DnaA-stimulated transcriptional activation of orilambda: Escherichia coli RNA polymerase beta subunit as a transcriptional activator contact site

Abstract

We present evidence that Escherichia coli RNA polymerase beta subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage lambda. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of lambda plasmids; this suppression was allele-specific. When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro. Therefore, we conclude that DnaA may contact the beta subunit of RNA polymerase during activation of the pR promoter. A new classification of prokaryotic transcriptional activators is proposed.

Figure 1

Map of bacteriophage λ replication region present in standard λ plasmids (also those used in this work). Genes cro, cII, O, P, and ren are marked (these plasmids do not contain a gene coding for the CI repressor). All promoters (p) and terminators (t) are indicated. The p_R promoter region contains the operator sites, and the p_M promoter (whose activity could potentially play a role in transcription initiated at p_R) is repressed by the cro gene product and cannot be activated in the absence of the CI protein. Transcripts are presented as arrows with arrowheads indicating directionality of transcription. Origin of λ DNA replication (oriλ) is located in the middle of the O gene. Restriction sites for HindIII, NsiI, BstXI, and DraI endonucleases (used for preparation of DNA fragments for templates in in vitro transcription reactions) are indicated.

Figure 2

Activation of the p_R promoter by DnaA protein in vitro. (A) Examples of autoradiograms after in vitro transcription experiments. (Left) In the control, 0.5 μg of bacteriophage T7 DNA was used as a template. (Right) In the experiment, 0.5 μg of a HindIII–DraI fragment of plasmid pKB2 was used as a template DNA. The reaction mixtures contained no DnaA (lanes −) or 35 ng (final concentration, 26.6 nM) of DnaA (lanes +). The position of p_R-derived transcripts (812 nucleotides long) is indicated. (B) Average data from six experiments of in vitro transcription. Template DNA (0.5 μg) and DnaA protein (as indicated) were used, and appropriate bands on the autoradiograms were quantified by densitometry. Results very similar to those obtained with the HindIII–DraI fragment of pKB2 were observed when NsiI–NsiI fragment of pKB2 or whole pKB2 plasmid linearized with BstXI were used as DNA templates (the lengths of p_R-derived transcripts were 288 and 269 nucleotides, respectively). The positions of appropriate restriction sites in λ plasmid DNA are depicted in Fig. 1.

Figure 3

(A Upper) Map of the replication region of bacteriophage λ (also present in λ plasmids) from the p_R promoter to oriλ. The scale is given in base pairs, the p_R promoter is marked by the thick arrow, putative DnaA boxes are represented by vertical rectangles, and sequences recognized by the O replication initiator (O boxes) and the A+T-rich region are also indicated. (A Lower) The fragment of λ DNA present in the lacZ fusions is indicated, and the orientation of the DnaA-box is marked by arrow (this part of the figure is not drawn to scale). (B) Sequences of the wild-type and scrambled DnaA boxes present in appropriate fusions. (C) The activity of β-galactosidase per single copy of the fusion is presented (in a table form) for both fusions in dnaA⁺ and dnaA-null hosts. It seems that p_R is activated only in the wild-type host harboring the “wild-type” fusion (YES in the table) and that the lack of either functional DnaA protein or DnaA-binding site immediately downstream of p_R results in no activation of the promoter (NO in the table), and the obtained values reflect residual p_R activity.

Figure 4

Activation of the p_R promoter by DnaA protein in vitro in the presence (•) and absence (▴) of the DnaA-binding sequence downstream of the promoter. Template DNA (0.5 μg) and indicated amounts of DnaA protein were used. Similar results were obtained when different templates, all derived from p_R–HG and its analogue bearing the scrambled DnaA box, were used: EcoRI–Bsu36I fragments, the plasmids linearized with Bsu36I, and the plasmids linearized with ClaI (the lengths of p_R-derived transcripts were 512, 512 and 1112 nucleotides, respectively). EcoRI site is located upstream of p_R and Bsu36I and ClaI sites are located in the lacZ gene of plasmid p_R–HG and its analogue. Average data from seven experiments are presented; appropriate bands on the autoradiograms were quantified by densitometry.

Figure 5

Model for activation of the p_R promoter by DnaA protein that assume a direct interaction between DnaA and RNA polymerase β subunit. The regions −35 and −10 of the promoter and location of the DnaA box important for the activation are marked. The scheme is not drawn to scale.