A novel penicillin-binding protein (PBP 5*) with D,D-carboxypeptidase activity is synthesized by Bacilus subtilis, beginning at about stage m of sporulation. The complete gene (dacB) for this protein was cloned by immunoscreening of an expression vector library and then sequenced. The identity of dacB was verified not only by the size and cross-reactivity of its product but also by the presence of the nucleotide sequence that coded for the independently determined NH2 terminus of PBP 5*. Analysis of its complete amino acid sequence confirmed the hypothesis that this PBP is related to other active-site serine D,D-peptidases involved in bacterial cell wall metabolism. PBP 5* had the active-site domains common to all PBPs, as well as a cleavable amino-terminal signal peptide and a carboxy-terminal membrane anchor that are typical features of low-molecular-weight PBPs. Mature PBP 5* was 355 amino acids long, and its mass was calculated to be 40,057 daltons. What is unique about this PBP is that it is developmentally regulated. Analysis of the sequence provided support for the hypothesis that the sporulation specificity and mother cell-specific expression of dacB can be attributed to recognition of the gene by a sporulation-specific sigma factor. There was a good match of the putative promoter of dacB with the sequence recognized by sigma factor E (oE), the subunit of RNA polymerase that is responsible for early mother cell-specific gene expression during sporulation. Analysis of PBP 5* production by various spo mutants also suggested that dacB expression is on a oE-dependent pathway.The penicillin-binding proteins (PBPs) are a family of membrane-bound enzymes that are active in the metabolism of prokaryotic cell walls (16). They are evolutionarily related to some of the soluble P-lactamases and D,D-peptidases secreted by bacteria and also have some structural features in common with a penicillin-binding transmembrane protein involved in signal transduction (24,46,62). The active-site serine of all of these penicillin-interactive proteins is part of the conserved domain S-X-X-K, which is located near the amino terminus of the 1-lactamases and the mature form of those PBPs with a molecular mass of less than 50 kDa but more towards the middle of the sequence in the larger PBPs (24). Closer to the carboxy terminus, all of these proteins also have the sequence K-T-G, H-T-G, or K-S-G, which is an essential part of the tertiary structure of the active site (24). The class A ,-lactamases and the PBPs also have a third domain (S-X-N) in common, which is located between the other two (46).Our studies have focused on PBP 5*, which is different from all of the other PBPs described so far because it is developmentally regulated. This protein, which has D,Dcarboxypeptidase activity in vitro, is not synthesized by Bacillus subtilis until about stage III of sporulation (43,49,50). Although it has a lower molecular weight than any of the six vegetative PBPs in this species, it is not a derivative of one of them (10, 50). Its locatio...

Initiation of RNA synthesis by the phage polymerases is abortive if the concentration of pyrimidine triphosphates is limiting. Under abortive initiation conditions the polymerases repeatedly initiate transcription but produce ribooligonucleotides that terminate just prior to the first occurrence of the limiting substrate. Abortive initiation is most severe if the limiting substrate occurs within the first 8-12 nucleotides of the nascent RNA chain and is particularly evident when UMP is limiting. The formation of stable elongation complexes (as determined by gel retardation experiments) occurs after the synthesis of an RNA product 8-12 nucleotides in length.

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