Transcription from the P1 promoters of Micromonospora echinospora in the absence of native upstream DNA sequences

Abstract

We demonstrated previously that the 0.4-kilobase DNA fragment from Micromonospora echinospora contains multiple tandem promoters, Pla, Plb, Plc, and P2, which are also functional when cloned into Streptomyces lividans. We now show by in vitro transcription with Streptomyces RNA polymerase that each of these promoters is an authentic initiation site, rather than a processing site for transcripts which initiate further upstream. The DNA sequence requirements for the closely spaced promoters Pla, Plb, and Plc, which are coordinately induced during stationary phase in M. echinospora, were examined by deletional analysis in S. lividans. The Pla and Plb promoters were functional despite deletion of native sequences 5 and 17 base pairs upstream of each initiation site, respectively. Thus, Pla and Plb had greatly reduced upstream DNA sequence requirements compared with typical procaryotic promoters. In contrast, transcription from promoter Plc was significantly decreased when native sequences 34 base pairs upstream were replaced.We have been studying promoter structure in the actinomycete Micromonospora echinospora (NRRL 15839), which grows as multicellular mycelia and has the capacity to form spores after the growing phase. During the stationary phase, M. echinospora produces the calicheamicins (19), a novel family of antitumor antibiotics that causes site-specific double-stranded cleavage of DNA (29).Previously, we defined a set of multiple tandem promoters within a 0.4-kilobase (kb) DNA fragment that has several novel features (1). The P1 promoter region consists of three transcriptional start sites separated by 12 and 17 nucleotides (nt), which are maximally active during stationary phase, concurrent with calicheamicin production. Promoter P2 is located 80 base pairs (bp) downstream from the P1 region and is maximally active during the exponential phase of the life cycle. The P1 and P2 promoters are potentially useful for the investigation of gene activation in actinomycetes during the transition from exponential to stationary phase, when many secondary metabolites are produced (22).The P1 and P2 promoters, and an additional site designated Ptk, are recognized by Streptomyces lividans transformants harboring the 0.4-kb fragment on a plasmid (1). We have therefore utilized the well-characterized genetic system of Streptomyces for more detailed transcriptional analysis. The P1 and P2 promoters belong to the major class of Streptomyces promoters that do not function in Escherichia coli (12,15,16). A consensus sequence has not been identified for this class of promoters. The occurrence of multiple forms of RNA polymerase holoenzyme in Streptomyces spp. which direct transcription from different promoters (6, 28) is consistent with the diversity of promoter sequences found in this organism. Recently, four different genes apparently encoding sigma factors were identified in S. coelicolor by homology to sequences derived from E. coli and Bacillus subtilis (26). In addition, the whiG locus also encodes a putative sigma facto...

The sapA promoter from Streptomyces coelicolor requires activation sites and initiator-like sequences but No -10 or -35 sequences

“…Perhaps the closest analogous promoters are P1a and P1b from M. echinospora. Baum et al (2,3) have shown that P1a and P1b do not require native DNA further than 5 and 20 bp upstream from the transcription start point, respectively. There are some intriguing parallels between these M. echinospora promoters and the sapA promoter.…”

Mutations in the P1 promoter region of Micromonospora echinospora

“…5, lane 1). In contrast, when RNA was isolated from the isogenic strain containing the 0.4-kb insert within plasmid pPP14 (lane 2), transcripts Pla, Plb, Plc, Ptk, and P2 were detected, as observed previously (1,2). The start site of each promoter has also been determined by using dinucleotides to prime transcriptional runoff assays with Streptomyces RNA polymerase (1).…”

“…Second, the Pla and Plb promoters lack upstream sequence requirements; i.e., they function in S. lividans even if native DNA sequences are replaced just 5 nucleotides before the Pla start site and 20 nucleotides before the Plb start site. In contrast, transcription from the Plc promoter located downstream of Plb is diminished in activity as a result of these substitutions 35 nucleotides upstream of its start site (1). The Plc promoter therefore appears to resemble many Escherichia coli promoters in requiring sequences that extend to at least 40 nucleotides upstream of transcriptional initiation (19).…”

“…First, the three transcriptional start sites of the P1 region are unusually close. The Pla initiation site, defined by in vivo studies (2) and by runoff transcription studies with Streptomyces RNA polymerase (1), is 15 bp upstream of Plb, which is 15 bp from Plc ( Fig. 1).…”

“…A transformation system has been described for M. echinospora, but problems with plasmid maintenance and/or replication are difficult obstacles in conducting genetic studies in that organism (16a). Therefore, we resorted to using the related actinomycete Streptomyces lividans for genetic studies (1,2). These promoters are expressed in a transformant of S. lividans carrying a plasmid that contains the 0.4-kb fragment, although in S. lividans all of these promoters are expressed constitutively (2).…”