A method for the identification of promoters recognized by RNA polymerase containing a particular-sigma factor:: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ

We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, P-REN40 recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sn from various developmental stages has shown a high degree of similarity of P-REN40 to consensus sequence of flagellar and chemotaxis promoters, The promoter was induced at the time of aerial mycelium formation, and was off in the So strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (M-I 30006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%). (C) 1998 Elsevier Science B.V.