Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster

Abstract

We isolated the gene encoding the alpha subunit of Bacillus subtilis RNA polymerase from a Agtll expression vector library by using anti-alpha antibody as a probe. Four unique clones were isolated, one carrying a lacZ-alpha gene fusion and three carrying the entire alpha coding region together with additional sequences upstream. The identity of the cloned alpha gene was confirmed by the size and immunological reactivity of its product expressed in Escherichia coli. Further, a partial DNA sequence found the predicted NH2 terminus of alpha homologous with E. coli alpha. By plasmid integration and PBS1 transduction, we mapped alpha near rpsE and within the major ribosomal protein gene cluster on the B. subtilis chromosome. Additional DNA sequencing identified rpsM (encoding S13) and rpsK (encoding Sll) upstream of alpha, followed by a 180-base-pair intercistronic region that may contain two alpha promoters. Although the organization of the alpha region resembles that of the alpha operon of E. coli, the putative promoters and absence of rpsD (encoding S4) immediately preceding the B. subtilis alpha gene suggest a different regulation.The function of the alpha subunit of the procaryotic DNA-dependent RNA polymerase is unclear. In the Escherichia coli system, there is insufficient genetic evidence to choose between a suggested structural role for alpha or a possible role in transcriptional selectivity (44). Four mutations have been identified in rpoA, the gene encoding E. coli alpha. The rpoA109 alteration prevents late gene expression of phage P2 but has no apparent effect on cell growth (8, 37). In contrast, the conditional rpoA101 and rpoA112 mutations prevent RNA synthesis in vivo at nonpermissive temperatures; these mutations also affect transcriptional fidelity in vitro but not in vivo (15). The phs alteration, which very likely lies within rpoA, has a pleiotropic phenotype which includes altered metabolism of melibiose, glutamate, sulfur, and arabinose, as well as a Cym auxotrophy (9, 32). Transcription of the araBAD operon is specifically affected in a phs background, leading Rowland et al. (32) to suggest that alpha is involved in transcriptional selectivity through interaction with either the promoter or with other transcriptional factors.We undertook a genetic analysis of the alpha gene of Bacillus subtilis, a procaryote which is evolutionarily distant from E. coli and which undergoes a simple developmental process, sporulation. By taking advantage of the available developmental phenotypes (26), we have additional means to characterize the interactions of alpha with other components of the B. subtilis transcriptase. We can also compare the organization and regulation of the B. subtilis rpoA region to that of E. coli, which comprises, in addition to alpha, at least 29 genes active in translation (23). E. coli rpoA lies in an operon with four ribosomal protein genes in the order promoter, rpsM (encoding ribosomal protein S13), rpsK (S11), rpsD (S4), rpoA, and rplQ (L17). Transcriptional and posttranscription...

Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis

“…Construction of a library of EcoRI-digested genomic DNA of B. subtilis in the Xgtll expression vector has been described previously (47). At the time this project was initiated, the library had a titer of 2.4 x 108 PFU/ml and 75% of the phage had inserts.…”

Bacillus subtilis mutants with alterations in ribosomal protein S4

“…The strR mutation maps to the far end of the ribosomal gene cluster near 120, since it is only 5% linked by transformation to rpsE, which is near the end of the spc operon. Genes known to be immediately downstream from spc are the a operon genes (3,31), the gene for ribosomal proteins S9 (8) and S20 (22), the attachment site for phage SPO2 (23), gerD (33), and three rRNA operons, rrnI, rrnH, and rrnG (17). The low transformation linkage with rpsE suggests that the location of strR is downstream of a, which is contiguous with spc, and probably downstream of the genes for S9 and S20, which show higher linkage to rpsE.…”

Glu-tRNA Gln amidotransferase: A novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation

“…The B. subtilis gt11 library was a gift of C. W. Price (University of California, San Diego) (12). B. subtilis 168 DNA was prepared from the wild-type Marburg strain.…”