RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: Its positive and negative effects on transcription (enhancer sequence/osmoregulation/porins)

The OmpR binding sequence (OBS) in the upstream region of the ompF promoter ofEscherichia coli was fused to 27 synthetic promoters. Transcription from a number of weak promoters, regardless of their sequences, was dramatically activated in the presence of OmpR, a transcriptional activator. In vivo DNA footprinting revealed that OmpR enhanced the binding ofRNA polymerase to the promoters. This enhancement was essential for transcription of weak promoters, while OmpR binding to the OBS fused to a strong promoter was inhibitory for transcription. These results indicate that OmpR stabilizes the formation of an RNA polymerasepromoter complex, possibly a closed promoter complex, and that a transcription activator can serve not only as a positive but also as a negative regulator for gene expression. Studies on transcriptional activation in eukaryotic cells have demonstrated that certain eukaryotic transcriptional activators for RNA polymerase II are able to function in heterologous systems as long as the test genes contain DNA sequences recognized by the transcriptional activators (for a review, see ref. 1). In contrast, no transcriptional activators in Escherichia coli have been shown to work in heterologous systems, and efforts to construct generalized activators have failed (1). In the present report, we demonstrate that OmpR, a transcriptional activator for ompC and ontpF of E. coli, the genes for major outer membrane porins (2), is able to function as a generalized activator for various unrelated promoters. There are a number of E. coli genes whose transcription is activated by a protein factor binding to a specific sequence upstream of the RNA polymerase recognition site (3-5). The RNA polymerase recognition sites for genes requiring transcriptional activators for "7O RNA polymerase are considerably different from the consensus sequence consisting of the -35 region (TTGACA) and the -10 region (TATAAT) relative to +1, the transcription initiation site (6). Because the sequences of these promoters diverge from the consensus sequence, RNA polymerase is either unable to bind to the promoters or to isomerize the closed complex of the promoter and RNA polymerase to the open complex without the aid of transcriptional activators. In the case of ompC and ompF, RNA polymerase is unable to transcribe these genes without OmpR because the -35 and -10 regions of these genes are quite different from the consensus sequence (7, 8). The OmpR binding sites for both genes have been shown to exist in the regions from -40 to -100 (9, 10). Recently, by in vivo DNA footprinting we demonstrated that within the OmpR binding regions there are two different motifs, the F and C boxes, and that OmpR binding to these motifs plays important roles in the regulation of ompF and ompC expression (11). We now examine whether OmpR is able to activate transcription from promoters with different sequences and whether OmpR binding to OmpR binding sequences (OBSs) enhances binding of RNA polymerase to a promoter sequence. For this purpose, the OBS from the ompF gene was fused to 27 different synthetic promoters and transcription from these promoters in vivo was examined in the absence and presence of OmpR with lacZ used as a reporter gene. MATERIALS AND METHODS Construction of Synthetic Promoters. Plasmid pTB0533 (pBR322 derivative) contains the Xba I/BamHI DNA fragment of the ompF promoter from -195 to + 118, which encompasses a part ofthe coding sequence from the initiation codon to the fourth codon. This fragment also contains four OBSs (Fa, Fb, Fc, and Cd boxes; see Fig. LA) as well as the -35 and -10 promoter sequence. The lacZ gene coding sequence starting from the eighth codon is fused in-frame to the above ompF sequence. By site-specific mutagenesis the 7-base-pair (bp) DNA sequence immediately after the Cd box (Fig. lA) of ompF, TCACGGf (asterisk indicates the first base in the -35 region of ompF), was replaced with the sequence AAGAEI to generate a unique Bgl II site (underlined). The DNA sequence between this Bgl II site and an original Pst I site at the + 1 position (transcription initiation site) of ompF was then replaced with synthetic oligonucleotides. For example, the sequence for promoter 1 consists of the consensus -35 sequence (TTGACA), an 18-bp spacer sequence (CTrTAAGCTTCCGGCTCG), the -10 sequence of the ompC promoter (GAGAAT), and a 9-bp sequence (GICGACAAT) after the -10 sequence to generate a unique Sal I site (underlined). The spacer sequence is identical to that of the lac promoter (6) except that the sixth T residue (indicated by an asterisk) was changed to A to create a unique HindIII site (underlined). The 9-bp sequence after the -10 sequence is from the lacZ promoter sequence, where the A residue with an asterisk indicates the transcription initiation site. Other synthetic promoters (from no. 2 to no. 27) were constructed by replacing DNA sequences either between the Bgl II and HindIII sites (for the -35 region) or between the HindIII and Sal I sites (for the -10 region) with their respective oligonucleotides. In all promoters except for promoter 10, the DNA sequences beside the -35 and -10 sequences are identical to those for promoter 1 described above. In promoter 10, the 2-bp sequence (CG) at the 3' end of the 18-bp spacer sequence was removed to reduce the promoter activity, since the promoter with the 18-bp sequence was lethal to cells. In all promoters, the -35 regions Abbreviation: OBS, OmpR binding sequence. 5940The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 87 (1990) 5941 are S bp downstream of the Cd box (see Fig. 1). Because of the difference of the first base in the -35 regions in different promoters, only those promoters whose -35 sequences start with a T residue retain the Bgl II site. An ompR' E. coli strain (MC4100; ref. 2) and its isogenic ompRstrain (MH1160; ref. 2) were then transformed with these plasmids constructed as described above. In Vivo Dimethyl Sulfate DNA Footprinting. Cells carrying the synthetic promoter plasmids were grown in L-broth medium (20 ml) supplemented with ampicillin (50 Ag/ml) to midlogarithmic phase. Dimethyl sulfate [final concentration, 0.1% (vol/vol)] was directly added to the cells under vigorous shaking at 370C. The treatment was continued for 20 sec before 2 ml of0.5 M EDTA (pH 8.0) was added to the culture. After 30 sec, 15 g of ice was added directly to the culture. The plasmid DNA was isolated and digested with BamHI, which cuts the plasmid DNA at 4120 bp downstream from the +1 position (transcription initiation site). The linear DNA fragment was then gel isolated and end labeled with [t-32P]ATP. The labeled DNA fragment was then digested with Xba I, which cuts at the -195 position. The resultant Xba I/BamHI fragment was again gel purified and cleaved with 1 M piperidine (G>A cleavage) and analyzed on a 6% sequencing gel. DNA manipulation is according to Maniatis et al. (12). f-Galactosidase Activity Assay. f3-Galactosidase assay was performed as described and expressed in Miller units (13). Since the promoter 10 plasmid was unstable, Lac cells were constantly generated. Therefore, the B-galactosidase activity was corrected by using the ratio of Lac' to Laccells in a given culture, which was obtained by plating the culture on Lac indicator plates.