The stimulation of Fnr-dependent transcription from the narG promoter by NarL-phosphate is known to require a cis-acting sequence, the NarL box, located approximately 195 bp upstream from the transcription start site, and the interaction of integration host factor (IHF) with a binding site in the intervening region (positions -110 to -140) between the NarL box and the transcription start site. By gel retardation and DNase I protection studies, we have demonstrated that NarL-phosphate, produced by the reaction of purified NarL with acetyl phosphate, specifically binds to a fragment derived from the upstream region of the narG promoter. The fragment was protected by NarL-phosphate binding to two distinct regions. One was an extended sequence of approximately 40 bp surrounding the NarL box at -195; the second was located downstream from the IHF-binding region and included a sequence extending from positions -80 to -120. Alteration by site-directed mutagenesis of a putative inverted NarL box sequence identified within the downstream protected region in a plasmid containing a narG-lacZ fusion eliminated the NarL-phosphate-mediated stimulation of transcription. NarL-phosphate bound to the two regions independently from IHF binding and it bound to each site independently when the two sites were separated by cleavage of the promoter fragment. Stimulation of transcription from the narG promoter by NarL-phosphate appears to result from the formation of a folded protein-DNA structure created by the binding of NarL-phosphate to multiple sites on either side of an IHF-induced bend in the upstream region of the promoter.

The effects of mutations in the -10, -35, and Fnr box regions of the narGHJl promoter of Escherichia coli were determined by assaying the expression of I8-galactosidase from narG::lacZ fusion plasmids under aerobic and anaerobic conditions. A 1-base change in the -10 hexamer completely abolished expression, whereas a 3-base change to create the consensus TATAAT resulted in significant aerobic as well as anaerobic expression. A mutation in the putative -35 hexamer did not affect anaerobic expression but reduced aerobic expression from the construction with the -10 consensus sequence. A mutation in the Fnr box severely reduced anaerobic expression but did not affect aerobic expression. When the complete 5' region of the nar operon including the NarL box was present, nitrate stimulated both aerobic and anaerobic expression. Stimulation of expression by nitrate occurred in an fnr mutant but not in a narL mutant. We conclude that the rate of transcription of the nar operon is dependent on two distinct modes of transcription. One mode, which occurs at low levels, depends on the -10 and -35 hexamer sequences and is dramatically enhanced by changing the -10 sequence to the consensus TATAAT. The second depends on the -10 and Fnr box sequences but is independent of the -35 sequence. This second mode occurs at a very high level under anaerobic conditions when Fnr is activated and is also enhanced by changing the -10 sequence to the consensus TATAAT. NarL, activated by nitrate, stimulated both modes of transcription, indicating that it does not act through Fnr but that it directly affects the interaction of RNA polymerase with the promoter.The expression of the narGHJI operon, which encodes the respiratory nitrate reductase of Escherichia coli, is controlled by two transacting factors, Fnr and NarL (5, 22). Fnr, activated by an unknown mechanism under conditions of oxygen depletion, induces the transcription of the narGHJI operon as well as a number of genes encoding enzymes of anaerobic metabolism (6,11,16,22,25). Fnr is thought to bind to a highly conserved palindromic DNA sequence (Fnr box) found in all Fnr-activated promoters centered at a position approximately 40 bp from the transcription start site (7,10,21). In the presence of nitrate, NarL further stimulates anaerobic transcription from the narGHJI promoter through interaction with a cis-acting sequence (NarL box) located approximately 200 bp upstream from the transcription start site (12).In the narGHJI promoter (Fig. 1), the Fnr box is centered -41.5 bp from the transcription start site (27) and, as generally found for positively regulated promoters (16) and other Fnr-dependent promoters (7), the -10 and -35 sequences are poorly conserved analogs of the -10 and -35 consensus sequences recognized by the u70-RNA polymerase of E. coli. On the basis of promoter reconstruction studies (27) we concluded that anaerobic transcription is dependent on the sequence of the -10 region and the critical positioning of the Fnr box and proposed that interaction of Fnr with the Fn...

No abstract

The nucleoside triphosphate of 5-(4',5'-dihydroxypentyl)uracil (DHPU) was detected in the acid-soluble extract from bacteriophage SP15-infected Bacillus subtilis W23. No uracil was found in the DNA of either replicating or mature phage. Labeled thymidine added during phage DNA synthesis was incorporated into phage DNA. The presence of DHPU as a nucleoside triphosphate in the acid-soluble pool and the incorporation of thymidine into phage DNA suggest that both DHPU and thymine are incorporated into SP15 DNA via their nucleoside triphosphates. 5-Fluorodeoxyuridine inhibited biosynthesis of SP15 DNA, and this inhibition was reversed by thymidine, resulting in the synthesis of a DNA containing reduced amounts of fully modified DHPU. It is proposed that 5-fluorodeoxyuridine, or its metabolic product, inhibits a step in the biosynthetic pathway to the nucleoside triphosphate of DHPU. (Bausch & Lomb Spectronic 20). Materials. Bases, nucleosides, and nucleotides were obtained from Calbiochem. Si nuclease was purchased from Miles Laboratories, Inc., and snake venom phosphodiesterase came from Worthington Biochemicals Corp. DNase I and Ti RNase were from Calbiochem, and pancreatic RNase A was from Sigma Chemical Co. 6-(p-Hydroxyphenylazo)uracil was a gift from B. W. Langley of Imperial Chemical Industries, Ltd. CsCl was obtained from Metallgesellschaft A.G., Frankfurt, West Germany. [6-3H]uracil, 32Pi, [5,6-3H]uridine, and [methyl-3H]thymidine were supplied by Schwarz/Mann.

The sequence requirements for Fnr-dependent transcription of the narGHJI operon of Escherichia coli were studied in plasmids carrying a narG::lacZ protein fusion with the 5' end of the promoter deleted so that expression was controlled exclusively by Fnr. These plasmids were subjected to in vitro mutagenesis, and beta-galactosidase activities were determined in transformed strains after aerobic and anaerobic growth. A single base-pair change in the Fnr box, a sequence which is highly conserved in all Fnr-dependent promoters, essentially abolished anaerobic induction of expression. Primer extension analysis located the transcription start site 57 bp upstream from the narG translation start site and placed the Fnr box at a position centred between -41 and -42 bases from the transcription start site. The position of the Fnr box relative to the transcription start site was critical for anaerobic induction of expression. The deletion of 2 bp or addition of 4, 6, 10, 14, 20, 22, 28, 30, and 40 bp immediately downstream from the Fnr box abolished anaerobic induction. Sequence changes between the Fnr box and the transcription start site had different effects, depending upon the region mutagenized. Base changes immediately downstream from the Fnr box, including bases -20 to -29, did not lead to any decrease in anaerobic induction of expression, but in most instances resulted in increased expression. Base changes further downstream prevented anaerobic induction of expression and suggested the requirement for a -10 hexamer which is partially homologous to the -10 consensus sequence for sigma 70-specific promoters of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)