G350 of Escherichia coli RNase P RNA contributes to Mg2+ binding near the active site of the enzyme

Gene 294 (2002) 177–185 www.elsevier.com/locate/gene

G350 of Escherichia coli RNase P RNA contributes to Mg 21 binding near the active site of the enzyme Terri A. Rasmussen 1, James M. Nolan* Department of Biochemistry-SL43, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112-2699, USA Received 4 April 2002; received in revised form 13 May 2002; accepted 6 June 2002 Received by D.L. Court

Abstract G350 of Escherichia coli RNase P RNA is a highly conserved residue among all bacteria and lies near the known magnesium binding site for the RNase P ribozyme, helix P4. Mutations at G350 have a dramatic effect on substrate cleavage activity for both RNA alone and holoenzyme; the G350C mutation has the most severe phenotype. The G350C mutation also inhibits growth of cells that express the mutant RNA in vivo under conditions of magnesium starvation. The results suggest that G350 contributes to Mg 21 binding at helix P4 of RNase P RNA. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Metal binding; Ribozyme; Holoenzyme; Major groove

1. Introduction Ribonuclease P (RNase P) is a ribonucleoprotein complex which cleaves 5 0 sequences from precursor tRNAs (pretRNAs) to yield mature tRNA products. The bacterial RNase P holoenzyme consists of a 140 kDa catalytic RNA and 14 kDa protein subunit. The protein subunit is required for viability in vivo, but in vitro it is dispensable for activity in the presence of high concentrations of monovalent or divalent cations. Mg 21 has been shown to be essential for folding of the RNase P RNA (Pan, 1995; Fang et al., 1999). In addition, Mg 21 is used as a cofactor for both the holoenzyme and RNA alone for hydrolysis of substrate (Perreault and Altman, 1993; Smith and Pace, 1993; Beebe et al., 1996; Kurz et al., 1998). Numerous chemical substitution and in vitro selection experiments have been utilized to define groups within the RNase P Abbreviations: Ap, ampicillin; Ap R, ampicillin resistance; Cm, chloramphenicol; cp332, circularly permuted RNase P RNA beginning with nucleotide 332; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IGEPAL, IGEPAL CA630 ((Octylphenoxy)polyethoxyethanol); kb, kilobase(s) or 1000 bp; kDa, kilodalton; Km, kanamycin; LB, Luria–Bertani medium; nt, nucleotide(s); P4, paired (helical) region 4; RNase P, ribonuclease P; rnpB, RNase P RNA gene; tRNA, transfer RNA; pre-tRNA, precursor transfer RNA; wt, wild-type * Corresponding author. Tel.: 11-504-584-2453; fax: 11-504-584-2739. E-mail address: [email protected] (J.M. Nolan). 1 Present address: Department of Pathology, Tulane Regional Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433, USA.

RNA (Hardt et al., 1995; Harris and Pace, 1995; Frank et al., 1996; Kazantsev and Pace, 1998; Heide et al., 1999; Christian et al., 2000), and the pre-tRNA substrate, that are important for Mg 21 binding and in catalysis. In the substrate, the RP oxygen at the scissile phosphate (Warnecke et al., 1996; Chen et al., 1997), the 2 0 -OH of the last precursor nucleotide (nt) (Perreault and Altman, 1992, 1993; Smith and Pace, 1993), and N7 of the first nt of mature tRNA (Kahle et al., 1990) have been shown to affect substrate cleavage by RNase P. In RNase P RNA, substitutions of phosphate oxygen and purine N7 in the P4 helix have demonstrated important Mg 21 binding sites necessary for substrate binding and cleavage and suggest that P4 may form part of the active site of RNase P RNA (Harris and Pace, 1995; Kazantsev and Pace, 1998). We describe here a mutation in the RNA subunit of the Escherichia coli RNase P RNA subunit adjacent to helix P4 that dramatically affects activity of both the RNase P RNA alone and in the holoenzyme. The mutation identifies potential Mg 21 coordination sites undetected in previous analyses.

2. Materials and methods 2.1. Host strains Most plasmids were grown and expressed in DH5a or XL1-Blue (Stratagene). Strain DW2 (Waugh and Pace, 1990) was a gift of James W. Brown, North Carolina

0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00766-7

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State University. Media and supplements were prepared as described (Ausubel et al., 1989). 2.2. Plasmid construction A 1.9 kilobase (kb) EcoRI/XhoI fragment, containing rnpB wild-type (wt) genomic sequence from plasmid pDW160 (Waugh and Pace, 1990) was subcloned into pQE30 (Qiagen) to form pQErnpB; this was used for testing viability of G350 mutants. Site directed G350 mutants of pQErnpB were made by the Quick-Change method (Stratagene (Weiner et al., 1994)). Plasmid p98SX (Sharkady and Nolan, 2001) was used as template for T7 RNA transcription for in vitro assays. G350 mutations were introduced into p98SX by the unique site elimination method (Deng and Nickoloff, 1992). Oligonucleotides used were: 350ACU(360rm), AAGCCGGGTTdTGTCGTGGAC; 350mutfor, GTCCACGACAhAACCCGGCTT; AflIII/BglII, CAGGAAAGAAgATcTGAGCAAAAG (used with 350mutfor for p98SX). Lowercase indicates sequences not found in the template plasmid DNA; h ¼ c, a, or t; d ¼ g, a, or t. All PCR amplified DNAs were sequenced. 2.3. Preparation of protein Protein was prepared by expression of pQE30EcoPprotein (Rivera-Leon et al., 1995), an N-terminal polyhistidine fusion to the E. coli RNase P protein coding sequence. Protein was purified by Ni-NTA (Qiagen) affinity of the his-tagged protein under denaturing conditions as recommended by the manufacturer and renatured by dialysis. Preparation of RNA: Labeled pre-tRNA substrate was prepared by T7 transcription of MvaI-linearized pDW152CCA (Oh and Pace, 1994) in the presence of [a- 32P]GTP (Amersham), and gel purified. E. coli RNase P RNA was prepared by T7 transcription of XhoI-linearized p98SX in the presence of [a- 32P]GTP, and gel purified. 2.4. Activity assays RNase P RNA was annealed in 25 mM HEPES (pH 8.0), 50 mM NH4Cl, 0.025% IGEPAL by heating to 80 8C for 2 min, room temperature 2 min, followed by 50 8C, 10 min (Pan, 1995). For holoenzyme experiments, RNase P RNA was diluted into 1£ reaction buffer (50 mM HEPES (pH 8.0), 100 mM NH4Cl, 10 mM MgCl2,, 0.05% IGEPAL) then mixed with RNase P protein in 1£ reaction buffer and incubated at 37 8C for 2 min. For RNA-alone experiments, RNase P RNA was diluted into 1£ RNA reaction buffer (50 mM HEPES (pH 8.0), 1 M NH4Cl, 10 mM MgCl2,, 0.05% IGEPAL) and incubated at 37 8C for 2 min. Substrate pre-tRNA was incubated separately in 1£ reaction buffer or 1£ RNA reaction buffer at 37 8C for 5 0 , then added to enzyme to start reactions. For magnesium titration experiments, MgCl2 concentration was varied as noted, single turnover reactions were performed at pH 6.0

to slow reactions to times compatible with manual mixing. Time courses of reactions were performed and the fraction cleaved was fit to the equation fraction cleaved ¼ 1 2 e2kt . Reactions (10 ml) were stopped by addition of an equal volume of urea-dye mix (1£ TBE, 50 mM EDTA, 8 M urea, 0.01% bromophenol blue, 0.01% xylene cyanol). For reactions performed at ½Mg21  . 10 mM, a Kin-Tech rapid quench instrument was used for accurate timing of reactions. In this case, 15 ml of substrate was reacted with 15 ml of RNase P RNA and quenched with 89 ml of 0.2 M EDTA; 10 ml of the quenched reaction was mixed with 10 ml urea dye mix. Reactions were heated to 80 8C, quickchilled on ice, then resolved on 8% acrylamide 8 M urea gels, fixed, dried and quantitated using a Fuji phosphorimager and MacBAS software. Statistical analysis was performed with StatView, version 4.51. 2.5. In vivo growth of mutant cells For plate assays, 1:100 or 1:1000 dilutions of overnight cultures were spread as droplets on LB plates supplemented with 3 or 5 mM EDTA and incubated overnight at 37 8C. For liquid cultures, 1:100 dilutions of overnight cultures were grown at 37 8C in LB containing 5 mM EDTA. Growth was monitored by A600 light scattering. Cell morphology and viability were studied by growing cells to late log phase in Tris media supplemented with 25 mM MgSO4 (St. John and Goldberg, 1980) and stained with the LIVE/DEAD bacterial viability kit (Molecular Probes, Eugene, OR). Cells were visualized by fluorescence and measured from video images using NIH Image, version 1.62 (http://rsb.info.nih.gov/nihimage/), on a Macintosh iMac 350MHz computer. 3. Results 3.1. Mutation of G350 affects activity of RNase P RNA and holoenzyme The G350A mutation (Fig. 1) was isolated serendipitously from the circularly permuted RNase P RNA template cp332 (Harris et al., 1994), during initial crosslinking studies of this RNA in the RNase P holoenzyme. cp332(G350A) holoenzyme was inactive in 400 mM NH4Cl, 10 mM MgCl2, but showed activity in the absence of protein at 1 M NH4Cl, 10 mM MgCl2 (data not shown). DNA sequence analysis revealed the G350A mutation. The G350A mutation, as well as G350U and G350C, were introduced into the native RNase P structure for further study. Since the initial phenotype was detected in the holoenzyme, the ability of the G350A mutant RNA to bind the RNase P protein subunit was tested by RNA competition experiments. Pre-tRNA cleavage assays were performed using wt RNase P RNA in the presence or absence of fourfold excess of G350A mutant RNA. If the G350A RNA is able to bind protein, yet remains inactive, its addition to wt RNA should inhibit the activity of the wt RNA; conversely, if the

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Fig. 1. RNase P RNA secondary structure. Secondary structure models for both Escherichia coli and minimal consensus bacterial RNA are shown (Brown, 1999). The G350 site is indicated by a black box. A summary of atomic substitution experiments are indicated on the consensus structure according to the legend; phosphorothioate interference sites (Harris and Pace, 1995; Kazantsev and Pace, 1998; Christian et al., 2000) and 7-deazaadenosine and 2 0 -deoxy interference sites (Kazantsev and Pace, 1998) were only detected in the conserved core of the RNA. Additional inosine interference sites (Heide et al., 1999) and purine interference sites (Siew et al., 1999) detected outside the conserved core of the RNA are not shown.

mutant RNA fails to bind protein, the activity of wt RNA should not be affected by the presence of excess G350A RNA. At 5 mM Mg 21, G350A holoenzyme alone shows less than 5% of wt holoenzyme activity (Fig. 2, lane 7, compare with lane 4). When G350A RNA was added in fourfold molar excess to both wt RNA and protein, cleavage activity was comparable to wt holoenzyme alone (Fig. 2, compare lanes 4 and 5). Under these conditions, the mutant RNA has little cleavage activity and does not appear to compete with wt RNA for binding to protein. These data indicate that the G350A mutant RNA binds protein only when it is in an active state. G350A, G350U, G350C mutant and wt RNAs were further assayed for pre-tRNA cleavage activity in the presence and absence of protein under multiple turnover conditions. Since G350A showed sensitivity to Mg 21 concentration and lies adjacent to the known Mg 21 binding sites in helix P4, the Mg 21 concentration was also varied, to

test the role Mg 21 binding might play in the phenotype of this mutation. The results are shown in Fig. 3A. Results are normalized to the fraction cleaved by the wt enzyme at 5 mM Mg 21 for each experiment; this facilitates comparison of the effect of mutations in the holoenzyme to those of the RNA-alone reaction. ANOVA analysis of the RNase P cleavage reaction results for mutant and wt RNAs in both the RNA alone and holoenzyme reactions revealed significant effects (P , 0:05) for all the G350 mutations on RNase P function. In the RNA alone reaction, when Mg 21 was varied between 5 and 10 mM, all the mutants showed statistically significant reductions in activity, varying from 16 to 35% of wt levels. G350C had the lowest activity, which was significantly lower than either of the other mutants. In the presence of equimolar RNase P protein, all mutants were again significantly reduced in activity, varying from 7 to 49% of wt activity. In this case, both the G350A and G350C mutants showed significantly lower activity than

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Fig. 2. Mutant RNase P holoenzyme competition experiment. Annealed wt and mutant RNAs were mixed and added to E. coli RNase P protein and preincubated before addition to uniformly 32P-labeled pre-tRNA. Reaction buffers contained 5 mM MgCl2 and 100 mM NH4Cl. Reactions were stopped and resolved on 8% acrylamide, 8 M urea, TBE gels, fixed, dried and quantitated by phosphorimager. Migrations of substrate pre-tRNA, product mature tRNA (mat-tRNA) and 5 0 -precursor RNA (5 0 pre) are indicated.

G350U. Additionally, both the G350A and G350C mutants showed significant recovery of activity at higher Mg 21 concentrations (8 and 10 mM) compared with the values at 5 and 6 mM. In contrast, both the wt and G350U mutants had relatively flat response to divalent cation concentrations in the holoenzyme. Activity of all RNAs was similar across a fourfold range of protein concentration (data not shown). Since the results shown in Fig. 3A for both holoenzyme and RNA alone are normalized to the fraction cleaved by the wt enzyme at 5 mM MgCl2, it is possible to compare the efficiency of the mutant RNAs in holoenzyme vs. the RNAalone context. For the G350A mutant, the holoenzyme shows a 2–4-fold greater defect compared with wt than does the RNA alone in reactions at 5 or 6 mM MgCl2. At higher MgCl2 concentrations, there is no statistically significant difference between holoenzyme and RNA alone. Similarly, the G350C mutant holoenzyme shows a 2-fold greater defect under holoenzyme conditions than in the RNA alone reaction; again, no significant difference was seen between RNA alone and holoenzyme in the 8 or 10 mM MgCl2 reactions. The G350U mutant did not show significant difference in defect between holoenzyme and RNA alone under any of the conditions tested. It appears that the activity defect of the G350A and G350C mutants is exacerbated

by the lower ionic strength reaction conditions used in holoenzyme reactions. The marked defect of the G350C mutant was investigated further by assaying activity under single-turnover conditions. This allowed us to monitor the effect of the G350C mutation on substrate binding and cleavage rather than the rate-limiting step of product turnover (Reich et al., 1988; Tallsjo¨ and Kirsebom, 1993). Mg 21 titration experiments were performed at pH 6.0. Since the catalytic step of the RNase P cleavage reaction is linearly dependent on [ -OH] (Smith and Pace, 1993), reducing the pH slows the reaction to allow for manual pipetting of reactions at lower [Mg 21], but has no effect on affinity of the RNase P RNA for substrate, Mg 21, or protein (Kurz et al., 1998). A rapid quench apparatus was used for ½Mg21  . 10 mM. Fig. 3B shows that the observed reaction rate for the G350C mutant is approximately 2-fold lower at low Mg 21 concentrations, but is ameliorated at higher concentrations of Mg 21. This indicates that the mutation affects the RNase P cleavage reaction at a step before product release. We presume that it is the catalytic step that is perturbed, since single-turnover activity was not affected by the G350C mutation when Ca 21 was used as divalent cation (data not shown). Ca 21 substitutes poorly for Mg 21 in the catalytic step of the RNase P cleavage reaction, but does not affect substrate binding (Smith et al., 1992). Thus it appears that neither substrate binding nor product release are affected by G350C mutation, but rather it is the cleavage step of the reaction that is inhibited in the mutant RNA. 3.2. In vivo phenotype In order to further assess the significance of the phenotype observed in vitro, in vivo complementation tests were performed using the DW2 tester strain (Waugh and Pace, 1990). DW2 has an interrupted genomic RNase P RNA gene (rnpB), marked by an insertion of a chloramphenicol resistance (Cm R) cassette. Viability of the DW2 cells is maintained by the presence of a wt copy of the rnpB gene on the pDW160 plasmid. This plasmid also carries a kanamycin resistance (Km R) marker and a temperature sensitive origin of replication that is compatible with pMB1-based plasmids (Meacock and Cohen, 1979; Waugh and Pace, 1990). DW2 is temperature-sensitive for growth due to the defective origin of replication of the pDW160 plasmid; an additional plasmid encoding active RNase P RNA must be present to allow growth of DW2 at restrictive temperatures (Waugh and Pace, 1990). Mutant tester plasmids were constructed by mutation of G350 in pQErnpB, which carries a 1.9 kb rnpB genomic DNA fragment in a pMB1, ampicillin-resistance (Ap R) plasmid. Mutant plasmids were then transformed into DW2 and cells were grown on Km, Ap Luria–Bertani medium (LB) plates at 30 8C to isolate transformants carrying both helper and tester plasmids. When transformed cells were streaked onto Ap plates at 42 8C, to select against the replication of the pDW160 helper plas-

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Fig. 3. Divalent cation titration experiments. (A) Steady-state cleavage experiments of wt and mutant holoenzyme or RNase P RNA alone. Multiple turnover reactions contained 0.5 nM holoenzyme (equimolar RNA and protein) or 1 nM RNA alone and 20 nM pre-tRNA; reactions were incubated 9 or 15 min. at 37 8C. Results shown are averages of 4–5 duplicate experiments and represent the fraction of substrate cleaved normalized to the fraction cleaved for wt RNA or holoenzyme at 5 mM MgCl2. Symbols are shown in the legend. Error bars show standard errors. (B) Plot of kobs vs. [Mg 21] for single turnover reactions of wt and G350C RNase P RNA. Reactions were 200 nM RNase P, 1 nM pre-tRNA, the pH of the HEPES buffer was 6.0. Reactions were incubated from 1 s to 45 min at 37 8C.

mid, all three mutants, G350A, G350U, and G350C, were able to grow at the restrictive temperature. The mutant genotype was confirmed by sequencing plasmids derived from colonies isolated at 42 8C. The cells grown at 42 8C lost the pDW160 plasmid, based upon their Km sensitivity, but were able to grow normally at both 37 8C and 42 8C on LB or Ap LB plates. Thus, the mutant RNAs were able to provide sufficient RNase P activity for growth on rich media. Since the in vitro data suggested a reduction of

Mg 21 affinity in the mutant RNAs, cells bearing mutant plasmids were further tested by growing them on LB plates containing either 3 or 5 mM EDTA (Fig. 4A). All mutants grew on 3 mM EDTA, although the amount of growth was noticeably lower for the G350C mutant; the G350C mutant showed virtually no growth overnight on 5 mM EDTA plates. Faint growth of G350C was detected after 2 days at 37 8C. The cells bearing only the G350C rnpB gene were deficient in growth under limiting Mg 21 concentra-

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tions. This growth deficiency was also seen in liquid culture experiments. Dilutions of wt and mutant cells into LB 1 EDTA were monitored for growth by optical density measurements (Fig. 4B). Although the G350C mutant was able to grow, the rate of growth was slower than wt, G350U, or G350A mutant cells. The G350C mutant also showed reduced growth rate in defined media (St. John and Goldberg, 1980) supplemented

with 10–25 mM MgSO4 (data not shown). Cell viability and morphology were investigated for mutant and wt cells grown in 25 mM Mg 21 and stained for viability using the LIVE/DEAD kit (Molecular Probes). There was no significant difference in viability of the strains (data not shown). As seen in Fig. 5A, all of the mutant cells appear smaller in diameter than the wt strain. This was confirmed by analysis of the long and short axis measurements of stained cells. As

Fig. 4. Growth of G350 mutants in vivo. (A) Growth on plates. 1:100 (5 mM EDTA plate and center of 3 mM plate) and 1:1000 dilutions (outer edge of 3 mM EDTA plate) of overnight cultures grown in LB media were dispensed on LB agar plates containing 3 or 5 mM EDTA and incubated overnight at 37 8C. (B) Liquid culture growth rate. Overnight cultures grown in LB media were diluted 1:100 into LB 1 5 mM EDTA. Growth was monitored by A600 light scattering. Optical density of starting overnight cultures varied by , 4% between strains.

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Fig. 5. G350 mutant cell morphology. (A) Microscopic analysis of cell viability and morphology. Late log phase cells were stained using the LIVE/DEAD bacterial viability test kit. No differences were seen in percent viable cells (data not shown). Wild-type cells appear plumper than mutant cells. (B) Numerical analysis of cell shape. Measurements of length and width in mm are graphed for wt (G) and the G350 mutants (A, C, and U). Bars indicate standard errors. P values of less than 0.05 for deviations from wt are indicated above each graph.

seen in Fig. 5B, there is no statistically significant difference in the length of mutant versus wt cells. However, all the mutants vary significantly from wt in cell width. This difference in width for the mutant cells was not seen in log phase cells grown in rich media (data not shown).

4. Discussion Numerous modification interference experiments, using self-cleaving RNase P RNA/pre-tRNA hybrid molecules, have indicated helix P4 as a metal binding site important

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for RNase P RNA activity. Phosphorothioate substitutions have implicated RP phosphate oxygen of residues A67, G68, A352, and SP phosphate oxygen of A67 as essential for cleavage activity (Harris and Pace, 1995; Kazantsev and Pace, 1998; Christian et al., 2000). 7-Deazaguanosine substitutions revealed potential Mg 21 coordination sites adjacent to P4 at N7 of residues A62, A65, A66, A249, and A351 (Kazantsev and Pace, 1998) (see Fig. 1). Together, these atoms appear to form coordination sites for at least two Mg 21 atoms in the major groove of P4. G350 appears to contribute to coordination of Mg 21 at one of these sites. The results of our mutational analysis of G350 mutants suggest that the O6 atom of G350 may contribute to metal binding. The most severe mutant phenotypes were seen with G350A and G350C, both of which place amino groups in the major groove in place of the carbonyl oxygen of the wt G350. In contrast, G350U has a less severe phenotype in the holoenzyme, and maintains the carbonyl oxygen of the wt RNA. Coordination of Mg 21 at O6 of G residues has been detected by crystallographic studies of tRNA (Quigley et al., 1978; Jovine et al., 2000), the Group I intron RNA (Cate and Doudna, 1996), and the hepatitis Delta ribozyme RNA (Ferre et al., 1998). Biochemical analysis of substitution mutants of domain V of the group II intron has also implicated O6 in coordination of Mg 21 (Konforti et al., 1998). The N2 amino group of G350 seems less likely to be involved, since inosine substitution at G350 has no effect on product (tRNA) binding (Heide et al., 1999). However, inosine substitutions have not been tested for cleavage activity. While our studies were nearing completion, nucleotide analog interference assays were reported which showed that substitution of the non-bridging oxygen of the homologous residue of the Bacillus subtilis RNase P RNA interferes with protein binding (Rox et al., 2002). It is not clear whether this effect represents a direct interference with protein binding, or is instead an indirect effect mediated by interference with Mg 21 binding. The solution structure of the isolated P4 helix, including one of the metal binding sites, has been determined recently (Schmitz and Tinoco, 2000). However, the contribution of G350 and adjacent residues to metal binding was not studied, since these residues were mutated to a stem-loop for technical reasons. Three-dimensional models of RNase P RNA structure differ on the placement of G350. In one model, it is placed in the major groove of P4 (Chen et al., 1998), and in the other, G350 resides in the P4 minor groove (Massire et al., 1998). The results of the present experiment will allow refinement of the placement of this residue near the active site of the enzyme. The results seem to indicate that the major groove face of G350 should be near the major groove of P4, to allow G350 to coordinate Mg 21 bound in the P4 major groove. The growth phenotype observed in vivo for G350 mutants is consistent with our in vitro observations. Cells bearing only G350C RNase P RNA grow poorly compared with wt cells under Mg 21 starvation conditions. The morphology of

the mutant cells was initially investigated to monitor viability and to determine if the growth phenotype was related to cell cycle control. Cell cycle defects typically affect cell length (Vinella and D’Ari, 1995), the difference in width observed instead is difficult to interpret in the context of available data. Cell width has been shown to be inversely related to cell doubling time (Trueba and Woldringh, 1980; Kubitschek, 1986). However, our results cannot be explained by doubling time alone, since all the mutants show a width phenotype, but only G350C shows a growth rate defect. The rnpB gene appears to be under growth rate dependent control (Dong et al., 1996). This may be related to the cell-width phenotype we observed for the mutant G350 mutant RNAs. The lack of mutant cell-width phenotype when cells were grown in rich media is consistent with a role for growth rate control of RNA expression. Further studies will be necessary to understand the mechanism by which the in vivo phenotype is manifest. G350 is a highly conserved nucleotide amongst all known bacterial RNase P RNA sequences (Fig. 1) (Brown, 1999). It is part of the highly conserved AGAA sequence motif. G350 is found in 83 of 84 known ‘A-type’ (ancestral) RNase P RNAs and 14 of 22 ‘B-type’ (Bacillus) RNAs, a total of 92% of all bacterial RNAs; all known natural G350 variants carry G350A. This high level of conservation argues that G350 is likely to be an important part of an essential Mg 21 binding site in the RNA. Our observation that G350 mutants were able to survive in vivo under optimal growth conditions is consistent with the lack of complete conservation of G350. The residual activity of the G350A RNase P would allow organisms carrying this mutation to survive and evolve compensatory Mg 21 coordination sites. Still, the striking phenotype of the G350C mutation, the high degree of conservation of G350, and its proximity to the magnesium coordination center at P4 suggests that it contributes to magnesium binding which is essential for activity. Acknowledgements This material is based upon work supported by the National Science Foundation under Grant no. MCB9631039 and a seed grant from the Cancer Association of Greater New Orleans. We thank Dan Hershlag for helpful suggestions, Gary Hoyle for assistance with statistical analysis, Joe Vaccaro for use of his rapid quench apparatus, Alexandra Peister for assistance with microscopy, and Darwin Prockop for use of the microscope. We thank Linda Hyman, Steve Sharkady, Sumit Borah, and Candace Timpte for their comments. References Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 1989. Current Protocols in Molecular Biology, 1st Edition. Wiley, New York.

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