The Streptomyces galP1 promoter has a novel RNA polymerase recognition sequence and is transcribed by a new form of RNA polymerase in vitro

Abstract

We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions ؊34, ؊36, and ؊11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The ؊35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.Sequences that signal transcription initiation in bacteria play an important role in gene regulation. Among the best studied of these sequences are those involved in RNA polymerase recognition. The observation that sequences centered around Ϫ10 and Ϫ35 bp upstream of the transcription start site are highly conserved in many promoters (35), the fact that these sequences are required for promoter activity (46), and the discovery of RNA polymerase heterogeneity in Bacillus subtilis (26) supported the model that in most cases the sigma subunit of RNA polymerase makes sequence-specific contacts in these two regions of bacterial promoters. The strongest evidence comes from experiments that show allele specificity of suppression between mutations in the sigma subunit of RNA polymerase and compensating base changes in the DNA sequence of promoters. Amino acid changes in various sigma proteins, H (8, 47), A (22), and E (43) of B. subtilis and 70 of Escherichia coli (13,38), show that these proteins contact the Ϫ10 and Ϫ35 regions of their cognate promoters.While the RNA polymerase holoenzymes that catalyze transcription in Streptomyces spp. are presumed to be functionally similar to those of other bacteria, relatively little is known about sequences that are required for RNA polymerase recognition in this complex bacterium. At least eight RNA polymerase holoenzymes have been identified in Streptomyces, and promoters with putative RNA polymerase binding sites similar to known consensus sequences have been described (reviewed in references 3 and 41). A consensus sequence prototypical of eubacteria (TTGACA centered around Ϫ35 and TATAAT centered around Ϫ10) appears in several Streptomyces promoters, and at least four genes whose predicted products resemble E. coli 70 have been cloned and characterized (2, 4, 42). One of these genes is apparently essential (4). The existence of an additional sigma factor that may be functio...

A New Piece of an Old Jigsaw: Glucose Kinase Is Activated Posttranslationally in a Glucose Transport-Dependent Manner in <i>Streptomyces coelicolor </i>A3(2)

“…Mutants lacking a functional glkA gene are unable to grow on glucose as sole carbon source and are deregulated in glucose repression of several catabolite-controlled genes and operons including those for the utilization of agar, glycerol, maltose and galactose [Angell et al, 1992;Brawner et al, 1997;Hindle and Smith, 1994;Kwakman and Postma, 1994]. Additionally, restoration of glucose kinase activity by introduction of a functional glucose kinase gene from Zymomonas mobilis failed to restore catabolite control [Angell et al, 1994].…”

StoPK‐1, a serine/threonine protein kinase from the glycopeptide antibiotic producer Streptomyces toyocaensis NRRL 15009, affects oxidative stress response

“…Homology searches with this +1 to −50 region showed strong similarity (>50%) to the promoter regions in nshP , which encodes a 23S RNA methylase in Streptomyces actuosus (Li et al , 1990). Both regions contain a putative class A element (Bourn and Babb, 1995) and a −35 region similar to the galP1 −35 region, which may be recognized by an alternative sigma factor (Brawner et al , 1997). In addition, homology was also noted to a fragment of S. coelicolor DNA (cosmid F12) containing a putative glucose‐6‐phosphate dehydrogenase and upstream of a D ‐3‐phosphoglycerate dehydrogenase.…”

“…Analysis of the DNA sequence upstream of stoPK‐1 reveals similarities to promoter regions in other Streptomyces loci such as the thiopeptide antibiotic resist‐ance gene nsh (Li et al , 1990) and the glucose‐repressed and galactose‐induced promoter from Streptomyces lividans , galP1 (Brawner et al , 1997). A class A‐type promoter element (TAGACT) typical of −10 regions in many Streptomyces genes (Bourn and Babb, 1995) is only 6 bp from the predicted initiation codon of stoPK‐1 , the exact spacing arrangement seen in the nsh promoter (Li et al , 1990), and a −35 region found to be critical in galP1 expression and thought to be recognized by a novel sigma factor (Brawner et al , 1997). Furthermore, a BLAST search of the putative promoter region of stoPK‐1 with the S. coelicolor sequence database revealed homology to a fragment of DNA containing a putative glucose‐6‐phosphate dehydrogenase and upstream of a D ‐3‐phosphoglycerate dehydrogenase.…”

Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction

“…The agarase gene, dagA, has four promoters (15) and is transcribed by at least three different RNA polymerase holoenzymes (16). The galP1 promoter, which directs glucose-sensitive, galactosedependent transcription of the Streptomyces galactose utilization operon, contains an unusual RNA polymerase binding site and is apparently transcribed by a new form of RNA polymerase holoenzyme (25). Mutational analysis of the promoter region of galP1 also revealed a complex operator that consists of hexamer and direct repeat sequences that overlap the RNA polymerase binding site (26).…”