Steve Busby

Promoter structure, promoter recognition, and transcription activation in prokaryotes.

Steve Busby’ and Richard H. Ebrightt ‘School of Biochemistry University of Birmingham Birmingham 615 2lT England TDepartment of Chemistry and Waksman Institute Rutgers University New Brunswick, New Jersey 08855 Escherichia coli RNA polymerase holoenzyme (RNAP) has been the object of intense study since its discovery. RNAP consists of core enzyme, with subunit composition

The Escherichia coli RNA polymerase alpha subunit: structure and function.

Recent work has established that the Escherichia coli RNA polymerase alpha subunit consists of an amino-terminal domain containing determinants for interaction with the remainder of RNA polymerase, a carboxy-terminal domain containing determinants for interaction with DNA and interaction with transcriptional activator proteins, and a 13-36 amino acid unstructured and/or flexible linker. These findings suggest a simple, integrated model for the mechanism of involvement of alpha in promoter recognition and transcriptional activation.

Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.

The functional subunit of a dimeric transcription activator protein depends on promoter architecture.

In Class I CAP‐dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase. In Class II CAP‐dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the ‐35 site. We have used an ‘oriented heterodimers’ approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase [CC(‐61.5) and CC(‐72.5)] and at one Class II promoter [CC(‐41.5)]. Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter‐proximal subunit of CAP. In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter‐distal subunit of CAP.

Definition of nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL

Transcription initiation at the Escherichia coli nirB promoter is induced by anaerobic growth and further increased by the presence of nitrite or nitrate in the growth medium. Expression from this promoter is totally dependent on the transcription factor, FNR, which binds between positions −52 and −30 upstream of the transcription startsite. The 20 base pairs from position −79 to −60 contain an inverted repeat of two 10‐base sequence elements that are related to sequences at the NarL‐binding site at the E. coli narG promoter. Comparison of these, and sequence elements at other promoters regulated by NarL, suggests a consensus NarL‐binding sequence. Mutations in the putative NarL‐binding site at the nirB promoter decrease FNR‐dependent anaerobic induction, suggesting that NarL acts as a helper to FNR during transcription activation. These mutations also suppress induction by nitrite: single mutations at symmetry‐related positions have similar effects, whilst double mutations have more severe effects, probably because two NarL subunits bind to the inverted repeat. Disruption of narL decreases nitrite induction of the nirB promoter whilst not suppressing induction by nitrate, suggesting that there may be a second nitrate‐responsive factor. Nitrate induction was, however, suppressed by double mutations at symmetry‐related positions in the NarL‐binding site, suggesting that this putative second factor may bind to sequences similar to those recognized by NarL.

Nitrite and nitrate regulation at the promoters of two Escherichia coli operons encoding nitrite reductase: identification of common target heptamers for both NarP‐ and NarL‐dependent regulation

Expression from both the Escherichia coli nir and nrf promoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium. The nir promoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite. The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 691/2 bp upstream of the transcript startpoint. The nrf promoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate. Mutational analysis of the nrf promoter has been exploited to corroborate the location of the ‐10 hexamer and the FNR‐binding site, and to find the sites essential for nitrite‐dependent activation and nitrate‐dependent repression. Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at the nir promoter, but centred 741/2 bp upstream from the transcript start. NarL‐dependent repression by nitrate is due to two heptamer sequences that flank the FNR‐binding sequence. We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from site to site.

Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers

We have constructed heterodimers consisting of two subunits: one CAP subunit that has a nonfunctional activating region but wild-type DNA binding specificity, and one CAP subunit that has a functional activating region but non-wild-type DNA binding specificity. We have oriented the heterodimers on lac promoter DNA by use of promoter derivatives that have DNA sites for CAP consisting of one wild-type half site and one non-wild-type half site, and we have analyzed the abilities of the oriented heterodimers to activate transcription. Our results indicate that transcription. Our results indicate that transcription activation requires the activating region of only one subunit of CAP: the promoter-proximal subunit. The oriented heterodimers method of this report should be generalizable to other dimeric transcription activator proteins.

Interconversion of the DNA‐binding specificities of two related transcription regulators, CRP and FNR

In Escherichia coli, FNR and CRP are homologous transcriptional regulators which recognize similar nucleotide sequences via DNA‐binding domains containing analogous helix‐turn‐helix motifs. The molecular basis for recognition and discrimination of their target sites has been investigated by directed amino acid substitutions in the corresponding DNA‐recognition helices. In FNR, Glu‐209 and Ser‐212 are essential residues for the recognition of FNR sites. A V208R substitution confers CRP‐site specificity without loss of FNR specificity, but this has adverse effects on anaerobic growth. In contrast, changes at two (V206R and E209D) or three (V208R, S212G and G216K) positions in FNR endow a single CRP‐site binding specificity. In reciprocal experiments, two substitutions (R180V and G184S) were required to convert the binding specificity of CRP to that of FNR. Altering Asp‐199 in FNR failed to produce a positive control phenotype, unlike substitutions at the comparable site in CRP. Implications for the mechanism of sequence discrimination by FNR and CRP are discussed.

Location and orientation of an activating region in the Escherichia coli transcription factor, FNR.

We have characterized a number of mutations in fnr that interfere with FNR‐dependent transcription activation at two promoters where the FNR‐binding site is centred around 41½ bp upstream from the transcription start site. The substituted residues in all but one of these FNR mutants are clustered around a presumed surface‐exposed beta‐turn containing G85 which, we suggest, forms an activating region that contacts RNA polymerase at these promoters. Using the‘oriented heterodimers’method described elsewhere, we show that this activating region on the promoter‐proximal subunit of the FNR dimer is sufficient to activate transcription initiation. In contrast, this region is not essential for activation of a third FNR‐dependent promoter where the FNR‐binding site is centred at 61 1/2 bp upstream from the transcription start site. However, a substitution at S73 interferes with FNR‐dependent activation at both this promoter and promoters in which the FNR site is located at 41 1/2 bp from the transcript start, suggesting that FNR may contain a second activating region.

Molecular genetic analysis of an FNR‐dependent anaerobically inducible Escherichia coli promoter

From the effects of 13 deletions and three linker‐scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR‐dependent induction of activity by anaerobiosis and further nitrite‐dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of ‘cassettes’ carrying different FNR‐binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR‐dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR‐binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR‐binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR‐binding sequences at the melR promoter is sufficient to confer fnr‐dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR‐binding sequence.