The basic features of the transcriptional machinery are remarkably conserved in all organisms. In particular, the , `, and 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 ` subunit of Escherichia coli shares eight regions of sequence similarity with the largest subunit of the eukaryotic enzymes(3) , and the subunit of E. coli was initially reported to share nine regions with the second largest subunit(4, 5) . Additional sequence alignment of homologues has further refined the boundaries of these nine regions into 12 colinear segments, which are conserved in both eubacteria and eukaryotes(6) . , 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 or ` 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 subunit(7, 8) . Here, we describe the genetic and transcriptional organization of the region containing rpoB, the gene encoding the subunit, and show that this organization differs substantially from that of the corresponding region in E. coli. Because 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 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 but nonetheless contained a common, 69-residue segment. We infer from these results that the common segment has a more fundamental role in function than previously believed.

MATERIALS AND METHODS

Bacterial Strains, Phage, and Genetic Methods

Location of the Alteration Caused by the rfm2103 Allele

()

Construction of Transcriptional Fusions

Figure 1: Genetic organization of the B. subtilis -` 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 and ` subunits of RNA polymerase, all of which are transcribed from left to right; the arrows adjacent to the L7/L12 and ` 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 and ` 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 -` 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-) interval. The sequence shown represents the C-terminal coding region for P23, the P23- intercistronic region, and the N-terminal coding region for . 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-) 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--D-galactoside. For quantitative estimates of promoter activity, we performed -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 A/min/ml/unit of optical density at 600 nm.

Mapping the 5`-Ends of rpoB Message

Computer Analysis

RESULTS

Isolation of gt11 Bacteriophages Encoding the Subunit

Genetic Organization of the -` Region

()

Identification of B. subtilis rpoB

Figure 2: Alignment of the primary sequences of the B. subtilis and E. coli subunits. The predicted sequence of B. subtilis (upper) is from this work and that of E. coli (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 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 subunit(42, 43, 44) . In E. coli , 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 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 catalytic function. Affinity labeling studies indicate that both Lys-1065 and His-1237 of E. coli 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 homologues characterized to date(10, 12) , and these residues are found at the expected positions in B. subtilis (Fig. 2). Genetic evidence underscores the importance of these residues in 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 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 , we conclude that we have isolated the gene encoding the B. subtilis 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 . Second, as an additional biological criterion, we mapped within the B. subtilis coding sequence an alteration conferring rifampicin resistance. In keeping with the standard genetic nomenclature, we will refer to the gene for the subunit as rpoB.

Transcriptional Organization of the -` Region

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- 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 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 -galactosidase activities directed by the four transcriptional fusions depicted in panelA. The strains carrying these fusions were grown and assayed for -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, (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 subunit under most growth conditions.

DISCUSSION

We have identified rpoB, the gene encoding the 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 , 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 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 ? 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 function in E. coli. However, it is the differences between the two 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 . 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)

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 , albeit at a different location, suggests that the segment is widespread among prokaryotes and therefore has a more fundamental role in function.

This notion is supported by work of Landick et al.(13) , who identified a series of substitutions in E. coli 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 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)

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

C-terminal Extension of Conserved Block 4

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.

()

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

()

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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