pattern between C55G and wild type tRNA. Only sites of increased sensitivity in the substitution mutants are shown.



The acceptor stem G4C substitution is cleaved with T1 more than wild type at nt 6, 10, 15, 31, and 56 (Fig.4, panel1B, lanes5-7, and Fig. 5B), suggesting global misfolding. T1 cleavage at anticodon loop positions 34, 36, and 37 was reduced. Additionally, G4C displays increased lead sensitivity at C and U in the anticodon stem and loop and at A in the variable loop instead of extending uniformly through C.

The D stem substitutions cause increased Pb cleavage at nt 19-20 (Fig.4, panel2, lanes2-4 in partsB-D) and increased T1 susceptibility at G^6, G, and G (Fig.4, panel2, lanes 5-7 in partsB-D; compare wild type (WT) in lanes5-7 of partA). The A29U substitution in the anticodon stem increased Pb cleavage at nt 20 and T1 sensitivity at nt 1, 4, 6, 31, and 56 (Fig.4, panel3B, lanes 5-7; compare panelA, lanes5-7). The T stem substitutions increased Pb sensitivity at nt 12 and 13 and at nt 49 close to the variant positions of 50 and 62 (Fig.4, panel4C, lanes2-4). In the same variants, T1 sites at nt 10, 15, and 56 became more prominent (Fig.4, panel4B and 4C, lanes5-7).

C55G greatly stimulated T1 sensitivity at nt 17, 18, 55, and 56 (Fig.4, panel5C, lanes5-7 (compare panelA; lanes5-7); Fig. 5C), indicating substantial disruption of the tertiary fold. The G at nt 55 is new with this substitution, so the intensity of T1 cleavage at this position cannot be evaluated relative to wild type, but the sites at nt 17 and 18 become more prominent than the strong site at nt 34 in the anticodon loop, showing that shielding of the D loop is largely due to the tertiary fold. G18C does not have a T1 site at nt 18, but the T1 sites at nt 17 and 56 are less pronounced than in C55G (Fig.4, panel5B, lanes5-7; compare panel5C), showing that this substitution (a CC apposition) does not disrupt the tertiary interaction as much. The 18/55 double substitution reduces T1 susceptibility of nt 17 (Fig.4, panel5D, lanes5-7).


DISCUSSION

Effects of Sequence Variation within tRNA on End Processing Reactions

The length and sequence of the 5`-end leader has no evident effect on Drosophila RNase P; nor does the length and sequence of the 3`-end trailer have a demonstrable effect on 3`-tRNase activity. For example, we efficiently processed yeast tRNA with a heterologous (polylinker) 5`-end leader using Drosophila RNase P (data not shown). Frendewey et al.(1985) 3`-end-processed two Drosophila tRNAs with very different 3`-end trailers. Castaño et al.(1985) processed human tRNA with Xenopus 3`-tRNase. We therefore chose to concentrate on the processing effects of sequence variation within tRNA.

A misinterpretation might arise from the use of different substrates (Fig.1, B and C) in variant processing and structure-mapping experiments. Leader and trailer sequences could interfere differentially with proper tRNA folding in sequence variants, inhibiting processing. Such differential interference could be missed unless structure probing was performed not only with the RNase P substrate (Fig.1B and 4) but also with the 3`-tRNase substrate (Fig.1C). In the variants investigated herein, differential interference probably did not occur, because where RNase P and 3`-tRNase processing differences were observed (Fig.3), RNase P was more sensitive than 3`-tRNase.

Some Helix Disruptions Inhibit Processing

Our mutagenesis strategy established that stem base pairing with a sequence change allows both 5`-end and 3`-end endonucleolytic processing (Fig.3, partD of allpanels; compare partsA), that stem single substitutions affect processing asymmetrically (Fig.3, partsB and C of panels1 and 6, 2 and 7, 4 and 9, and 5 and 10), that sequence and base pairing changes in the middle of the anticodon stem do not affect processing (Fig.3, partsB and C of panels3 and 8), and that the tRNA tertiary fold must be stabilized for processing (Fig.3, panels5 and 10, partsB and C). Processing is prevented or strongly inhibited by the stem substitutions G4C, U11A, and U62A (Fig.3, partB of panels1 and 6, partB of panels2 and 7, and partC of panels4 and 9, respectively). In each case, one apposition severely inhibited processing (4/68 C/C, 11/24 A/A, and 50/62 A/A). These inhibitory effects are not sequence-specific, since all double substitutions (partD of the above panels in Fig.3) restored processing to the wild type level. They are, however, sequence-dependent, since the opposite single substitutions (4/68 G/G, 11/24 U/U, 50/62 U/U; Fig.3, part C of panels1 and 6 and 2 and 7 and partB of panels4 and 9) were less inhibitory. The disruptive effects of unfavorable appositions are often local, since large structure changes were seldom observed (see below).

Some stem nucleotides also participate in tertiary folding through the formation of base triples (Holbrook et al., 1978), complicating the experimental design and interpretation. We cannot generalize concerning the processing effects of specific nucleotide appositions, which could be context-dependent.

Global tRNA Folding and Processing

Drosophila tRNA has a more open structure than yeast tRNA (Fig.5A) (Behlen et al., 1990; data not shown). Lead cleavage has been used as a structure probe for 5 S RNA (Brunel et al., 1990) and M1 RNA, the catalytic component of E. coli RNase P (Ciesiolka et al., 1994). Unlike the exceptional specificity in the yeast tRNA system, cleavages were widely distributed through the loops.

Defective RNA folding was suggested by widespread changes in RNase T1 susceptibility observed with G4C (Fig.4, panel1B; Fig. 5B), which evidently disrupts folding and also prevents processing (Fig.3, panels1C and 6C). Two variants, A24U and A29U, display altered T1 cleavages (Fig.4, panels2C and 3B) and are processed well (Fig. 3, panels2C and 7C, 3B and 8B). One variant, U11A, cannot be processed (Fig.3, panels2B and 7B), while its Pb and T1 cleavage patterns are not significantly different from other members of the 11/24 variant set (Fig.4, panel2B; compare partsC-D). Thus processing inhibition is probably due to misfolding of G4C, but U11A and U62A are probably folded correctly, the processing defects arising from local helix deformations.

Tertiary Folding

C55G causes increased T1 susceptibility of G and G^18 (Fig.4, panel 5C; Fig. 5C), prevents RNase P processing, and strongly inhibits 3`-tRNase (Fig.3, panels5C and 10C). G18C disrupts tertiary structure (Fig. 4, panel5B, lanes5-7) and processing (Fig. 3, panels5B and 10B) less severely. These are not local or sequence-specific effects, since the double substitution that restores base pairing (as suggested by the reduction of G T1 sensitivity to close to the wild type level; Fig.4, panel5D, lanes5-7) also restores processing (Fig.3, panels5D and 10D). Disruption of the D/T loop interaction coincides with processing inhibition, suggesting that the tertiary fold is required for substrate recognition.

Comparison of Variant Sequence Effects on RNase P and 3`-tRNase

The effect of substrate variation on RNase P and 3`-tRNase was similar in every case (Fig.3, compare panels1-5 with 6-10). Where differences were evident (Fig.3, panels4B and 9B, and panels5B and 10B), RNase P was more sensitive than 3`-tRNase. Although the structure and mechanism of the two enzymes are probably different, common substrate identity elements appear to be dispersed in the D, T, and acceptor stems and the tertiary fold.

The interpretation that Drosophila tRNA has dispersed processing determinants, including those in the D stem, and a requirement for tertiary folding, is consistent with results obtained using other systems. Full-length tRNAs require many tertiary contacts for proper folding (Dirheimer et al., 1995), unlike simplified substrates, which are presumably locked into a suitable conformation for catalysis (McClain et al., 1987). Human RNase P has substrate determinants in the D stem (Hardt et al., 1993). Kinetics of RNA folding have been dissected as a combination of secondary and tertiary structure elements in the more complex Tetrahymena self-splicing intron system (Zarrinkar and Williamson, 1994).

On the basis of our broad survey, Drosophila RNase P and 3`-tRNase apparently have similar substrate determinants in the structure of mature tRNA. These functional determinants probably reduce processing efficiency in sequence variants by acting at three distinguishable levels: local helix deformation, global misfolding, and disruption of tertiary contacts. A detailed study of mutation effects on folding and processing could help refine the definition of substrate structure.


FOOTNOTES

*
This work was supported by grants from the York College/CUNY Office of Sponsored Programs, the PSC/CUNY, and the National Institutes of Health (Minority Biomedical Research Support S06 GM08153, Minority Access to Research Careers T34 GM08498, and Academic Research Enhancement Award R15 GM04603). 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.: 718-262-2704; Fax: 718-262-2652.

Dept. of Biology, New York University, New York, NY 10003.

**
Dept. of Biology, St. Johns University, Jamaica, NY 11439.

§§
Howard University School of Medicine, Washington, D. C. 20059.

¶¶
Dept. of Pharmacology, Cornell University Medical School, New York, NY 10021.

^1
The abbreviations used are: nt, nucleotide(s); MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We thank I. Arjun, N. Persaud, and S. Whyte for skillful technical assistance; D. Engelke, J. Jendrisak, P. Preiser, and O. Uhlenbeck for helpful discussions and a critical reading of the manuscript; and L. Behlen for the gift of plasmid DNA.


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