(Received for publication, February 9, 1995; and in revised form, May 25, 1995)
From the
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.
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.
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 (
)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).
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.
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.
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, 175 mM KCl. Results were independent of a wide range of MgCl
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.
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).
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).
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.
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.
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
, 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).
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 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.
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.
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.