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Volume 270, Number 26, Issue of June 30, pp. 15908-15914, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Maturation of Pre-tRNA by Escherichia coli RNase P Is Specified by a Guanosine of the 5`-Flanking Sequence (*)

Thierry Meinnel (§) , Sylvain Blanquet

From the (1)Laboratoire de Biochimie, Unité de Recherche Associée 1970, CNRS, Ecole Polytechnique, F-91128 Palaiseau Cédex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The C/A base pair at the top of the acceptor stem of Escherichia coli tRNA accounts for several of the specialized roles of this tRNA in translation initiation. According to the rules of RNA substrate recognition by RNase P, the C/A pair is likely to disfavor the 5`-maturation of pre-tRNA. Indeed, in contrast to other E. coli tRNA species, tRNA was not properly matured when overproduced from a multicopy expression vector. Half of the recovered tRNA retained an extension at the 5` side. Such a defect of tRNA processing could be cured by changing bases C and A by a Watson-Crick base pair or by non-paired bases, provided one of them was a G. It could also be compensated by either (i) overexpression of RNase P or (ii) introduction within the plasmid of one out of the three 5`-flanking sequences naturally occurring in the four E. coli tRNA genes. The effect of these flanking sequences on the maturation of tRNA could be accounted for by the presence of a G located 2 bases upstream from C. Notably, this G is the only residue that is conserved in the 5`-flanking sequences of all four E. coli tRNA genes.

INTRODUCTION

The biosynthesis of functional tRNAs involves a series of post-transcriptional events, including maturation and modification processes. Several dozen tRNA modifications have already been described (Sprinzl et al., 1989). Although the role of most modifications remains unknown, some of them are involved in the recognition by aminoacyl-tRNA synthetases (Muramatsu et al., 1988; Sylvers et al., 1993). Maturation events include the processing of the 5` and 3` ends of pre-tRNAs in all organisms as well as pre-tRNA splicing and editing in eukaryotes (see Deutscher(1990) for a review on the processing of the 3` end).

The 5` maturation of tRNAs depends on the action of a single and ubiquitous enzyme, RNase P (see Altman(1989, 1993) and Pace and Smith (1990) for reviews). In Escherichia coli, this enzyme (EC 3.1.16.5) is composed of a 377-base RNA moiety (M1 RNA) and of a 14-kDa protein subunit (C5 protein) encoded by the rnpB and rnpA genes, respectively. Although both genes are essential in vivo, the RNA moiety is active in vitro in the absence of the C5 protein (Guerrier-Takada et al., 1983). The regions of immature tRNAs influencing RNase P recognition are the acceptor stem, the CCA end, and the T-stem and loop (Carrara et al., 1989; Forster and Altman, 1990; Holm and Krupp, 1992; Kahle et al., 1990; Kirsebom and Svärd, 1992; Krupp et al., 1991; McClain et al., 1987; Reilly and RajBhandary, 1986; Svärd and Kirsebom, 1992, 1993; Thurlow et al., 1991). The position of cleavage of a pre-tRNA by RNase P is directed by the occurrence of a G at position +1 of the mature tRNA, as guiding nucleotide, and by the lengths of the acceptor and T-stems, which play the role of rulers setting the distance between this G and the T-loop. The absence of a G at position +1 and the modification of the length of the ruler were shown to cause aberrant cleavage of a pre-tRNA (Svärd and Kirsebom, 1992, 1993). In the case of the maturation of E. coli tRNA and tRNA, whose acceptor stems are one base pair longer, it was found that, in addition to the above determinants, the nature of base +73 was important for the kinetics of cleavage (Burkard et al., 1988; Green and Vold, 1988; Kirsebom and Svärd, 1992; Krupp et al., 1991). Interestingly, E. coli tRNA is characterized by the absence of a G and by the occurrence of a C/A unpairing. However, the structural motifs enabling specific pre-tRNA maturation have not yet been described.

This work establishes that, in E. coli, a G at position -2 in the 5`-flanking sequence of pre-tRNA compensates for the negative effect caused by the unusual C/A unpairing on the maturation process.

EXPERIMENTAL PROCEDURES

Materials

All chemicals were from Merck. Enzymes were purchased from Boehringer Mannheim, except T4 DNA ligase, which was from Life Technologies, Inc. Buffers used were those indicated by the suppliers.

E. coli isoleucyl-, leucyl-, lysyl-, methionyl- and valyl-tRNA synthetases and methionyl-tRNA formyltransferase were from the laboratory's stock. Oligonucleotides (18-38-mers) were synthesized on a Pharmacia Gene Assembler and purified by anion-exchange chromatography (Mono Q, Pharmacia Biotech, Inc.).

Synthesis of tRNA Genes

tRNA numbering is as follows. (i) The first base in 5` of the matured tRNA is base +1 and the last is base +76; (ii) the bases belonging to the 5`-flanking sequence are numbered from -1 to -9. Base -1 is located immediately upstream from base +1 (see Fig. 1). tRNA species are named as follows. (i) The nature of the 5`-flanking sequence is indicated immediately after the name of the tRNA; (ii) a mutated position in the sequence of a pre-tRNA is indicated by the substituting base followed by its position. For instance, tRNA (U) stands for a tRNA in which the transcribed sequence upstream from base +1 is that of the metZ gene (see Fig. 1B) with position -2 replaced by a U (see Fig. 1B for the numbering of the positions upstream from base +1 in the case of the different sequences). The label ``fasm'' designates a tRNA that has been given the acceptor stem of tRNA (see Guillon et al., 1992b; Meinnel et al., 1993).

Figure 1: Construction of tRNA genes with different 5`-flanking sequences. PanelA, pBStRNA gene was restricted in the presence of XhoI and SacII. Six oligonucleotides (numbered 1-6) were ligated together and inserted within the two corresponding restriction sites of pBStRNA. The directions of the oligonucleotides are indicated. The -35 and -10 sequences of the whole lpp promoter (underlined on top) and the first transcribed nucleotide (boldface and indicated with a verticalarrow) are shown. The XhoI-SacII fragment of the original pBStRNA with the EcoRI restriction site immediately upstream from the SacII site is indicated. To produce different 5`-flanking sequences, both overlapping oligonucleotides 3 and 4 were varied. Upon this step, both the EcoRI and SacII sites usually disappeared. The sequence of tRNA itself was invariant. PanelB, the numbering of each base is indicated. The five 5`-flanking sequences used throughout this work are boldface. The five first 5` nucleotides of matured tRNA are also indicated in one case. The restriction sites are boxed. The L sequence corresponds to that of the original pBStRNA vector. The Y, Z, or Z sequences are those that are transcribed from the E. coli metY, metZ, and metZ genes, respectively. The S sequence with the newly created SacII restriction site is also indicated. The consensus sequence derived from these four sequences (Y, Z, Z, or S) is shown at the bottom of the figure. N means A, C, G, or T; S means C or G; R means A or G.


tRNA, tRNA, tRNA, tRNA, tRNA, and tRNA genes and their derivatives were constructed by assembling six overlapping oligonucleotides as described (Meinnel et al., 1988). tRNA genes were ligated between the EcoRI and PstI sites of the pBSTNAV2 expression vector (Meinnel et al., 1992). To modify the 5`-flanking sequence of pre-tRNAs, a specialized shuttle vector, pBStRNA, was constructed. This vector features (i) a deletion of the XhoI-SacII fragment of pBStRNA (this deletion overlaps the lpp promoter, the 5`-flanking sequence and the nucleotides +1 and +2 of the tRNA gene, see Fig. 1A) and (ii) a SmaI restriction site inserted between XhoI and SacII. To construct the different variants, six oligonucleotides corresponding to the lpp promoter and to the desired flanking sequence (see Fig. 1A) were ligated together and cloned between the XhoI and SacII restriction sites of pBStRNA. The sequence of the six oligonucleotides and of several sequences introduced upstream from base +1 are shown in Fig. 1. tRNA gene sequences were verified by dideoxy-sequencing of single-stranded DNA obtained by using the R-408 helper phage (Stratagene, La Jolla, CA). The XhoI-BamHI fragment of pBStRNA was also cloned between the SalI and BamHI sites of vector pUC19, yielding pUCtRNA. In addition, the XhoI-HindIII fragment of pBStRNA was cloned between the SalI and HindIII sites of vector pBR322, yielding pBRtRNA.

tRNA Purification

Freshly transformed bacteria were grown for 20 h at 37 °C in 1 liter of 2 TY medium containing 50 mg of ampicillin. The cultures were harvested by centrifugation and resuspended in 8.5 ml of buffer A (1 mM Tris-HCl (pH 7.4), 10 mM magnesium acetate). 10 ml of phenol previously saturated with buffer A were added, and the tube was vigorously shaken for 30 s. The mixture was then centrifuged for 1 h at 12,000 rpm at 20 °C (JA17 rotor; Beckman). Thereafter, the aqueous phase was collected and ethanol-precipitated. The precipitate was resuspended in 4 ml of 1 M NaCl and spun for 30 min at 10,000 rpm in a JA17 rotor. The supernatant was ethanol-precipitated, resuspended in 4 ml of 2 M Tris-HCl (pH 8) and left for 2 h at 37 °C. After ethanol precipitation, the pellet was dissolved in 2 ml HO. Usually, 30-50% of such RNA preparations corresponded to the overproduced tRNA, i.e. bulk tRNA preparations accepted 500-800 pmol of amino acid/A unit.

tRNA variants were further purified on a Q-Sepharose column (1.6 20 cm; Pharmacia). The tRNA extract in 10 ml of buffer B (20 mM Tris-HCl (pH 7.6), 8 mM MgCl, 0.1 mM EDTA, 0.2 M NaCl) was loaded onto the column previously equilibrated in the same buffer; thereafter, a 0.36-0.48 M NaCl gradient in buffer B (2.5 ml/min; 0.10 M/h) separated the tRNAs. Collected fractions were assayed for tRNA by measuring the incorporation of a radioactive amino acid (Meinnel et al., 1993). Formylation assays were as described previously (Blanquet et al., 1984; Guillon et al., 1992b). Fractions of interest were then pooled and ethanol precipitated. The pellet was redissolved in 0.2 ml of HO and stored at -20 °C. 10-20 mg of pure tRNA were routinely recovered from a 1-liter culture. Amino acid acceptances of the final tRNA preparations ranged from 1,350 to 1,650 pmol/A unit.

Construction of Vectors Overexpressing the RNase P Holoenzyme

The rnpA region was amplified from chromosome DNA of strain K37 (Miller and Friedman, 1980) with the help of two oligonucleotides bordering the rnpA gene. The first one, 5`-CGTTCTCACGGGATCCGTGCTCGT created a BamHI restriction site at the 5` side of the gene. The second one, 5`-TCAATTCCGTATCTAGAACAGGTTG introduced a XbaI restriction site at the 3` end of the gene. The 570-base pair amplified fragment was then restricted in the presence of BamHI and XbaI and introduced within pUC18 under the control of the lac promoter, yielding pUCrnpA. The nucleotide sequence of the resulting rnpA gene was verified. In contrast to control plasmid pUC18, pUCrnpA relieved the thermosensitivity of strain N2020 (rnpA49,trpA36,argA52,glyA34, rpsL,lacZ,ValR) (a kind gift of B. Bachmann, Genetic Stock Center, Yale University, New Haven, CT; see also Schedl and Primakoff(1973) for the description of the rnpA49 allele) as measured at 44 °C on LB plates supplemented with 0.5 mM isoproyl-1-thio--D-galactopyranoside and 50 µg/ml ampicillin.

rnpB was amplified by using the following two oligonucleotides. The first one, 5`-CGATTGGAATTCGCAACGCGG, was complementary to a sequence located just upstream from the -35 region of the promoter of the rnpB gene and created an EcoRI restriction site. The second oligonucleotide, 5`-TGAAACTGATGGCAACCGCAAAAATGC, was complementary to a region located immediately after the StuI restriction site downstream to the rnpB gene. The DNA of 515 (a kind gift of O. Fayet, LMGM, Toulouse, France), a bacteriophage of the Kohara library (Kohara et al., 1987), which contains a single copy of the rnpB gene (Komine and Inokuchi, 1991), was chosen as template. The 950-base pair amplified fragment was then hydrolyzed in the presence of EcoRI and StuI and cloned within the EcoRI and EcoRV sites of pBluescript SK+, yielding pBSrnpB. The whole sequence of the rnpB gene was verified. In contrast to pBluescript SK+, pBSrnpB allowed strain N2020, which has a rnpA(Ts) phenotype (see above), to grow at 44 °C on LB plates containing 50 µg/ml ampicillin. Since an excess of wild-type M1 RNA is able to cure the effect of the rnpA49 mutation (Motamedi et al., 1984), this experiment demonstrated that vector pBSrnpB indeed overexpressed this RNA.

To construct a pUC derivative overexpressing both the C5 protein and the M1 RNA, the XbaI-SalI fragment of pBSrnpB, which has the rnpB gene under the control of its own promoter, was cloned between the same sites of pUCrnpA, yielding pUCrnpAB. This vector was able to complement the N2020 strain at 44 °C. Finally, the SphI-NdeI fragment of pUCtRNA, which contains the tRNA gene under the control of the lpp promoter (see above) was cloned in pUCrnpAB, yielding pUCrnpABtRNA. This vector overexpressed the M1 RNA and C5 protein components of RNase P as well as tRNA (see Fig. 2).

Figure 2: Construction of a vector overexpressing both the rnpA and rnpB gene products and tRNA as well. The figure is not to scale. The three genes (rnpA, rnpB, and tRNA) are boxed, and the directions of their transcription are indicated. The three promoters (Pr) are indicated with verticalarrows. The transcription terminator (TrrrnC) located immediately downstream to the tRNA gene is shown. The main restriction sites are indicated with arrows and labeled just beyond. The cloning vector was pUC18.


Analysis of tRNA Processing

tRNA (0.25-0.5 pmol in 0.5-1 µl) was mixed with 5 µl of a denaturing buffer containing 7 M urea, 10 mM EDTA, 10% glycerol, 0.05% bromphenol blue, and 0.05% xylene cyanol. The mixture was boiled for 1 min, chilled on ice, and loaded on a 6% polyacrylamide denaturing sequencing gel (0.8-mm thickness, 50-cm length) previously preheated at 50 °C. After a 3-h run at 1,500 V, the part of the gel containing tRNA was soaked for 30 min in 0.1 mg/liter ethidium bromide. Gel stainings were recorded with an imager (Appligene). Reverse images were stored on disk (TIFF format) and further processed with Image software (version 1.49, Macintosh).

Promoter Mapping by Primer Extension

1 µg of DNA from plasmid pBStRNA was incubated for 15 min at 37 °C with 4 units of E. coli RNA polymerase for in vitro transcription in the conditions indicated by the supplier (Boehringer Mannheim). The reaction was quenched by the addition of 50 mM EDTA, phenol/chloroform extraction, and ethanol precipitation. For primer extension analysis (see Lévque et al.(1991), the 31-mer oligonucleotide 5`-GTGGTTGCGGGGGCCGGATTTGAACCGACGA-3`, corresponding to the sequence starting 47 bases downstream from the EcoRI restriction site located just before the first base (+1) of the tRNA, was used (Fig. 1). Two picomoles of this oligonucleotide were 5`-labeled with 0.37 MBq of [-P]ATP (370 MBq/ml and 111 MBq/mmol; DuPont NEN). The P-labeled extension products were analyzed on a 6% polyacrylamide sequencing gel. A nucleotide sequence performed with the same oligonucleotide as primer and pBStRNA double-stranded DNA as the template was run in parallel.

RESULTS

tRNAExpressed in Vivo from the pBStRNA Vector Is Incompletely Matured at the 5` Side

Derivatives of the pGFIB plasmid, a vector allowing overexpression downstream to the lpp promoter (Masson and Miller, 1986), have been successfully used in the course of producing many wild-type or mutant tRNAs in E. coli cells (see for instance Meinnel et al.(1988, 1992, 1993) and Normanly et al.(1986)). In the case of pBSTNAV2, the tRNA genes could be inserted between the EcoRI and PstI sites (Meinnel et al., 1988, 1992). Transcription was likely to start 3 nucleotides upstream from the EcoRI site (Fig. 1A) and to stop immediately downstream to the PstI site at the rrnC transcription terminator. It is noteworthy that a vector expressing tRNA species with an EcoRI site at the same position was used in several studies dealing with the enzymatic properties of RNase P (see the pAT derivatives used in Guerrier-Takada and Altman(1993) and McClain et al.(1987)).

The extent of E. coli tRNA overexpression from vector pBSTNAV was of the order of that routinely observed with other tRNAs, i.e. methionine acceptance of the crude tRNA extract was of about 600 pmol/A unit. The overproduced tRNA species was easily purified by anion-exchange chromatography, and pure species could be obtained, as estimated by the measurement of the formylmethionine acceptance (1, 650 pmol/A unit). However, upon analysis by denaturing PAGE, 5`-labeled tRNA reproducibly showed two major bands. The same two bands could be detected by ethidium bromide staining, while only one band was observed with either tRNA, tRNA, tRNA, tRNA, or tRNA prepared with the same method (Fig. 3).

Figure 3: Analysis of the extent of steady-state maturation of several E. coli tRNAs and of variants of these tRNAs modified in the acceptor stem. tRNAs were produced in vivo from the corresponding pBStRNA vectors. Purified tRNAs were separated by polyacrylamide gel electrophoresis. The gel was then stained with ethidium bromide and analyzed on an imager. Lane1, tRNA; lane2, tRNA; lane3, tRNA; lane4, tRNA C/A; lane5, tRNA; lane6, tRNA; lane7, tRNA (C /A); lane8, tRNA; lane9, tRNA; lane10, tRNA; lane11, tRNA; lane12, tRNA.


Large amounts of the two tRNA subspecies were prepared by PAGE.()Surprisingly, both species shared the same Michaelis-Menten parameters in the reactions catalyzed by E. coli methionyl-tRNA synthetase or methionyl-tRNA formyltransferase (data not shown). According to enzymatic RNA sequencing (Kuchino and Nishimura, 1989) and analysis of the modified nucleosides (Kuchino et al., 1987), both tRNA species had the expected sequence and modifications (data not shown). Sequencing was also monitored by means of reverse transcriptase (Hahn et al., 1989) to solve the last bases at the 5` side (data not shown). The most rapidly migrating species had the correct 5`-sequence, while the other one showed a 5` extension of 3 bases, 5`-UUC-3`.

The Defect in tRNAMaturation Is Compensated by an Overexpression of RNase P in Vivo

The additional UUC trinucleotide, which matched the sequence of the EcoRI restriction site at the 5` side of the tRNA gene (see Fig. 1B), could result from an alternative transcription start point at the level of base U. To probe this idea, pre-tRNA was transcribed in vitro by using pBStRNA as the template. Its 5` end was mapped by a primer extension experiment (Fig. 4). A single start (G), which exactly corresponded to that of the natural lpp promoter (Nakamura and Inouye, 1979), was found. This demonstrated that the expected homogeneous pre-tRNA was actually produced, at least in vitro.

Figure 4: Mapping of the transcription start of the tRNA gene expressed from plasmid pBStRNA. Transcription of pBStRNA was performed in vitro. RNA products were then incubated in the presence of a 5`-labeled oligonucleotide complementary to tRNA in the presence of reverse transcriptase and of dNTPs and finally hydrolyzed with RNase A. Primer extension products were then analyzed on a sequencing gel (laneE) run in parallel with a sequencing reaction performed with the above oligonucleotide as primer and pBStRNA as DNA template (lanesT, C, G, and A). The sequencing gel was exposed for 3 h to an x-ray film. The resulting autoradiography was then analyzed on an imager.


A second possibility was that the produced pre-tRNA was not a specific and/or efficient substrate of RNase P. Aberrant cleavage sites of several other pre-tRNAs have already been observed in vitro. However, the immature longer tRNAs were believed to still be substrates of RNase P (Burkard et al., 1988; Kirsebom and Svärd, 1992, 1993). To probe whether an increase of RNase P concentration could improve the extent of 5` maturation of tRNA, the two components of RNase P as well as tRNA under the control of the lpp promoter were overexpressed from pUCrnpAB-tRNA (see ``Experimental Procedures'' and Fig. 2for details of the construction). The extent of tRNA overproduction in JM101Tr cells from pUCrnpAB-tRNA was identical to that from pBStRNA (600 pmol/A unit) but smaller than that obtained from pUCtRNA (800 pmol/A unit). Comparison by PAGE analysis of each purified tRNA showed that the immature tRNA no longer accumulated when RNase P was overexpressed (Fig. 5). Clearly, the extended tRNAs could be recognized and cleaved by RNase P.

Figure 5: Influence of an increase of the in vivo steady-state of RNase P on the extent of tRNA maturation. tRNAs expressed from different overproducing plasmids were purified and analyzed by PAGE as described in Fig. 4. tRNA products originated from pBStRNA (lane1), pUCtRNA (lane2), pUCrnpAB-tRNA (lane3), or pBRtRNA (lane4).


This experiment suggested that, reciprocally, the relative extent of maturation of pre-tRNAin vivo might be improved, provided its expression was lowered. tRNA was therefore expressed under the control of the lpp promoter from pBR322, a lower copy number plasmid than pBSTNAV2. The relative amount of tRNA in a crude tRNA extract obtained from cells harboring plasmid pBRtRNA corresponded to 120 pmol/A unit, as compared to 30 pmol/A in the case of the control pBR322 plasmid. tRNA originating from pBRtRNA was nearly fully matured. However, a minor species containing one additional base was still observed (Fig. 5). This result also favored the idea that the relative intracellular concentrations of pre-tRNA and of RNase P influenced the extent of maturation of tRNA. To know whether immature tRNA could also be detected in non-overproducing cells, a crude tRNA extract was prepared from strain JM101Tr, analyzed by PAGE, transferred onto nitrocellulose, and hybridized with a P-labeled oligonucleotide probe specific for tRNA. A single band was observed on the autoradiography (data not shown), thus establishing that immature tRNA did not accumulate under physiological conditions.

The 1-72 Base Pair of tRNA Directly Influences the 5` Maturation in Overproducing Cells

Because the other tRNAs studied under identical conditions (Fig. 3) were correctly matured, the cause of the defect of maturation of tRNA had to be searched in its nucleotide sequence. The sequence of the acceptor stem of the pre-tRNA, which plays a role in the processing by RNase P (see references in the Introduction), could be suspected. Four tRNA chimeras expressed from pBStRNA were therefore studied. All four tRNAs shared the acceptor stem of tRNA in common, but the rest of the nucleotide sequence was that of tRNA, tRNA, tRNA, or tRNA. In each case, a defect in maturation similar to that observed with tRNA was obtained (Fig. 3).

Because the acceptor stem of tRNA has the 1-72 unpairing as special feature, the genes of tRNA and tRNA were given the C/A pair of tRNA. The resulting tRNA species exhibited a defect in maturation (Fig. 3). In a second set of experiments, all 16 possible combinations of bases were created at positions +1 and +72 of the tRNA gene carried by vector pBStRNA. In each case, the extent of overexpression was similar to that measured with wild-type tRNA. However, full maturation could only be observed when a base pairing had been created between base +1 and base +72 (CG, AU, GC, GU, UA, and UG), or when a G occurred at either position (AG, GG, or GA) (Fig. 6). tRNA species with 5`-extensions of 2 or 3 bases occurred in the other cases (CA, CC, CU, AA, AC, UC, UU). Notice that the extent of processing was particularly reduced in the case of the variants with CU, UC, or UU. In the case of the UC variant, a species with a deletion of base +1 was also produced (Fig. 6).

Figure 6: Influence of bases +1 and +72 of tRNA on its maturation in vivo. Variants of tRNA with different nucleotides at positions +1 and +72 were produced from plasmid pBStRNA and analyzed by PAGE as described in Fig. 4. The bases at position +1 and +72 for each lane are as follows. Lane1, C/A (corresponds to wild-type tRNA); lane2, C/C; lane3, C/G; lane4, C/U; lane5, A/A; lane6, A/C; lane7, A/G; lane8, A/U; lane9, G/A; lane10, G/C; lane11, G/G; lane12, G/U; lane13, U/A; lane14, U/C; lane15, U/G; lane16, U/U.


The Base Composition of the 5`-Flanking Sequence Influences the 5` Maturation of tRNA

The availability of a system measuring the extent of tRNA maturation allowed us to search for the motifs ensuring the most efficient processing of tRNAin vivo. In particular, since the 5`-flanking sequence proved important in the maturation of several pre-tRNA species, changes were carried out in the 5`-flanking sequence of pre-tRNA.

In E. coli, four tRNA genes (metY, metZ, metZ, and metZ) occur, with three different 5`-flanking sequences (the two genes metZ and metZ share the same sequence; Ishii et al., 1984; Kenri et al., 1994). We first substituted the 5`-flanking sequence in pBStRNA (called L) by each of the three wild-type sequences, yielding pBStRNA, pBStRNA, and pBStRNA (see the sequences of the corresponding constructions in Fig. 1B). In each case, a fully homogeneous mature tRNA was produced. Clearly, the extent of maturation did not depend on the length of the introduced 5`-flanking sequence, since full processing occurred similarly with 5 (Z), 8 (Y), or 9 (Z) additional bases. To evidence, if any, the nucleotide(s) favoring the in vivo maturation of tRNA, bases -5 to -1 in the Y sequence were systematically substituted by those found in the L sequence (A, A, U, U, or C). Analysis of the corresponding tRNA (Fig. 7A) showed that an incomplete maturation occurred only upon the substitution of base G by U. The same conclusion could be reached in the context of the Z sequence. We also substituted base G of the Y and Z sequences by a C. Incomplete maturation was observed in either case. However, when an A was introduced at position -2 of the Y, Z, or Z sequences, full processing occurred. Clearly, a pyrimidine at position -2 disfavored the maturation.

Figure 7: Influence of the 5`-flanking sequence on the maturation of tRNA in vivo. tRNA were analyzed by PAGE as described in Fig. 4. The nomenclature of the different 5`-flanking sequences is defined under ``Experimental Procedures'' as well as in Fig. 1B. PanelA, lane1, tRNA; lane2, tRNA (C); lane3, tRNA (A); lane4, tRNA (C); lane5, tRNA (U); lane6, tRNA (U); lane7, tRNA (U); lane8, tRNA (A); lane9, tRNA (C); lane10, tRNA (C); lane11, tRNA (A); lane12, tRNA (U); lane13, tRNA (C/U); lane14, tRNA; lane15, tRNA (A); lane16, tRNA. PanelB, lane1, tRNA; lane2, tRNA (G); lane3, tRNA (U); lane4, tRNA (A); lane5, tRNA (G); lane6, tRNA (G); lane7, tRNA (C); lane8, tRNA (G); lane9, tRNA (U); lane10, tRNA (A); lane11, tRNA (U); lane12, tRNA (U/U); lane13, tRNA (G); lane14, tRNA (C/G); lane15, tRNA.


To further evidence the role of the 5`-flanking bases in the maturation process, each of the first 6 bases of the L sequence (bases -1 to -6) were systematically substituted by the corresponding ones of Y. The resulting tRNAs are shown in Fig. 7B. Full maturation could only be obtained upon replacement of base -2 by a G or of base A by a C. Incomplete maturation still occurred with an A, or with G or U at position -4. Clearly, base G appeared to be of utmost importance for the maturation. Interestingly, this base is the only one that is conserved in the 5`-flanking sequences of all E. coli tRNA genes.

One possibility to explain the importance of G was that it could pair with C of pre-tRNA, compensating thereby for the atypical 1-72 pair. To probe this idea, bases U, G, U and G were separately introduced in the context of pBStRNA to create base pairings U/A, C/G, G/C, or U/A. The four resulting tRNA species were incorrectly processed (Fig. 7B). This demonstrated that the creation of an additional base pairing in the region of the acceptor stem was not enough to correct the defect of maturation of tRNA. Finally, because a guanosine at position -2, +72, or +1 appeared to guide the cleavage position of tRNA, we also assessed the influence of the introduction of a G in the L flanking sequence. The corresponding tRNA showed however incorrect maturation (Fig. 7B).

To finally determine whether the occurrence of G alone was enough to direct the full maturation of tRNA, a pBStRNA derivative with a 5`-flanking sequence sharing as little resemblance as possible with the ones already studied, i.e. L, Y, Z, and Z, was synthesized. This sequence, called S (see Fig. 1B), was given a G plus an unique restriction site appropriate for further cloning of derivatives of tRNA. The chosen restriction site was SacII with the last G of the site at position -1. Purified tRNA expressed from pBStRNA could be shown to be fully matured, thus confirming the crucial role of G in the maturation of pre-tRNA.

DISCUSSION

The most recent conclusions dealing with the specificity and efficiency of E. coli RNase P in the removal of the flanking sequence of a pre-tRNA involve (i) the occurrence of a G at position +1, (ii) the integrity of the CCA 3` end, (iii) the sequence of the T-loop, and (iv) the number of base pairs in the acceptor and T-stems (see Altman(1989, 1993) and Svärd and Kirsebom(1992, 1993), and references therein). In this context, the maturation of pre-tRNA deserved interest. This tRNA species is indeed featured by a C at position +1 and, in addition, C is faced by A, which causes thereby a reduction of the length of the acceptor stem helix. Such a feature supports at least two critical functions of tRNA in the initiation step of translation, i.e. the capacity of methionyl-tRNA to be formylated (Guillon et al., 1992a, 1992b, 1993; Lee et al., 1991) and the resistance of formylmethionyl-tRNA to cleavage by peptidyl-tRNA hydrolase (Dutka et al., 1993; Schulman and Pelka, 1975).

According to the rules that govern the processing of a tRNA by RNase P, two possibilities could be a priori considered for the maturation of pre-tRNA. Either the yield of processing of pre-tRNA is constitutively smaller than that of the other pre-tRNAs or some features along the sequence of pre-tRNA can compensate for the atypical C and A bases and for the absence of G. In this context, it is noteworthy that incompletely processed tRNA cannot be detected in E. coli cells.

In this report, we took advantage of the incomplete maturation of tRNA overexpressed from the lpp promoter to search for the nucleotides governing the efficiency of 5` maturation of this peculiar tRNA. It is shown that the absence of pairing between bases +1 and +72, as well as that of a G at either position, are indeed responsible for the accumulation of immature tRNAin vivo. This conclusion is in good agreement with the aforementioned model of substrate recognition by RNase P. Furthermore, the extent of maturation of tRNA can be improved by either an increase of the intracellular concentration of RNase P or a lowering of the concentration in pre-tRNA. The defect in maturation can also be overcome if any of the three genuine 5`-flanking sequences of E. coli tRNA (called Y, Z, or Z) is introduced instead of that occurring in the pBStRNA expression vector (the different sequences are shown in Fig. 1B). Interestingly, several reports have already indicated that an additional sequence feature may help the specific processing of pre-tRNAs missing one of the canonical structural requirements for efficient maturation by E. coli RNase P. In the cases of pre-tRNA or pre-tRNA, specific maturation depends on the occurrence of a C at position +73 (Kirsebom and Svärd, 1992; Krupp et al., 1991). The present study establishes that the maturation of wild-type pre-tRNA requires the occurrence of a G at position -2 in the flanking sequence and that the introduction of a pyrimidine at this position disfavors the processing. A C at position -4 appears to compensate, at least partly, for the absence of a G in pre-tRNA. It must be underlined that G, which is the only conserved nucleotide in the three flanking sequences of the E. coli tRNA genes (see Fig. 1B), is only randomly found at this position in the other tRNA genes (see Komine et al., 1990).

The importance in the reaction catalyzed by RNase P of the 5`-flanking sequence of pre-tRNAs has already been suggested in several cases (Guerrier-Takada and Altman, 1993; Kirsebom and Svärd, 1993; Svärd and Kirsebom, 1992; Thurlow et al., 1991). In this study, the absence of G in the 5`-flanking sequence of tRNA caused the accumulation of tRNA species with 1-3 extra bases at the 5` side. These immature species behaved as substrates of RNase P, since an increase of the in vivo concentration of this enzyme restored full maturation. An interesting question to address is whether they correspond to abnormally accumulating intermediary reaction products or whether these immature species originate from aberrant cleavages induced by the absence of a signal such as G. Two mechanisms accounting for either model may be proposed. First, whatever the 5`-flanking sequence, RNase P would trigger the maturation of pre-tRNAs through an initial endonucleolytic cleavage, at position -3 for instance. This reaction could be followed by a trimming activity of RNase P on the already enzyme-complexed pre-tRNA. The rate of this step would be significantly slowed down in the absence of the correct flanking sequence, thereby leading to premature dissociation of the enzyme from the immature tRNAs. In the second mechanism, G of pre-tRNA would be the major initial signal directing the first specific endonucleolytic cleavage. This nucleotide would therefore compensate for the missing G as guiding nucleotide. In the absence of G, alternative cleavage sites might occur, either at C or at U. Although neither of the above mechanisms can be ruled out, it is noteworthy that the maturation of several pre-tRNAs by RNase P usually involves a single endonucleolytic cleavage with the release of a full-length 5`-flanking sequence (Robertson et al., 1972).

The interactions between the M1 RNA and the pre-tRNA complex have also to be considered (Guerrier-Takada et al., 1989; Kirsebom and Svärd, 1993). For instance, base C of the M1 RNA cross-links with base -3 of pre-tRNA (Guerrier-Takada et al., 1989). Furthermore, a mutant RNase P devoid of C aberrantly cleaves its substrates at positions -4, -5, or -6. In this context, one might speculate that, to compensate for the absence of a base pair at positions +1 and +72 or of a G at either position, an interaction occurs between G of pre-tRNA and C of the M1 RNA. Such an interaction specific for tRNA would possibly prevent the occurrence of an aberrant cleavage site at position -3.

FOOTNOTES

*
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. 33-1-69-33-48-80; Fax: 33-1-69-33-30-13; E-mail: titi@botrytis.polytechnique.fr.

The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENTS

We are indebted to B. Bachmann and O. Fayet for gifts of bacterial strain and of bacteriophage. We also thank F. Dardel, J.-M. Guillon, and Y. Mechulam for critical reading of the manuscript; S. Gillet for the gift of tRNA; and C. Lazennec for expert technical help.

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