Transcription of the Rhodobacter sphaeroides cycA P1 promoter by alternate RNA polymerase holoenzymes

Abstract

These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroides cytochrome c2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterized Escherichia coli heat shock (sigma32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Esigma32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of -7. A point mutation at position -34 that is towards the E. coli Esigma32 -35 consensus sequence (G34T) increased cycA P1 activity approximately 20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed -10 or -35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes, cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Esigma37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Esigma38). (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10-19, 1998).

FIG. 1

In vitro transcription of cycA P1 templates by different RNA polymerase holoenzymes. (A) Transcription assays were performed as described in Materials and Methods with plasmid templates which lack (ΔP) or contain the wild-type cycA P1 promoter (w.t.) cloned upstream of the spf transcriptional terminator. The RNA polymerases used (+) are indicated above each set of samples. Eς^D and Eς³⁷ are different R. sphaeroides holoenzymes that can be separated by Q-Sepharose chromatography (10). Where indicated, recombinant R. sphaeroides His-ς^D was added to increase the saturation of RNA polymerase preparations. The arrows indicate the positions of RNA1 and cycA P1 transcripts. (B) Analogous assays were performed with a template containing the G34T mutant cycA P1 promoter (−34T).

FIG. 2

Upstream or downstream mutations that do or do not alter cycA P1 activity. The sequence of wild-type cycA P1 (plasmid pCM241) is shown at the top with potential −10 and −35 elements set off by spaces. Bases that differ in individual mutant promoters are shown in lowercase letters and in bold type. Small deletions are indicated by dots. Vector sequences contributed by the deletions are underlined twice in panel A. Bases that are identical to those of the wild-type cycA P1 sequence are shown in uppercase letters. The reported β-galactosidase (LacZ) activities are averages of at least three independent cultures and are shown with standard deviations. LacZ activities that are significantly lower than the wild-type activity (∗) or significantly higher than a wild-type reporter gene (‡) are indicated.

FIG. 3

Transcription of wild-type cycA P1 and G34T templates by individual RNA polymerase holoenzymes. Templates which lack the cycA P1 sequence (ΔP), the wild-type cycA P1 promoter (w.t.), or the G34T (−34T) mutation in the cycA P1 promoter were used as indicated directly above the gel. Above each set of lanes (ΔP, −34T, and w.t.) is indicated the source of RNA polymerase holoenzyme (w.t. holo, holoenzyme preparations from wild-type cells; ΔrpoH holo, analogous material from a ΔRpoH mutant [11]; ΔrpoH core, core RNA polymerase from a ΔRpoH mutant). For assays where core RNA polymerase was reconstituted with potential sigma subunits, ς³⁷ and ς^D were obtained from a wild-type RNA polymerase sample (fractions 9 and 19, respectively, in Fig. 4A). ς³⁸ was obtained from the ΔRpoH RNA polymerase sample (fraction 20 in Fig. 4B). The positions of transcripts (arrows) are indicated as follows: RNA1, the Eς^D-dependent transcript from the oriV promoter present on all templates (10); cycA P1, the specific cycA transcript produced by templates that contain wild-type or G34T mutant promoters.

FIG. 4

Transcription of the G34T mutant promoter by reconstituted RNA polymerase holoenzymes. (A) Transcripts produced when the G34T promoter is incubated with different R. sphaeroides RNA polymerase samples. The positions of transcripts are indicated to the right of the rightmost gels as follows: RNA1, the Eς^D-dependent oriV transcript from the transcription plasmid template (10); cycA P1, the product of the G34T promoter. Reactions in panel A used renatured proteins from a wild-type RNA polymerase sample (shown on the left) as a source of potential sigma factors for addition to core RNA polymerase (shown in panel B) from the ΔRpoH mutant (11). Reactions in panel B used renatured proteins from a RpoH null mutant as a source of potential sigma factors (11) for reconstitution with the same ΔRpoH core RNA polymerase. In panels A and B, numbers above the rightmost gels denote the SDS-polyacrylamide gel fraction that was added to core RNA polymerase as a source of potential sigma factor. Lane 0 contains transcripts produced by core RNA polymerase alone. Lane His-ς^D in panel A shows transcripts produced when recombinant R. sphaeroides His-ς^D is added to core RNA polymerase. To the left of the leftmost gels in panels A and B is shown the polypeptide composition of RNA polymerase samples used for in vitro transcription reactions. These samples include RNA polymerase samples from wild-type cells (w.t. RNAP in panels A and B) or a ΔRpoH mutant (11) (ΔrpoH RNAP in panel B) and core RNA polymerase from an RpoH null strain (ΔrpoH core in panel B) separated alongside prestained [MW (p)] or unstained [MW (u)] protein molecular weight standards (molecular weights [in thousands] indicated on the left margin). Numbers to the right of the leftmost gel indicate how this gel was sliced to obtain proteins to be tested for sigma factor activity. Individual RNA polymerase subunits are indicated to the left.

FIG. 5

Effects of point mutations on cycA P1 function. Below the consensus promoters for E. coli Eς⁷⁰ and Eς³² (top) are listed mutations that increase (in bold type and above the wild-type promoter) or decrease (below the wild-type promoter) cycA P1 activity (normalized to wild-type activity of 1.00; see Fig. 2B to D for primary data). Relative activities were corrected for LacZ levels produced from a control plasmid lacking cycA P1 sequences (pBM4A [Fig. 2]). Asterisks indicate mutations that significantly alter promoter activity. In cases where the cycA P1 mutation is not identical to the name of the mutant promoter, its name is provided in parentheses. Bases shown in bold type in the cycA P1 sequence are identical to the bases in the E. coli Eς⁷⁰ and Eς³² consensus promoters.