Nucleic Acids Research, Vol. 19, No. 21 5957-5964

..) 1991 Oxford University Press

Analysis of the gene encoding the RNA subunit of ribonuclease P from T.thermophilus HB8 Roland K.Hartmann and Volker A.Erdmann Institut fOr Biochemie, Freie Universitat Berlin, Thielallee 63, 1000 Berlin 33, FRG Received July 10, 1991; Revised and Accepted October 10, 1991

ABSTRACT The gene for the RNA subunit of ribonuclease P from the extreme thermophilic eubacterium T. thermophilus HB8 was cloned using oligonucleotide probes complementary to conserved regions of RNase P RNA subunits from proteobacteria. The monocistronic gene and its flanking regions were sequenced. The gene is enclosed by a promoter and a rho-independent terminator. Nuclease S1 protection analyses showed that the primary transcript is identical with the mature RNA, i.e. no processing events are involved. The stem and loop structure of the terminator remains part of the mature molecule. In vitro transcription of the cloned gene with purified RNA polymerase from T. thermophilus yields the same RNA product as in vivo, indicating that no other components except RNA polymerase are involved in the synthesis of the RNA. RNase P RNA from T. thermophilus cleaved a pretRNATYr from E. coli with highest efficiency between 550C and 650C. The T. thermophilus RNA, which has a G-C content of 86% in helical regions, displays several structural idiosyncrasies, although its secondary structure is similar to that of proteobacteria. Numerous invariable nucleotides in the structural core of eubacterial RNase P RNAs are also conserved in the RNA from the extreme thermophilic eubacterium.

EMBL accession no. X60463

eukaryotic nuclear and mitochondrial RNase P RNA subunits (17); however, a so-called cage-shaped tertiary structural domain (18) is found in all RNA subunits of RNase P enzymes and RNase MRP, a mitochondrial RNA processing enzyme involved in mitochondrial replication (19, 20), which shares antigenic determinants with human nuclear RNase P (21). We analyzed the RNase P RNA subunit from the extreme thermophilic eubacterium T. thermophilus HB8 to get insight into the molecular adaptations of the catalytic RNA subunit due to an anticipated elevated thermostability of the enzyme. Transcription of the gene and maturation of the catalytic RNA were investigated, as well as conditions for in vitro RNA catalysis in the absence of the protein subunit using a pre-tRNATYr from E. coli as substrate.

MATERIALS AND METHODS Enzymes were purchased from Boehringer Mannheim, Pharmacia, New England Biolabs and Bethesda Research Laboratories. Radioactive nucleotides were obtained from Amersham.

INTRODUCTION

Bacterial strain T. thermophilus HB8 cells (ATCC 27634) were grown at 70 to 75°C in medium D, as previously described (22), supplemented with 5g of tryptone (Difco Laboratories), 4g of yeast extract, 2g of NaCl and ig of glucose per liter. Cells were harvested in the mid-log phase.

Ribonuclease P (RNase P) cleaves precursor tRNAs at the 5'-end of mature tRNAs. The enzyme is composed of RNA and protein in the majority of activities investigated so far (1-14). There is no evidence for an RNA component in RNase P from chloroplasts (15). RNA components of enzymes from E. coli and B. subtilis were shown to be catalytically active in the absence of the protein subunit (16). So far, catalytic activities of RNA subunits alone from eukaryotic nuclear, mitochondrial or archaebacterial RNase P enzymes could not be demonstrated (17). Eubacterial RNase P molecules, compared to archaebacterial and eukaryotic enzymes, show the highest RNA/protein ratio (1, 3, 7, 10, 13, 17), indicating that the protein moiety has been reduced to a minimum size required for functionality of the enzyme. Secondary structures of RNase P RNAs from proteobacteria and Bacilli spp. show substantial differences, although several structural features are conserved (9). No convincing sequence similarities have been hitherto found between eubacterial,

Cloning of the gene encoding the RNase P RNA DNA was isolated from T. thermophilus HB8 cells by standard procedures (23). For cloning of the RNase P RNA gene, two oligonucleotides (5 '-ATAAGCCGGGTTCTGTC-3' and 5 '-GCCCTATGGAGCCCGGACTTTCCTCCCC-3 ') complementary to conserved segments of RNase P RNAs from proteobacteria (9) were used as hybridization probes. Thereby, a 2.6 kbp genomic BamHI fragment was cloned into pT7T318U (Pharmacia), yielding plasmid pM1HB8. Oligonucleotides were 5 '-end-labeled utilizing T4 polynucleotide kinase and [-y-32P]ATP as described (24). Competent cells of E. coli BMH 71/18 (25) were prepared and transformed as described previously (26). Southern hybridizations were carried out at room temperature for 48 h in 50% formamide, 0.75 M NaCl/75 mM sodium citrate (pH 7.0), 0. 1 % SDS, 5 x Denhardt solution (24), 50 mM sodium phosphate (pH 6.5), 100 jig/ml sonicated herring

5958 Nucleic Acids Research, Vol. 19, No. 21 sperm DNA and 25.000 cpm/cm2 (Cerenkov) of 32P-labeled oligonucleotide using 25 I1 per cm2 of nitrocellulose filter. Filters were washed three times in 0.3 M NaCl/30 mM sodium citrate (pH 7.0), 0.1% SDS for 10 min. Colony filters were prehybridized in 0.75 M NaCl/75 mM sodium citrate (pH 7.0), 0.1% N-lauroylsarcosine (Na-salt, Sigma), 0.02% SDS, 1% (w/v) blocking reagent (Boehringer, Digoxygenin nonradioactive DNA labeling and detection kit) at 68°C for at least two hours until colony spots lost their brownish colour. Hybridization was carried out at room temperature for 48 h in the same buffer (50 gl/cm2), but containing 5% (w/v) blocking reagent and, in addition, 50% formamide, 35 yg/ml sonicated calf thymus DNA and 100.000 cpm/ml (Cerenkov) of each of the two 32P-labeled oligonucleotides. Nitrocellulose filters were washed three times in 0.3 M NaCl/30 mM sodium citrate (pH 7.0), 0.1% SDS for 10 min.

Primer synthesis and DNA sequencing For dideoxy sequencing (27), the 2.6 kbp fragment was cloned in both orientations into pT7T318U/19U (Pharmacia) vectors. Single-stranded plasmid DNA was prepared by superinfection with phage M13 K07 (Pharmacia). Oligonucleotides used for sequencing were produced by the phosphoramidite method as described previously (28), and DNA sequencing was performed with T7 DNA polymerase (Pharmacia) and [35S]dATP[aS]. For unequivocal sequence determination, both strands were completely sequenced by using consecutive primers (each 25 to 35 bases long). Ambiguous sequence stretches were resolved by using deaza c7dGTP mixes (Pharmacia). 5'- and 3'-labeling of DNA fragments used for nuclease Si protection analysis The 1.4 kbp BamHI-BstEII fragment, covering the 5'-region of the RNase P RNA gene up to the 5'-end of the cloned DNA fragment (Fig. 1), was dephosphorylated, extracted with phenolchloroform followed by ethanol precipitation, redissolved in double destilled water, digested with XhoIl and 5'-end-labeled with T4 polynucleotide kinase (24). The 475 bp XhoIl-BstEII fragment (Fig. 1, probe 1), exclusively labeled at the BstEIl site, was excised from a 5 % polyacrylamide gel, eluted from the gel, and used as hybridization probe. The 186 bp AvaIl fragment (Fig. 1, probe 2), covering the 3'-portion ofthe gene, was 3'-endlabeled by filling in the recessed 3'-ends in the presence of [a1-32P]dGTP utilizing KLENOW polymerase (24). Nuclease Si mapping Preparation of total RNA from exponentially growing T. thermophilus cells according to the hot phenol procedure and nuclease S1 protection analyses were performed as described (29). Alternatively, total RNA was isolated according to the sodium acetate method: T. thermophilus cells were harvested at an OD6W of 1.0. Cells from 10 ml of cell suspension were resuspended in 1 ml of 0.3 M sodium acetate pH 5.0, centrifuged and resuspended in 0.3 ml of 0.3 M sodium acetate pH 5.0. The suspension was extracted with an equal volume of phenol, followed by phenol-chloroform extraction and ethanol precipitation. RNA was dissolved in 60 g1d of sterile double destilled water. The hybridization temperature was 50°C. In controls, equal amounts (100 ,ug) of bulk tRNA from E. coli MRE 600 (Boehringer) instead of total RNA from T. thernophilus were incubated with the labeled DNA probe.

Transcription with T. thermophilus RNA polymerase in vitro For synthesis of 32P-labeled transcripts (Fig. 3), T thermophilus HB8 RNA polymerase (3-10 units/assay, [30]) was incubated for 30 min at 65°C in a final volume of 100 yd with 0.5-3 itg of template DNA in buffer A (50 mM glycine/NaOH [pH 8.5], 0.1 mM EDTA, 5 mM dithioerythritol, 18 mM MgCl2, 200 mM KCI, 1 mM spermidine, 1 mM thermine) using 0.6 mM each ATP, GTP and UTP, 1 jl [a-32P]CTP (3000 Ci/mmol, 10 tCi4dl) and 0.2 mM CTP. The reaction was stopped by phenolchloroform extraction followed by ethanol precipitation. For preparative transcription of RNase P RNA by T. thermophilus RNA polymerase, approximately 10 itg of template DNA, i.e. the cloned 2.6 kbp BamHI linear fragment, and 50 units of T. thernophilus RNA polymerase (30) were incubated for 30 min at 65°C in a final volume of 300 Al in buffer A, but with the following modifications: Incubation was startd with 0.6 mM each ATP, CTP, GTP, UTP and 3 Al of [ai-32P]CTP (3000 Ci/mmol, 1 ACi/Al); after 15 min, 0.3 mM each ATP, CTP, GTP, UTP were added. The reaction was stopped by phenolchloroform extraction followed by ethanol precipitation. The RNase P RNA transcript was purified on a 5 % polyacrylamide-8M urea gel, detected by autoradiography, excised from the gel and eluted over night at 40C in 0.5 M ammonium acetate pH 4.8, 10 mM magnesium acetate and 0.1 mM EDTA, followed by ethanol precipitation. For synthesis of cold in vitro transcripts used for nuclease S1 mapping (Fig. 4B), approximately 5 yg of template DNA carrying the RNase P gene, i.e. the 2.6 kbp BamHI linear cloned fragment or the circular recombinant plasmid pM1HB8, were incubated for 30 min at 65°C in a final volume of 100 td in buffer A using 10 units of T. thermophilus RNA polymerase and 0.8 M each ATP, CTP, GTP and UTP. Four id of RNase-free DNase I (27 U/4l, Boehringer) were added, followed by incubation at 37°C for 15 min. The reaction was stopped by phenol/chloroform-extraction, followed by ethanol precipitation.

Subcloning of the RNase P gene The sequence encoding the RNase P RNA was excised from the recombinant plasmid pMlHB8 as a 402 bp SmaI fragment and cloned adjacent to the EcoRI site of the T7 expression vector pT7T318U (Pharmacia). The resulting plasmid, called pT7M1HB8, was linearized with NarI, which cuts in the stem of the terminator hairpin, to yield a T7 runoff transcript which was identical to the wildtype RNA (Fig. 2) in the range of nucleotides 5-376. The in vitro transcript differed from the wildWpe RNA by the 5'-terminal sequence 5'-GGGAATT instead of 5'-UGGC, and by the lack of extraneous nucleotides 377-392 at the 3'-end (Fig. 2). In vitro cleavage of E. coli pre-tRNATYr by T. thermophilus RNase P RNA E. coli precursor tRNATYr was transcribed from the T7 expression vector pUC19TyrT (31). The plasmid was digested with Fold and transcribed with T7 RNA polymerase (Gibco/BRL) in the presence of [a-32P]UTP, [a-32P]CTP or [ca-32P]GTP as recommended by the manufacturer, yielding a runoff transcript of 131 nucleotides which carried the tRNA moiety with three additional nucleotides beyond the CCA end, and 43 nucleotides of 5'-flanking sequence. T. thermophilus RNase P RNA and 32P-labeled pre-tRNATYr were incubated at indicated temperatures in 50 mM glycine/NaOH pH 8.0, 100 mM MgCl2, 100 mM NH4CI, 4%

Nucleic Acids Research, Vol. 19, No. 21 5959 PEG 6000 (Merck) and 0.5% (w/v) SDS in a standard volume of 20 1l, followed by ethanol precipitation. Cleavage products were separated on 10% polyacrylamide-8M urea gels and visualized by autoradiography. Radioactive bands were excised from the gel and Cerenkov counted.

RESULTS We cloned a 2.6 kbp BamHI fragment encoding the RNase P RNA from the extreme thermophilic eubacterium T. thermophilus HB8 (Fig. 1) using two oligonucleotide probes complementary to conserved sequences of RNase P RNAs from proteobacteria (9). Southern hybridization analysis gave clear evidence for a single copy gene (data not shown). The gene and approximately 340 nucleotides of the 5'- and 190 nucleotides of the 3'-flanking region were sequenced (data not shown). Promoter -10 and -35 hexanucleotide sequences, separated by 17 nucleotides and similar to E. coli promoter consensus sequences (32), were identified upstream from the region homologous to RNase P RNAs from proteobacteria (9). The coding region is flanked by a typical rhoindependent terminator hairpin followed by a stretch of seven thymidines (data not shown).

(Fig. 2). The latter two helices have been deleted or largely reduced in a minimal RNase P RNA which shows reduced but still significant activity (36). In contrast to RNAs from proteobacteria and Bacilli spp., the helix enclosed by nucleotides 26-47 is short (7 bp) and continuous, and includes no G-U base pairs in the T. thermophilus RNA. Further idiosyncrasies of the T. thennophilus RNA are a noncanonical A-C base pair in helix 175-181/189-195, nine potential base pairs instead of eight in most proteobacterial sequences in the helix confined by nucleotides 195 and 218, and the absence of a noncanonical AC or G-A base pair at the end of the terminal helix (corresponding to positions 15 and 361, Fig. 2), found hitherto in all RNAs from proteobacteria and Bacillus spp. (9, 33). In conclusion, the structure of RNase P RNA from the extreme thermophilic eubacterium T. thermophilus HB8 displays idiosyncrasies, which are absent in RNAs from proteobacteria and Bacilli spp. So far,

Structure of T. thermophilus RNase P RNA Due to the strong bias for G-C base pairs in helices, i.e. a reduction of A-U and G-U base pairs, the T. thernophilus RNase P RNA provides a valuable set of compensating base changes to verify the occurrence of helical segments. As a result, the sequence of T. thermophilus RNase P RNA fits well into the secondary structure model proposed for RNA subunits from proteobacteria (9, 33, Fig. 2). Secondary structures of proteobacterial RNAs and T. thermophilus RNase P RNA deviate significantly from that of Bacilli spp. (9, 33). As the ,3 proteobacterium Thiobacillus ferrooxidans and the y proteobacteria E. coli, Pseudomonasflourescens and Chromatium vinosum, the T. thermophilus RNA lacks a variable expansion segment in the loop between nucleotides 340 and 360 at the junction to helix 17-23/333-339 (Fig. 2), which is found in RNAs from Bacilli spp., in a proteobacteria and in the ,B proteobacterium Alcaligenes eutrophus (33). Pronounced differences between the T. thermophilus and proteobacterial structures are found in variable helical regions, i.e. the helices confined by nucleotides 26-47, 131-169 and 242-297 B|at

BamHI

XhoIl

BemHI

AvuNlAvul

200 bp

0D

* 5 32p

3'. 3p *

0

=

promoter

j

=

terminator

Figure 1. Schematic representation of the 2.6 kbp BamHI fragment encoding the catalytic RNA of ribonuclease P from T. thermophilus. The coding region (hatched area), promoter, start and direction of transcription (arrow), and terminator hairpin are indicated. Only restriction sites relevant for the nuclease SI analysis have been included. Probes 1 and 2 used for nuclease SI analysis are shown underneath.

Figure 2. Proposed secondary structure of the RNase P RNA from T. thermophilus according to (9, 33). Sequences connected by the arc indicate a potential pseudoknot structure. A further pseudoknot forned between nucleotides 70-73 and 266-269 (indicated by lines) was proposed recently (34). A potential base pairing between nucleotides 115-118 and 218-221 (35) has not been included. For computation of helical G-C content in RNAse P RNAs from T. thermophilus and E. coli (9), sequences extending the conserved terminal helix (nucleotides 5-15/361-371) and pseudoknot interactions were omitted from the comparative analysis. Potential G-U base pairs at the end of helical segments were neither defined as singlestranded nor helical.

.-

5960 Nucleic Acids Research, Vol. 19, No. 21 the T. thermophilus RNA shows highest similarities to RNAs from y proteobacteria and the fi proteobacterium Thiobacillus ferrooxidans. However, sequences of other subphyla as Thermotoga, the deinococci and the green non-sulfur bacteria will give a more comprehensive set of data to define phylogenetic relations among eubacterial RNase P RNAs. The T. thermophilus RNase P RNA has a G-C content of 86.3% in helical and 53.8% in single-stranded regions, compared to 68.6% and 49.1% in the E. coli RNA (9), without consideration of potential tertiary interactions (Fig. 2). G-U base pairs are reduced in the T. thermophilus RNA (3 versus 8). The total G-C content of the T. thermophilus RNA (71.4%) is similar to the average of approximately 69% determined for chromosomal DNA (37). In E. coli, the overall G-C content of RNase P RNA (61.9%) significantly exceeds that of chromosomal DNA (52%, 38). This supports the suggestion that even in the mesophile E. coli the catalytic RNA subunit requires stabilization

by increased G-C base pairing due to the minimized size of the protein subunit in eubacterial RNase P enzymes. On the other hand, the percentage of G and C in single-stranded regions is very similar (53.8 % versus 49.1 %) and relatively low in both RNAs. Particularly, the total number of adenines is almost identical (63 versus 62) in single-stranded regions of T. thermophilus and E. coli RNase P RNAs, and most of the conserved nucleotides in the single-stranded core regions of RNase P RNAs (33) are also found in the T. thermophilus RNA. As more RNA sequences become available, a distinct set of strictly conserved positions will emerge. These nucleotides, crucial to function as the two adenines at position 241 and 242 (33, Fig. 2), will be appropriate targets for defined mutational and modification studies.

M I 2M0

94

im .30. -

A

1

4

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._

t

a

i

Figure 3. In vitro transcription of the gene coding for the RNA subunit of RNAase P from T. thermophilus using the homologous RNA polymerase (30). The linear DNA fragments (1 and 2) used as templates are shown at the top. (a) . Corresponding transcripts (lanes 1 and 2) were run on 5% polyacrylamide-8M urea gels and visualized by autoradiography. DNA sequencing ladders (lanes M) only yield approximate values of transcript lengths due to incomplete denaturation of the RNase P RNA transcript in 8M urea. (b). A similar transcription assay as shown in Fig. 3a was scaled up tenfold, and the transcript with an apparent length of 370 nucleotides shown in lane 1 of Fig. 3a was excised from the gel, eluted and incubated with a 32P-labeled transcript of the pre-tRNATYr from E. coli (31) at 650C (see Materials and Methods). The fate of the pre-tRNATYr was analyzed on a 8% polyacrylamide-7M urea gel: lanes 3 and 4, the pre-tRNATYr incubated in the absence (lane 3) or presence (lane 4) of the in vitro transcript from the RNase P RNA gene of T. thennophilus; lengths (in bases) of specific cleavage products are marked by arrows next to lane 4; lane M, 5 _32P-labeled DNA fragments (pGEM, Promega) used as size marker.

Figure 4. Nuclease SI mapping of the RNase P RNA gene from T. thermophilus. The size of single-stranded DNA fragments was determined by gel electrophoresis in 6.5 % polyacrylamide-8M urea. (A). For the analysis of the 5'-region, the 475 bp XhoII-BstEI1 fragment (Fig. 1, probe 1), including the proximal 104 nucleotides of the coding sequence, was 5'-end-labeled at the BstEII site (see Materials and Methods), denatured, hybridized to 100 yg of total RNA from T. thermophilus HB8 and digested with nuclease Si. Lanes 1 and 2, probe hybridized at 50°C (lane 1) or 37°C (lane 2). In control lanes 3-5, the probe was incubated with 100 Ag of bulk tRNA from E. coli MRE 600 instead of total RNA from T. thermophilus at 50°C (lanes 3 and 5) or 37°C (lane 4) and subjected to the identical procedure; lane 3, no nuclease SI added; lanes M, dideoxy sequencing ladders used as size markers, which lack, in contrast to the single-stranded probe fragments protected from nuclease SI digestion, the 5'-terminal phosphate; in marker lanes M, lengths of bands (in bases) relevant for size determination were indicated. (B). For the analysis of the 3-region, the 186 bp AvaIl fragment (Fig. 1, probe 2) was labeled by filling in the recessed 3'-ends with Klenow polymerase in the presence of [ce-3 P]dGTP. Lanes 1 - 3, NarI, CfoI and HaeIH digests of the probe used as size markers (in bases); lanes 4 and 5, probe hybridized at 50°C to 100 tsg of total RNA from T. therrnophilus HB8 isolated by the sodium acetate (lane 4) or hot phenol procedure (lane 5); lane 6, control, but omission of nuclease SI; lanes 7 and 8, probe hybridized at 50°C to 40 ,ug RNA (lane 7) or 10 lig RNA (lane 8) transcribed in vitro from the RNase P RNA gene using the homologous RNA polymerase (see Materials and Methods); for in vitro transcription, the recombinant plasmid pMiHB8 (lane 7) or the linear 2.6 kbp BamHI fragment (lane 8) was used as template. As controls for nuclease SI protection analyses shown in lanes 4, 5, 7 and 8, equal amounts of bulk tRNA from E. coli MRE 600 (instead of total RNA from T. thermophilus) were incubated with the probe and subjected to the identical procedure; the corresponding lane, however, was omitted since no unspecific bands of protection were detectable; open arrow next to lane 8, signals of protection by in vitro transcripts.

Nucleic Acids Research, Vol. 19, No. 21 5961 In vitro transcription of the RNase P RNA gene The 2.6 kbp BamHI fragment encoding the RNase P RNA from T. thermophilus, uncut or cleaved with BstEll in the coding region (Fig. 3, upper part, templates 1 and 2) was used for in vitro transcription utilizing the homologous RNA polymerase. Both templates directed the synthesis of discrete transcripts as shown in Fig. 3a. Based on approximate size determinations using dideoxy sequencing ladders (Fig. 3a, lanes M), it was likely that the runoff transcript synthesized from template 2 (Fig. 3a, lane 2) originated from the conjectural promoter found immediately upstream of the gene (Fig. 1). The discrete transcript obtained with the uncut 2.6 kbp fragment (Fig. 3, top, template 1), roughly estimated to be 370 nucleotides in size (Fig. 3a, lane 1), had an approximate length expected for an eubacterial RNase P RNA. The result further suggested that the potential rho-independent terminator identified at the end of the gene functioned in in vitro transcription. To investigate if the full-length transcript derived from the 2.6 kbp fragment (Fig. 3a, lane 1) represented the catalytically active subunit of the T. thermophilus RNase P, a large scale transcription assay (see Materials and Methods) based on the one shown in Fig. 3a, lane 1, was loaded on a 5% polyacrylamide-8M urea gel. The transcript with an approximate size of 370 nucleotides

*.

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IL Ib

0.6

*.

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-

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0.2

-

20

30

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40

Temperature

9X0

70

(CC)

1 2 3 4 5 6 7 8 910 111213 131_

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was visualized by autoradiography, excised from the gel, eluted, desalted on sephadex G-25 chromatography, and precipitated with ethanol. As can be seen in Fig. 3b, lane 4, the RNA was able to cleave a 32P-labeled pre-tRNATYr (31) from E. coli at 65°C, yielding the tRNA with the mature 5'-end, 88 nucleotides in length, and the 5'-precursor fragment (43 nucleotides) as products (arrows next to lane 4). The position of cleavage by T. thermophilus RNase P RNA was verified by enzymatic RNA sequencing (data not shown), and was found to be identical to that of E. coli RNase P RNA (31).

Nuclease Si mapping of the RNase P RNA gene SI mapping studies were performed to identify the in vivo initiation site of transcription, potential processing intermediates, and the site of transcriptional termination. To analyze the 5'-flanking region, the Xholl-BstEll fragment, approximately 475 bp in length, was 5'-end-labeled (Fig. 1, probe 1), denatured, and hybridized at 37°C (Fig. 4A, lane 2) or 50°C (Fig. 4A, lane 1) to 100 ,zg of total RNA isolated from exponentially growing T. thermophilus HB8 cells. The lengths of protected singlestranded DNA fragments were determined by coelectrophoresis of dideoxy sequencing ladders (Fig. 4A, lanes M). Only one signal of protection was observed (Fig. 4A, lanes 1 and 2) which was assigned to the primary transcript initiated five ( one) nucleotides downstream from the -10 box of the promoter. Conclusively, no further 5 '-processing of the primary transcript occurs. For the analysis of the 3'-flanking region, the 186 bp Avall fragment, covering the 3'-portion of the gene, was 3'-end-labeled with Klenow polymerase (Fig. 1, probe 2). The probe was denatured and hybridized at 50°C to 1001tg of two independent preparations of total RNA isolated from exponentially growing T. thermophilus cells (Fig. 4B, lanes 4 and 5), as well as to 40,tg (lane 7) or 10ktg (lane 8) of cold in vitro transcripts of the RNase P RNA gene synthesized by T. thermophilus RNA polymerase. Narn, CfoI and Hae II digests of the probe were used as size markers (Fig 4B, lanes 1-3). With both, RNA isolated from cells (lanes 4 and 5) and RNA transcribed in vitro (lanes 7 and 8), one clustered signal was observed, which could be assigned to transcripts including almost the entire hairpin (Fig. 2). The data give clear evidence that transcription immediately stops as RNA polymerase has traversed the terminator hairpin.

.........

43_

Figure 5. Temperature dependence of pre-tRNATYr cleavage catalyzed by T. thermophilus RNase P RNA. Relative activity denotes the fraction of the maximum cleavage activity obtained at 55°C. At the bottom, one of several sets of cleavage assays performed at different temperatures was chosen to illustrate the data shown at the top; cleavage products were separated on 10% polyacrylamide-8M urea, visualized by autoradiography, excised from the gel and Cerenkov counted; arrows next to lane 1 mark the lengths (in bases) of pre-tRNATYr (131 bases), 5'-matured tRNA (88 bases) and the 5'-precursor fragment (43 bases); the position of cleavage by T. thermophilus RNase P RNA was verified by enzymatic RNA sequencing (data not shown), and was found to be identical to that of E. coli RNase P RNA (31). In lanes 1-3, 5, 7, 9, 11 and 13, approximately 100 ng of pre-tRNATYr were incubated in a volume of 20 Zl for 10 minutes in the presence of 100 ng T. thermophilus RNase P RNA (see Materials and Methods) at 25°C (lane 1), 35°C (lane 2), 45°C (lane 3), 57°C (lane 5), 66°C (lane 7) 74°C (lane 9), 84°C (lane 11) and 94°C (lane 13); lanes 4, 6, 8, 10 and 12, identical conditions as in lanes 5, 7, 9, 11, and 13, respectively, but omission of RNase P RNA.

Table I. Preincubation of cleavage assays at 80°C for 10 min in the absence of RNase P RNA or the pre-tRNATYr substrate.

2

pre-tRNATyr

RNase P RNA

refolding

% activity

+

-

+

100

+

-

-

80.6

+

+

41.6

+

-

5.1

3 4

-

After preincubation samples were transferred on ice immediately (-, no refolding) or allowed to cool down slowly from 80°C to room temperature in a beaker containing 200 ml of water (+, refolding). After preincubation and refolding in the case of samples 1 and 3, the lacking component, i.e. pre_tRNATYr or RNase P RNA, was added, and samples were assayed at 55°C for 10 min, followed by ethanol precipitation, electrophoresis in 10% polyacrylamide-8M urea, autoradiography, excision of cleavage products and Cerenkov counting. Cleavage activity of assay 1 was arbitrarily defined as 100%.

5962 Nucleic Acids Research, Vol. 19, No. 21 The hairpin, possibly lacking a few terminal bases of the stem (Fig. 2), remains part of the RNase P RNA, i.e. no 3'-processing events occur in vivo. In addition, the clustered signal (Fig. 4B, lanes 5, 6, 7 and 8) is indicative of microheterogeneous 3'-ends.

Conditions for catalysis by RNase P RNA For synthesis of large quantities of RNase P RNA from T. thermophilus, the gene was subcloned into the T7 expression vector pT7T318U (see Materials and Methods). Optimal conditions for the cleavage of pre-tRNATYr by T thermophilus RNase P RNA were similar to those determined for the E. coli RNA (39). Assays were performed in 50 mM glycine/NaOH pH 8.0, 100 mM MgCl2, 100 mM NH4Cl, 4% PEG 6000 (Merck) and 0.5% SDS. In the presence of 100 mM NH4Cl and 4% PEG, optimal cleavage efficiency was achieved at 100-200 mM MgCl2. In the presence of 100 mM MgCl2 and 4% PEG, NH4Cl had no stimulatory effect on cleavage. However, a 1.5 to 2-fold stimulation by 100-200 mM NH4Cl was observed in the presence of 100 mM MgCl2 and absence of PEG. Stimulation by PEG at 100 mM MgCl2 and 100 mM NH4Cl was 1.5 to 2-fold at 4 to 10% PEG. Figure 5 shows the influence of temperature on the reaction catalyzed by T. thermophilus RNase P RNA using the E. coli pre-tRNATYr as substrate. Highest cleavage rates are obtained between 55 and 65°C. At 78°C activity totally collapses, which could be due to denaturation of pre-tRNATYr and/or RNase P RNA. Melting temperatures (Tm-values) of several tRNA species, determined at 10 mM Mg++, were reported to be predominantly in the range of 75 -78°C for E. coli species and 84-87°C for equivalent T. thermophilus tRNAs (40). To analyze the structural stability of the catalytic RNA, preincubations at 80°C for ten minutes in the presence or absence of substrate or enzyme were performed (Table 1). Preincubated samples were transferred on ice (Table 1, assays 2 and 4, no refolding) or allowed to cool down slowly from 80°C to room temperature (refolding, assays 1 and 3), and the lacking component was added, followed by incubation at 55°C for ten minutes. As can be seen from values for relative cleavage activities in Table 1, the catalytic RNA, compared to the pre-tRNATYr, undergoes a complicated refolding process after thermal denaturation at 80°C. The data, however, leave the possibility open that denaturation of E. coli pre-tRNATYr occurs as well and therefore influences the temperature dependence of the cleavage reaction (Fig. 5). Catalytic parameters for the cleavage of E. coli pre-tRNATYr by RNase P RNA from T thermophilus at 55°C were determined to be 1.3 AM for Km and 0.4 for keat (min-1) [data not shown]. The Km of 1.3 [tM is higher than values determined with E. coli RNase P RNA at 37°C under similar salt conditions (0.03 and 0.04 ItM, 31 and 36), whereas an identical value for kca, was reported for the E. coli RNase P RNA (31, 36).

DISCUSSION Structure of the gene The RNase P RNA subunit of T. thermophilus is transcribed from a single copy monocistronic transcription unit. No further processing of the primary transcript occurs as shown by nuclease S1 mapping, and the terminator hairpin remains part of the RNA (Fig. 2 and 4). Single copy genes have been reported for eubacterial and eukaryotic RNase P RNAs (7,11, 41, 42). In E. coli and

Salmonella typhimurium, transcription as well starts with mature 5'-ends (42, 43). However, evidence was presented that two additional promoters located more upstream direct some synthesis of 5'-precursor molecules of RNase P RNA in E. coli, implicating a 5'-processing pathway for the primary transcript in this organism (43). Transcription terminates at a rho-independent terminator, but, in contrast to T. thermophilus RNase P RNA, approximately 40 nucleotides have to be removed from the primary transcript to yield mature RNase P RNA. This indicates that a processing activity is involved in 3'-end maturation of E. coli RNase P RNA (43, 44). The RNase P RNA gene is a further example that documents the tendency in T. thermophilus to reduce precursor sequences and processing events in genes encoding stable RNAs as ribosomal RNAs (45, 46) or 4.5S RNA (47). In E. coli and Salmonella typhimurium, a potential open reading frame (ORF) is located immediately downstream from the terminator (42, 44). In addition, DNA repeat structures, approximately 110 nucleotides in size, are reiterated 3.5 in E. coli (44) and 4.5 times in S. typhimurium (42) in the downstream regions of RNase P RNA genes. The repeats begin with the last 23 nucleotides of the coding regions and include the terminator sequences. In E. coli, several potential overlapping open reading frames for small basic proteins are found within these repeats (44). In Bacillus subtilis, a potential 80-amino acid ORF is located approximately 40 nucleotides downstream from the terminator of the RNase P RNA gene, overlapping with a second ORF which starts approximately 230 base pairs distal to the terminator (48). Inspecting the T. thernophilus RNase P RNA gene for potential open reading frames and similar repeat structures, we found one potential reading frame 61 nucleotides downstream from the uridine cluster following the terminator hairpin (data not shown). The reading frame is not interrupted by a stop codon within the first 126 nucleotides of sequence determined hitherto. No extended repeat structures, as found in 3'-regions of RNase P RNA genes of E. coli and Salmonella typhimurium, could be detected in the downstream region of the RNase P RNA gene of T. thennophilus. One common theme of the hypothetical open reading frames in 3'-regions of RNase P RNA genes from T. thermophilus, E. coli and B. subtilis is an elevated content of lysine and arginine residues.

Mature 5'- and 3'-ends Based on the data shown in Fig. 4B, the major 3'-end was assigned to G392 (Fig. 2) located in the lower part of the terminator stem, lacking the last two cytidines of the stem and the uridine cluster following the hairpin. The result indicates that transcritption authentically stops in vivo and in vitro at this nucleotide (and possibly at the two preceding and following positions), but leaving the uridine cluster untranscribed. The possibility cannot be excluded, however, that transcription virtually stops at the uridine cluster, but some nibbling by nuclease SI occured due to a melting of the RNA-DNA hybrid at the terminal U-A rich region. A trimming of the 3'-end by 3'-exonucleases in vivo seems unlikely since identical signals of protection were obtained with total RNA from T. thermophilus and RNA synthesized in vitro with purified RNA polymerase from T. thermophilus (Fig. 4B, lanes 4, 5, 7 and 8). Nevertheless, the appending terminator hairpin in T. thermophilus RNase P RNA, which is unlikely to have an effect on activity of RNase P RNA (33, 49), might provide some protection against 3'-exonucleases in vivo.

Nucleic Acids Research, Vol. 19, No. 21 5963 Structure and function of the catalytic RNA With the T. thermophilus RNase P RNA, highest cleavage rates were observed between 55 and 65°C, but activity is completely abolished at 78°C (Fig. 5). Using identical conditions, we determined a temperature optimum of 40°C for the E. coli RNase P RNA, and activity was insignificant above 50°C. This reflects the more compact structure of the T. thermophilus RNA due to increased G-C base pairing, which might include stabilizing tertiary interactions in addition to the pseudoknots shown in Fig. 2, which are also valid for the E. coli RNA. Since the T thermophilus RNA subunit alone denatures at 80°C (Table I), a temperature which still allows growth of T. thermophilus, the precise role of the protein subunit in stabilizing the active conformation of the RNA subunit has to be determined. It has to be considered, however, that the high temperature for optimal cleavage efficiency by RNase P RNAs from T. thermophilus might not only reflect an optimal folding ofthe RNA between 55 to 65°C, but may also result from a better product release at higher temperatures, especially in view of the relatively high Km-value of 1.3 mM determined at 55°C (see Results section). A kinetic analysis of the reaction catalyzed by RNase P RNA from B. subtilis (48) showed that the relatively slow overall catalytic rate (kcar) of the RNA alone reaction compared to catalysis by the holoenzyme is caused by a decreased dissociation of cleaved tRNA from RNase P RNA in the absence of the protein subunit. As a consequence, the RNase P RNA reaction, in contrast to the reaction catalyzed by the holoenzyme, is strongly inhibited by mature tRNA (48). It was therefore proposed that the small basic protein decreases anionic repulsion between RNA enzyme and substrate at the substrate binding site, which is compensated for by higher cation concentration in the RNA alone reaction. However, a mere electrostatic role of the protein subunit seems questionable since a better substrate binding due to decreased anionic repulsion should also lead to slower dissociation of the product (cleaved tRNA) provided that only features of mature tRNA are recognized by RNase P. However, the holoenzyme is not inhibited by the product, i.e. 5'-matured tRNA (48). Conceivably, the protein might induce and/or stabilize a conformational switch in the catalytic RNA following high affinity substrate (pre-tRNA) binding that leads to low affinity binding of tRNA and therefore rapid product release after cleavage. The conformational change occurs, independenly if cleavage takes place or not in the case of bound pre-tRNA or bound 5'-matured tRNA. After dissociation of tRNA from the enzyme, the high affinity binding site is restored. The conformational switch might not occur or might be less defined in the RNA alone reaction, thus causing decelerated product dissociation due to constant high affinity binding of the tRNA moiety. The model would explain why Km values for the reactions catalyzed by RNA alone and holoenzyme are similar, although product release and therefore kcat is severely reduced in the RNA alone reaction (31, 50).

ACKNOWLEDGEMENTS Financial support for these studies from the Deutsche Forschungsgemeinschaft (SFB 9/B5 and SFB 344/C2, and Gottfried Wilhelm Leibniz-Programm) and the Fonds der Chemischen Industrie e.V. is acknowledged. We thank Sabine Schultze for the synthesis of oligonucleotides, Dr. Cecilia Guerrier-Takada for providing the plasmid pUC19TyrT, Anita

Marchfelder for performing the cleavage reaction, gel electrophoresis and autoradiography shown in Figure 3b, Holger Ebeling and Angela Schreiber for the photographic work, Judith Schlegl for preparation of RNA polymerase from T. themiophilus and Drs. Rolf Bald and Jens Peter Fuirste for inspiring discussions.

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