©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Genetic and Transcriptional Organization of the Region Encoding the Subunit of Bacillus subtilis RNA Polymerase (*)

(Received for publication, December 23, 1994; and in revised form, June 5, 1995)

Kathryn J. Boor (§) Marian L. Duncan (¶) Chester W. Price (**)

From the Department of Food Science and Technology, University of California, Davis, California 95616

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The gene encoding the beta subunit of Bacillus subtilis RNA polymerase was isolated from a gt11 expression library using an antibody probe. Gene identity was confirmed by the similarity of its predicted product to the Escherichia coli beta subunit and by mapping an alteration conferring rifampicin resistance within the conserved rif coding region. Including the rif region, four colinear blocks of sequence similarity were shared between the B. subtilis and E. coli beta subunits. In E. coli, these conserved blocks are separated by three regions that either were not conserved or were entirely absent from the B. subtilis protein. The B. subtilis beta gene was part of a cluster with the order rplL (encoding ribosomal protein L7/L12), orf23 (encoding a 22,513-dalton protein that is apparently essential for growth), rpoB (beta), and rpoC (beta`). This organization differs from the corresponding region in E. coli by the inclusion of orf23. Experiments using promoter probe vectors and site-directed mutagenesis located a major rpoB promoter overlapping the 3`-coding region of orf23, 250 nucleotides upstream from the beta initiation codon. Thus, the B. subtilis rpoB region differs from its E. coli counterpart in both genetic and transcriptional organization.


INTRODUCTION

The basic features of the transcriptional machinery are remarkably conserved in all organisms. In particular, the beta, beta`, and alpha subunits that comprise the catalytic core of the eubacterial RNA polymerase are similar to subunits of the three nuclear RNA polymerases of eukaryotes (see (1) and (2) for reviews). The beta` subunit of Escherichia coli shares eight regions of sequence similarity with the largest subunit of the eukaryotic enzymes(3) , and the beta subunit of E. coli was initially reported to share nine regions with the second largest subunit(4, 5) . Additional sequence alignment of beta homologues has further refined the boundaries of these nine regions into 12 colinear segments, which are conserved in both eubacteria and eukaryotes(6) . alpha, the third largest subunit of the eubacterial core enzyme, also has similarity to the third subunit of RNA polymerase II and to the fourth subunits of RNA polymerases I and III, although this similarity is less striking than that found among the beta or beta` homologues (reviewed in (1) and (2) ). This conservation of primary amino acid sequence suggests common functions for the shared regions.

Although the well characterized RNA polymerase from E. coli is available to represent the Gram-negative lineage of eubacteria, there has been less information regarding RNA polymerases from Gram-positive bacteria. With the view that the transcriptional apparatus of a genetically amenable Gram-positive bacterium could contribute to a structure-function analysis of RNA polymerases, we began a study of the genes encoding the core subunits from the spore-forming bacterium Bacillus subtilis. We earlier reported the isolation and characterization of rpoA, the gene for the alpha subunit(7, 8) . Here, we describe the genetic and transcriptional organization of the region containing rpoB, the gene encoding the beta subunit, and show that this organization differs substantially from that of the corresponding region in E. coli. Because beta is involved in most of the catalytic functions of RNA polymerase, including nucleotide binding(9, 10) , transcription initiation, elongation, and termination(10, 11, 12, 13, 14, 15) , and interactions with both the subunit (16, 17) and the NusA protein(18, 19, 20) , we also compared the likely functional domains of the B. subtilis and E. coli subunits. This comparison revealed two regions of E. coli beta that were entirely absent from the B. subtilis protein and thus are likely dispensable for function. Significantly, another region of 186 residues, which is known to be dispensable in E. coli, shared little overall sequence similarity with B. subtilis beta but nonetheless contained a common, 69-residue segment. We infer from these results that the common segment has a more fundamental role in beta function than previously believed.


MATERIALS AND METHODS

Bacterial Strains, Phage, and Genetic Methods

E. coli Y1090 was used as host for the gt11 expression vector, grown as previously described(21) . B. subtilis strains used are shown in Table 1. For strain constructions, B. subtilis was made competent for natural transformation as described by Dubnau and Davidoff-Abelson (23) . Standard recombinant DNA methods were performed as described by Davis et al.(24) , polymerase chain reactions were done according to published protocols(25) , and DNA sequencing was done by the dideoxynucleotide chain termination method with reactions primed on double-stranded DNA templates, using the Sequenase enzyme and protocols from U. S. Biochemical Corp. We sequenced 6130 base pairs of the rpoB region on both strands, using either nested deletions, made as previously described(8) , or custom oligonucleotide primers (Operon Technologies, Alameda, CA) as necessary.



Location of the Alteration Caused by the rfm2103 Allele

A fragment of rpoB bearing strong sequence similarity to the rif region of E. coli rpoB was amplified from the chromosome of B. subtilis strain PB355 (rfm2103), together with a control fragment from the PB2 wild type strain. These PCR (^1)products were cut at the PvuII and StyI sites, which flank the rif region, yielding 817 nt fragments that were subcloned into pUC19 for further analysis. The resulting plasmids were linearized and transformed into PB2 with selection for resistance to rifampicin (200 µg/ml). The fragment amplified from strain PB355 conferred rifampicin resistance at a frequency 7-fold higher than the appearance of spontaneous resistance, determined from transformations using the control fragment isolated from strain PB2. The rfm2103 alteration was confirmed by directly sequencing the rif chromosomal region of PB300, the parent of PB355, using the fmol system (Promega).

Construction of Transcriptional Fusions

Promoter activity was detected using single-copy transcriptional fusions to the lacZ reporter gene of the pDH32 vector(26) . As shown in Fig. 1, we first used three fragments to locate activity in the L7/L12-P23-beta interval. pKB3 contained a 1.1-kb EcoRI-SpeI fragment that spanned the entire interval. pKB13 contained the upstream portion of this interval on a 0.7-kb EcoRI-HindIII fragment, and pKB4 contained the downstream portion on a 0.4-kb HindIII-SpeI fragment. All three plasmids were linearized and transformed into B. subtilis strain PB2, whereupon they integrated into the amyE chromosomal locus by means of the amy ``front'' and ``back'' sequences carried by the pDH32 vector(26) .


Figure 1: Genetic organization of the B. subtilis beta-beta` region. The chromosome in the rpoB region is represented by the heavyline and kilobase scale. The rectanglesabove the physical map indicate the open reading frames encoding ribosomal protein L7/L12, the 22,513-dalton protein P23, and the beta and beta` subunits of RNA polymerase, all of which are transcribed from left to right; the arrows adjacent to the L7/L12 and beta` reading frames indicate that they extend beyond the cloned region. The restriction map shows the sites used in the recombinant DNA constructions described under ``Materials and Methods.'' (R), EcoRI linkers derived from the construction of the gt11 library(7) ; H, HindIII; S, SpeI; P, PvuII; and Y, StyI. The regions of the chromosome used in some of the constructions are indicated by the horizontallinesbeneath the restriction map. pKB10 was made by subcloning the indicated 1.1-kb EcoRI-SpeI fragment into the pCP115 integration vector(27) . Upon integration into the B. subtilis chromosome, pKB10 would prevent any transcription initiating upstream of the L7/L12 gene from entering the beta and beta` genes. pKB3, pKB4, and pKB13 were made by sucloning the indicated fragments into the single-copy transcriptional fusion vector pDH32 (26) . The two fragments conferring promoter activity are labeled +. The physical map of the beta-beta` region was derived from the restriction map and DNA sequence of the chromosomal inserts carried by the two gt11 phages.



The promoter activity manifested by pKB4 was further localized by subcloning PCR-generated fragments into pDH32. pKB14 carried a fragment extending from nt 829 to the SpeI site at nt 1101 ( Fig. 3and 5), pKB15, a fragment from nt 935 to the SpeI site, and pKB16, a fragment from nt 829 to 989. DNA sequencing confirmed that no alterations had been introduced by the PCR reactions and that all three fragments were oriented with the gene direction toward lacZ. These three plasmids were then linearized and integrated into the amyE locus of strain PB2 to yield strains PB365, PB366, and PB367.


Figure 3: Nucleotide sequence of the orf23-rpoB (P23-beta) interval. The sequence shown represents the C-terminal coding region for P23, the P23-beta intercistronic region, and the N-terminal coding region for beta. This includes the 0.4-kb HindIII-SpeI fragment that contains rpoB promoter activity (Fig. 1). The two 5`-ends of rpoB message detected in the primer extension experiment described in Fig. 4are indicated by the verticallines marked A at nt 876 and B at nt 1012. The locations of the three complementary primers used to analyze the rpoB message are indicated by <P973, <P1065, and <P1150, with (<) denoting the 3`-end of the primer. The proposed -35 and -10 recognition sequences of the rpoB promoter are doubleunderlined at nt 840-845 and 863-868, respectively; the indicated mutations change the -35 sequence from TTGACT to TAAACT in pKB17. The proposed ribosomal binding site for the rpoB message is underlined at nt 1076-1083.




Figure 4: Mapping the 5`-end of the rpoB message by primer extension. Wild type strain PB2 was grown in 2 SG sporulation medium and harvested during logarithmic growth; the RNA was then extracted. Primer extensions were done using a molar excess of three different synthetic primers, P973, P1065, and P1150 (described under ``Materials and Methods''). These primers are complementary to message synthesized using the orf23-rpoB (P23-beta) intercistronic region as template; their locations are shown in Fig. 3. For each experiment, samples containing 75 µg of RNA were loaded onto lane1 of a sequencing gel. A sequencing ladder was run in parallel using the same primer employed in the extension reactions; the letters A, C, G, and T indicate the dideoxynucleotide used to terminate the reaction. The sequences indicated on the right are from the non-transcribed strand and are the complement of the sequences that can be read from the ladder. Reactions using primers P973, P1065, and P1150 all gave a 5`-signal centered around the thymidine complementary to adenosine 876 (Fig. 3); the experiment using primer P1065 is shown in panel A. Reactions using primers P1065 and P1150 both gave a 5`-signal centered around the thymidine complementary to adenosine 1012 (Fig. 3); the experiment using primer P1150 is shown in panel B.



Sequences necessary for the promoter activity manifested by pKB16 were further defined by site-directed mutagenesis. A mutation in the proposed -35 recognition sequence of the rpoB promoter carried by pKB16 was created using the primer 5`-GCAAAAAAAGTTAAACTCGGTATTTTAACTATG, the same as nt 829-861 except for the underlined residues, and extended to nt 989 using the same second primer employed to amplify the pKB16 insert. DNA sequencing confirmed that this mutagenized fragment was identical to that carried by pKB16 except for the alteration of the proposed -35 sequence from TTGACT to TAAACT. The mutagenized fragment was cloned into pDH32 to yield pKB17, which was linearized and integrated into the amyE locus of PB2 to yield strain PB368.

Initial screening for promoter activity of the integrated fusions was on tryptose blood agar base plates (Difco Laboratories) containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. For quantitative estimates of promoter activity, we performed beta-galactosidase assays essentially as described by Miller(28) . B. subtilis cells were grown to late logarithmic stage in 2 SG sporulation medium (29) and then diluted 1:25 into fresh medium. Samples were taken throughout the logarithmic and stationary phases of growth. Activity was expressed in Miller units, defined as 1000 DeltaA/min/ml/unit of optical density at 600 nm.

Mapping the 5`-Ends of rpoB Message

RNA was prepared essentially by the method of Igo and Losick(30) , using the modifications described by Varón et al.(31) . Strain PB2 was grown in 50 ml of 2 SG sporulation medium and harvested during exponential growth. RNA was extracted as described (30, 31) and precipitated by overnight incubation at -20 °C with 2 volumes of ethanol. For primer extension reactions, three different oligonucleotide primers were used to analyze the orf23-rpoB intercistronic region: P973, a 17-mer (5`-TTAAGAAAACCACATCC-3`) complementary to nt 973-989 in Fig. 3; P1065, a 21-mer (5`-ATTCACCCCTCAAATCATGCG-3`) complementary to nt 1065-1085; and P1150, a 19-mer (5`-GGTAATTCTAACACTTCGC-3`) complementary to nt 1150-1168. Each primer was 5`-end labeled with [-P]ATP (3000 Ci/mmol, Amersham Corp.) and T4 polynucleotide kinase (Promega). Annealing and primer extension were done using the Promega primer extension kit according to the manufacturer's instructions, except that 75 µg of RNA and 5 ng of primer were used in a 20-µl reaction volume.

Computer Analysis

The statistical significance of protein sequence comparisons was evaluated with the FASTA and RDF programs of Pearson and Lipman(32) , using the National Biomedical Research Foundation data bases. Highly related sequences usually have an optimized alignment score exceeding 100 and a z value greater than 10 standard deviations above the mean alignment of a shuffled sequence.


RESULTS

Isolation of gt11 Bacteriophages Encoding the beta Subunit

Antibody raised against B. subtilis RNA polymerase holoenzyme was used to screen gt11 libraries for phages that might carry core subunit genes(21) . From a random library constructed with chromosomal DNA cut with AluI, HaeIII, or RsaI(7) , we found nine positive clones, one of which was subsequently shown to encode the alternative transcription factor ^B(21) . Among the remaining eight phages, epitope selection (33) identified five that might encode either of the two largest core subunits, beta or beta`. The restriction maps of the five presumptive beta and beta` clones indicated that they fell into two classes and carried overlapping regions of the B. subtilis chromosome. These two classes are represented by the phages gt11-15 and gt11-17, shown in Fig. 1. Subsequent characterization established that the chromosomal inserts carried by these phages contained the entire beta coding region.

Genetic Organization of the beta-beta` Region

We determined the DNA sequence on both strands of the 6.1-kb region isolated on the gt11 clones. We then identified the genes within this region by aligning the predicted B. subtilis gene products with their E. coli counterparts, using the FASTA program of Pearson and Lipman(32) . As shown in Fig. 1, we found that B. subtilis had a gene order similar to E. coli from the rplL homologue (encoding ribosomal protein L7/L12) through the rpoC homologue (encoding the beta` subunit), confirming the results of previous genetic mapping experiments(34) . However, our analysis revealed that the gene order in B. subtilis differed from E. coli by the inclusion of an open reading frame (orf23) that could code for a protein of 22,513 daltons (P23). The predicted sequence of P23 was significantly similar to the products of two unidentified open reading frames sequenced as part of the E. coli genome project directed by F. R. Blattner (University of Wisconsin, Madison). (^2)The first of these (GenBank accession number U14003 283) is located at 99 min on the E. coli chromosome and encodes a predicted product of 343 residues (optimized alignment score with P23 is 217; z value is 26.1 S.D. above the mean). The second (GenBank accession number U18997 12) is located at 76 min and encodes a predicted product of 388 residues (optimized alignment score is 214; z value is 24.5). Both E. coli products also significantly resemble each other (optimized alignment score is 282; z value is 29.9), and both reading frames lie distant from the E. coli rpoB region at 88 min. Notably, our attempts to disrupt B. subtilis orf23 by plasmid integration failed to yield viable transformants (data not shown). Because we demonstrate below that B. subtilis rpoB is expressed from a promoter immediately downstream from orf23, an orf23 disruption would not significantly affect the integrity of the rpoB transcriptional unit. Thus, the observation that orf23 could not be readily disrupted suggests that it encodes an essential product.

Identification of B. subtilis rpoB

The proposed beta reading frame encodes a predicted 1,193-residue product that is 56% identical to the E. coli beta subunit (Fig. 2). The two proteins share their highest sequence conservation in four large blocks, and these are separated by three regions that are either variable or entirely absent in B. subtilis beta (see Table 2). Within the four large blocks, the 12 segments that are highly conserved among the second largest subunits of eubacterial and eukaryotic RNA polymerases (6) are found in the expected order (see Fig. 2). Moreover, individual residues that are known to be important for E. coli beta function are found in the corresponding locations in the B. subtilis protein. As shown in Fig. 2, these include two key residues that lie near the active center that binds the transcript-initiating nucleotide (10) , as well as 15 of the 16 residues at which single substitutions confer rifampicin resistance(12, 13, 18, 37, 38) .



Figure 2: Alignment of the primary sequences of the B. subtilis and E. coli beta subunits. The predicted sequence of B. subtilis beta (upper) is from this work and that of E. coli beta (lower) is from Ovchinnikov et al.(35) . Each of the four conserved blocks was separately aligned by means of the FASTA program of Pearson and Lipman(32) , with identical residues indicated by a colon (:) and conserved substitutions (36) by a period (.). The underlinedsegments denote the 12 regions that are highly conserved in the second largest subunits of RNA polymerases from eubacteria and eukaryotes(6) . The four conserved blocks are separated by three variable regions that either are not conserved or are absent entirely from the B. subtilis protein. Key residues of E. coli beta that have been altered by mutation are shown below the E. coli sequence. These include residues at which single substitutions cause either rifampicin resistance (*) or an altered termination phenotype (ˆ) (see (12) , (13) , (18) , (37) , (38) ). Arrows indicate one particular substitution that confers rifampicin resistance on both organisms; this is the site of the E. coli rpoB2 alteration (H526Y) (18) and the corresponding B. subtilis rfm2103 alteration (H482Y) (this work). Other arrows indicate E. coli substitutions, which render RNA polymerase insensitive to the Alc termination factor of bacteriophage T4 (R368H (paf32)(39) ) or which alter promoter clearance capabilities of the mutant polymerase (K1065R (12) and H1237A(10) ). Affinity labeling studies have shown that Lys-1065 and His-1237 lie near the binding site for the transcript-initiating nucleotide(10) .





Rifampicin is an antibiotic that traps prokaryotic RNA polymerases in the initial transcribing complex, preventing elongation of the nascent transcript beyond three or four nucleotides(40, 41) . All known mutations resulting in rifampicin resistance map to the gene encoding the beta subunit(42, 43, 44) . In E. coli beta, most alterations conferring rifampicin resistance lie between residues 512 and 573 in conserved block 2(12, 13, 18, 37, 38) . We used PCR to isolate this region from the chromosome of B. subtilis strain PB355 (rfm2103) and found that it contained only one alteration from the wild type sequence: a C to T transition that would change residue 482 of B. subtilis beta from a histidine to a tyrosine. Transformation of strain PB2 with the fragment bearing the rfm2103 allele conferred the ability to grow in the presence of high levels of rifampicin, indicating that the H482Y alteration alone is sufficient for resistance. As shown in Fig. 2, this is the identical change in primary sequence caused by the E. coli rpoB2 allele(18) , which elicits severe defects in transcription termination and is incompatible with the rho-15, nusA10, nusA11, and dnaA46 mutations (11, 19, 45, 46) . Indeed, most rifampicin-resistant mutations are highly pleiotropic, causing altered initiation, elongation, and termination phenotypes(9, 11, 15, 19, 46, 47, 48) . Because rifampicin-resistant mutants in conserved block 1 have the same in vivo and in vitro phenotypes as mutants in conserved block 2, Jin and Gross (18) and Severinov et al.(49) have suggested that these two blocks perform a common catalytic function in the core enzyme.

Conserved blocks 3 and 4 are also thought to comprise part of the active center for beta catalytic function. Affinity labeling studies indicate that both Lys-1065 and His-1237 of E. coli beta lie near the site that binds the transcript-initiating nucleotide, although neither residue appears to be directly involved in subsequent phosphodiester bond formation(10) . Notably, Lys-1065 and His-1237 are conserved in all prokaryotic and eukaryotic beta homologues characterized to date(10, 12) , and these residues are found at the expected positions in B. subtilis beta (Fig. 2). Genetic evidence underscores the importance of these residues in beta function. Alteration of E. coli Lys-1065 to arginine (K1065R) results in a dominant lethal mutant enzyme that is blocked in promoter clearance(12) , and alteration of His-1237 to alanine (H1237A) also results in a mutant polymerase with impaired promoter clearance(10) . Other single residues surrounding E. coli Lys-1065 are also important for beta function, because the R1069A, G1071A, and K1073A substitutions result in dominant lethal mutants with decreased promoter clearance capabilities and aberrant response to pause sites(50) . All three of these residues are conserved in B. subtilis.

On the basis of the strong similarity of its predicted product to E. coli beta, we conclude that we have isolated the gene encoding the B. subtilis beta subunit. This conclusion is reinforced by two independent criteria. First, the initial antibody screening and epitope selection indicated that the gt11 clones encoded a protein with the antigenic properties of B. subtilis beta. Second, as an additional biological criterion, we mapped within the B. subtilis beta coding sequence an alteration conferring rifampicin resistance. In keeping with the standard genetic nomenclature, we will refer to the gene for the beta subunit as rpoB.

Transcriptional Organization of the beta-beta` Region

We used a combination of plasmid integration experiments, primer extension studies, and fusion constructions to determine the location of both the rpoB promoter and a possible site of processing for the rpoB message. Plasmid integration (51) allowed us to determine that the cloned region contained a promoter capable of sustaining normal cell growth. We first constructed the integration plasmid pKB10, which contained the 1.1-kb EcoRI-SpeI fragment from the rpoB region shown in Fig. 1, then transformed B. subtilis wild-type strain PB2 with selection for the chloramphenicol resistance encoded by the plasmid. In all the resulting transformants tested, PCR confirmed that pKB10 had in fact integrated into the rpoB region of the genome via a single homologous crossover event, leading to a tandem duplication of the 1.1-kb EcoRI-SpeI fragment separated by plasmid sequences (data not shown). Such an integration event would prevent any transcription that originates upstream of rplL from entering the rpoB gene(8, 31, 51) . Because many viable transformants were obtained from this experiment and because the transformants that carried the integrated pKB10 plasmid in the expected configuration were indistinguishable from wild type cells in terms of growth rate and sporulation frequency (data not shown), we conclude that the 1.1-kb EcoRI-SpeI fragment contains a promoter that can supply the cells with a functional level of beta protein.

To confirm the presence of a promoter within this 1.1-kb fragment, we moved this region into the single-copy transcriptional fusion vector pDH32(26) . Upon introduction of the resulting fusion into the B. subtilis chromosome at the amyE locus, this fragment showed clear promoter activity (Fig. 1). To more precisely locate this activity, we first subcloned two portions of the 1.1 kb-fragment into the pDH32 vector and introduced these two fusions into the amyE locus. As shown in Fig. 1, the upstream 0.7-kb EcoRI-HindIII fragment, which included the rplL-orf23 intercistronic region, had no detectable promoter activity. In contrast, the downstream 0.4-kb HindIII-SpeI fragment, which included the orf23-rpoB intercistronic region, had strong promoter activity. The nucleotide sequence of this region is shown in Fig. 3. Primer extension experiments (Fig. 4) located two 5`-ends of the rpoB message within the region. One signal was centered near nt 876 (labeled A in Fig. 3, 4, and 5) and the other near nucleotide 1012 (labeled B in Fig. 3-5).

To establish whether the 5`-ends detected in the primer extension experiment correlated with regions that contained promoter activity, we made a series of additional transcriptional fusions in the pDH32 vector, as shown in Fig. 5. From the activities manifested by these fusions, we concluded that whereas the 5`-end centered around nt 876 lay near sequences conferring promoter activity, the 5`-end centered around nt 1012 did not. Instead, it seems likely that this latter 5`-end represented a site at which the rpoB message was processed. Because we detected this same signal near nt 1012 using two different primers, we think it less likely that it was an artifact of the extension reactions.


Figure 5: Activity of lacZ reporter genes transcriptionally fused with fragments from the P23-beta interval. Panel A depicts the HindIII-SpeI fragment that contains rpoB promoter activity (Fig. 1). The C-terminal coding region of P23 and the N-terminal coding region of beta are indicated by the rectanglesabove the nucleotide scale. The locations of the 5`-ends of rpoB message detected in the primer extension experiments of Fig. 4are shown by the arrows, with the signal centered at nt 876 marked A and the signal centered at nt 1012 marked B. The fragments shown beneath the nucleotide scale were subcloned into the transcriptional fusion vector pDH32 and integrated in single copy at the amyE loci of the indicated strains. The fragments carried by strains PB367 and PB368 are identical except for the mutation of the proposed -35 recognition sequence (TTGACT to TAAACT), indicated by the filledtriangle. Panel B shows the beta-galactosidase activities directed by the four transcriptional fusions depicted in panelA. The strains carrying these fusions were grown and assayed for beta-galactosidase activity as described under ``Materials and Methods.'' The fusion carried by strain PB367 had strong promoter activity, and this activity was abolished by the -35 mutation in the otherwise identical fusion carried by strain PB368. We therefore conclude that the A signal centered at nt 876 represents the likely site of initiation for the rpoB message. In contrast, the fusion carried by strain PB366 had no promoter activity, suggesting that the B signal represents a processing site.



Inspection of the region necessary for promoter activity in the fusion experiments revealed the sequence TTGACT-(17 base pairs)-TAATAT just upstream from the 5`-end near nt 876 (Fig. 3). This sequence and spacing closely match the consensus recognized by RNA polymerase holoenzyme containing the major factor of B. subtilis, ^A(52) . To determine whether this sequence was required for rpoB promoter activity, we made a double mutation in the proposed -35 recognition sequence, changing it from TTGACT to TAAACT (Fig. 3). When the fragment containing this mutation was fused to the lacZ reporter gene of the pDH32 vector and integrated into the chromosome, promoter activity was completely abolished (Fig. 5).

On the basis of promoter activity measurements, primer extension experiments, and mutational analysis, we conclude that a major rpoB promoter partly overlaps the 3`-coding region for the preceding orf23 gene. Our experiments further suggest that the estimated 213-nt leader of the message originating from this promoter is processed near nt 1012. In E. coli, the rpoB message is processed at an RNaseIII site located within a region of secondary structure in the 321-base pair rplL-rpoB interval(53, 54) . In B. subtilis, there is no stable secondary structure apparent in the sequence of the orf23-rpoB interval, and the mechanism of the inferred processing remains to be established. Because there is no obvious factor-independent terminator sequence separating orf23 and rpoB, transcription originating upstream from the gene for r-protein L7/L12 may also contribute to rpoB expression. However, as shown by the plasmid integration experiments, this upstream transcription is not required for normal growth rate and sporulation frequency. Thus, the rpoB promoter we identified is sufficiently strong to provide adequate levels of beta subunit under most growth conditions.


DISCUSSION

We have identified rpoB, the gene encoding the beta subunit of B. subtilis RNA polymerase. The evidence for this identification includes (i) the antigenic properties of the rpoB product, (ii) the high similarity of the predicted rpoB product with E. coli beta, and (iii) the presence within the rpoB coding region of an alteration conferring rifampicin resistance. We have further shown that B. subtilis rpoB is transcribed from a promoter that overlaps the 3`-end of the preceding orf23 coding region and that this promoter is sufficiently active to support wild type growth rate and sporulation frequency.

The transcriptional organization of the B. subtilis rpoB region stands in sharp contrast to that of E. coli, in which the primary transcripts originate from the promoters of the upstream ribosomal protein operons L11 and L10(54, 55, 56, 57) . In E. coli, differential regulation of r-protein and RNA polymerase subunit expression results partly from the action of a transcriptional attenuator that lies between the gene for r-protein L7/L12 and rpoB(53, 58) and partly from specific translational feedback mechanisms operating on the r-protein and polymerase subunit messages(59, 60, 61, 62) . We do not yet know whether B. subtilis rpoB expression is subject to the same translational regulation as has been proposed for the enteric system. However, because B. subtilis has a strong promoter immediately upstream from rpoB, for which no counterpart exists in E. coli, it is possible that differential regulation of r-protein and RNA polymerase gene expression could be accomplished largely at the transcriptional level.

The E. coli beta subunit plays a key role in transcriptional initiation, elongation, and termination. What information regarding structure-function relationships can be derived from comparison of the predicted sequences of B. subtilis and E. coli beta? As shown in Fig. 2, the two proteins share highest sequence conservation in four large blocks. This degree of conservation between such highly divergent organisms suggests that the four large blocks mediate common functions, and there is ample biochemical and genetic evidence that key residues within these blocks are critical for beta function in E. coli. However, it is the differences between the two beta subunits that provide our most significant results. On the one hand, the four conserved blocks are separated by three regions that are either variable or absent in B. subtilis beta. On the other hand, we find that the B. subtilis protein has a C-terminal, 46-residue extension of the fourth conserved block that is not present in E. coli. These differences appear to be characteristic of the Gram-positive and Gram-negative lineages of eubacteria.

Variable Region 1 (Amino Acid Residues 211-368 in B. subtilis and 224-409 in E. coli)

The deletions Delta(166-328) and Delta(186-433) created within E. coli beta by Severinov et al.(39) had no obvious effect on beta function in vitro, and the Delta(166-328) alteration was non-lethal in vivo, leading these authors to conclude that much of this region was dispensable for minimal beta function. As shown in Fig. 2, there is little overall conservation between these regions in E. coli and B. subtilis beta, and B. subtilis variable region 1 also has 43 fewer residues, supporting the notion that this region is not critical for function. However, as first pointed out to us by Benjamin Hall, this region does contain a common, 69-residue segment that is located near the C-terminal part of variable region 1 in E. coli (corresponding to residues 339-409) and near the N-terminal part in B. subtilis (corresponding to residues 211-279). This common segment shares 48% identity between the two organisms.

In E. coli, this 69-residue segment contains the paf32 alteration (39) and is therefore thought to define part of a contact site with the Alc protein, a site-specific termination factor encoded by bacteriophage T4 which acts as a block to the transcription of host genes(63, 64) . On the basis of these results, Severinov et al.(39) proposed that non-essential regulatory proteins specific to the E. coli system may have evolved to target this dispensable region. However, the conservation of the 69-residue segment within variable region 1 of B. subtilis beta, albeit at a different location, suggests that the segment is widespread among prokaryotes and therefore has a more fundamental role in beta function.

This notion is supported by work of Landick et al.(13) , who identified a series of substitutions in E. coli beta that affects transcription termination in vivo. As shown in Fig. 2, four of these substitutions map within the common 69-residue segment, and a fifth maps in the region immediately adjacent in the E. coli protein. Furthermore, we note that the common segment is also conserved in the beta subunits of Pseudomonas putida(65) , Buchnera aphidicola(66) , Mycobacterium leprae(67) , and Mycobacterium tuberculosis(44) , lending further support to the suggestion of a more universal role. In Pseudomonas and Buchnera, the segment lies toward the C-terminal portion of variable region 1, as is the case in E. coli. In contrast, the mycobacterial segment lies toward the N-terminal portion of the region, as is the case in B. subtilis. Thus, it appears that the difference in location of this segment within variable region 1 reflects a difference in domain organization between Gram-positive and Gram-negative bacteria. The importance of this common, 69-residue segment may also extend to the eukaryotic lineage. Shaaban et al.(6) found that alterations mapping in or near the corresponding region of the second largest subunit of yeast RNA polymerase III also affected termination properties of the enzyme.

Variable Region 2 (Amino Acid Residues 897-898 in B. subtilis and 939-1038 in E. coli)

The phenotypes of deletion and insertion mutations have shown that amino acid residues 940-1040 are dispensable for normal beta function in E. coli(68, 69, 70) . This region is entirely missing from B. subtilis beta, as well as from the homologous RNA polymerase subunits from mycobacteria, chloroplasts, eukaryotes, and archaebacteria(44, 67, 69) , supporting the notion that these residues are not important for function. Because variable region 2 is retained but not highly conserved in the beta subunits of P. putida and B. aphidicola(65, 66) , the possibility remains that some of these residues mediate a dispensable function unique to Gram-negative bacteria.

Variable Region 3 (Amino Acid Residues 984-985 in B. subtilis and 1126-1179 in E. coli)

The complete absence of these 54 amino acid residues in the B. subtilis protein suggests that this region is not critical for beta function. This view is supported by the complete absence of the corresponding region in the beta homologues of chloroplasts and eukaryotes (6, 71) and its significant reduction in the beta subunits of mycobacteria(44, 67) . However, as was the case for variable region 2, variable region 3 is fully retained but not highly conserved in other Gram-negative bacteria (65, 66) .

C-terminal Extension of Conserved Block 4

In vitro reconstitution studies (16, 17) suggest that sequences within conserved block 4 (residues 985-1144 in B. subtilis and 1180-1339 in E. coli) are necessary for holoenzyme formation. When the C-terminal portion of E. coli beta was truncated by as few as 42 residues, it retained the ability to associate with the beta` and alpha subunits to form complexes of the size expected for RNA polymerase core enzyme. However, these complexes did not include the subunit, which confers promoter recognition specificity on the core(52) , leading to the proposal that the C-terminal region of E. coli beta is important for binding (16, 17) . In B. subtilis beta, the corresponding C-terminal region is highly conserved but extends an additional 46 residues beyond the terminus of the E. coli protein (see Fig. 2). This 46-residue, C-terminal extension is highly charged, with 21 acidic residues and 4 basic residues. Notably, the C-terminal regions of the beta subunits from M. leprae(67) and M. tuberculosis(44) also extend an additional 38 residues beyond the terminus of the E. coli protein, and these mycobacterial extensions share 20% identity with the B. subtilis extension (data not shown). Thus, a C-terminal extension of beta is likely characteristic of the Gram-positive lineage rather than specific to B. subtilis. Whether this C-terminal extension has any influence on the binding of factors to core has yet to be determined.


FOOTNOTES

*
This research was supported by Public Health Service Grant GM42077 from NIGMS, National Institutes of Health. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].

§
Supported by Public Health Service Training Grant GM08343 from NIGMS, National Institutes of Health. Current address: Dept. of Food Science, Cornell University, Ithaca, NY 14853.

Current address: Stanford University Genome Center, 855 California Ave., Palo Alto, CA 94304.

**
To whom correspondence should be addressed. Tel.: 916-752-1596; Fax: 916-752-4759; cwprice{at}ucdavis.edu.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; kb, kilobase(s); nt, nucleotide.

(^2)
F. R. Blattner, unpublished results.


ACKNOWLEDGEMENTS

We thank Benjamin Hall and Konstantin Severinov for helpful discussions and for communicating results prior to publication, David Dubnau for providing strain DR1010, Carl Batt for the gift of oligonucleotide primers, and Susan Thomas for technical assistance.


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