Nobuyuki Fujita

The mediator for stringent control, ppGpp, binds to the β‐subunit of Escherichia coli RNA polymerase

Inhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure.

Solution Structure of the Activator Contact Domain of the RNA Polymerase α Subunit

The structure of the carboxyl-terminal domain of the Escherichia coli RNA polymerase α subunit (αCTD), which is regarded as the contact site for transcription activator proteins and for the promoter UP element, was determined by nuclear magnetic resonance spectroscopy. Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins. The UP element binding site was found on the surface comprising helix 1, the amino-terminal end of helix 4, and the preceding loop. Mutation experiments indicated that the contact sites for transcription activator proteins are also on the same surface.

Promoter Selectivity of Escherichia coli RNA Polymerase E and E Holoenzymes EFFECT OF DNA SUPERCOILING

The functional specificity was compared between two factors, (the major at exponentially growing phase) and (the essential at stationary growth phase), of Escherichia coli RNA polymerase. The core enzyme binding affinity of was less than half the level of as measured by gel filtration column chromatography or by titrating the concentration of required for the maximum transcription in the presence of a fixed amount of core enzyme. In addition, the holoenzyme concentration required for the maximum transcription of a fixed amount of templates was higher for E than E. The transcription by E was, however, enhanced with the use of templates with low superhelical density, in good agreement with the decrease in DNA superhelicity in the stationary growth phase. We thus propose that the selective transcription of stationary-specific genes by E holoenzyme requires either a specific reaction condition(s) or a specific factor(s) such as template DNA with low superhelical density.

Systematic sequencing of the Escherichia coli genome: analysis of the 0 – 2.4 min region

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.

Transcription factor recognition surface on the RNA polymerase alpha subunit is involved in contact with the DNA enhancer element.

The carboxy‐terminal one‐third of Escherichia coli RNA polymerase alpha subunit plays a key role in transcription regulation by a group of protein transcription factors and DNA enhancer (UP) elements. The roles of individual amino acid residues within this regulatory domain of the alpha subunit were examined after systematic mutagenesis of the putative contact regions (residues 258–275 and 297–298) for the cAMP receptor protein (CRP). The reconstituted RNA polymerases containing the mutant alpha subunits were examined for their response to transcription activation by cAMP‐CRP and the rrnBP1 UP element. Mutations affecting CRP responsiveness were located on the surface of the putative CRP contact helix and most of these mutations also influenced the response to the rrnB UP element. These observations raise the possibility that the CRP contact surface is also involved in contact with the DNA UP element, although some amino acid residues within this region play different roles in molecular communication with CRP and the UP element. Among the amino acid residues constituting the contact surface, Arg265 was found to play a major role in response to both CRP and the DNA UP element. Judged by DNase I footprinting analysis, alpha mutants defective in transcription from the CRP‐dependent lacP1 promoter showed decreased activity in the cooperative binding of CRP. Likewise, mutants defective in rrnBP1 transcription showed decreased binding to the UP element. The amino acid residues important for contact with both CRP and DNA are conserved in the alpha subunits of not only bacterial, but also chloroplast RNA polymerases.

Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two‐component system sensing external magnesium availability

PhoP is a response regulator of the PhoQ‐PhoP two‐component system controlling a set of the Mg(II)‐response genes in Escherichia coli. Here we demonstrate the mode of transcription regulation by phosphorylated PhoP of divergently transcribed mgtA and treR genes, each encoding a putative Mg(II) transporter and a repressor for the trehalose utilization operon respectively. Under Mg(II)‐limiting conditions in vivo, two promoters, the upstream constitutive P2 and the downstream inducible P1, were detected for the mgtA gene. Gel‐shift analysis in vitro using purified PhoP indicates its binding to a single DNA target, centred between –43 and –24 of the mgtAP1 promoter. This region includes the PhoP box, which consists of a direct repeat of the heptanucleotide sequence (T)G(T)TT(AA). Site‐directed mutagenesis studies indicate the critical roles for T (position 3), T (position 4) and A (position 6) for PhoP‐dependent transcription from mgtAP1. DNase I footprinting assays reveal weak binding of PhoP to this PhoP box, but the binding becomes stronger in the simultaneous presence of RNA polymerase. Likewise the RNA polymerase binding to the P1 promoter becomes stronger in the presence of PhoP. For the PhoP‐assisted formation of open complex at the mgtAP1 promoter, however, the carboxy‐terminal domain of α subunit (αCTD) is not needed. For transcription in vivo of the treR gene, four promoters were identified. The most upstream promoter treRP4 divergently overlaps with the mgtAP1 promoter, sharing the same sequence as the respective –10 signal in the opposite direction. In vitro transcription using mutant promoters support this prediction. In the presence of PhoP, transcription from the promoter treRP3 was repressed with concomitant activation of mgtAP1 transcription. The PhoP box is located between −46 and –30 with respect to treRP3, and the αCTD is needed for this repression.

Mapping the cAMP receptor protein contact site on the α subunit of Escherichia coli RNA polymerase

The C‐terminal region (amino acid residues 236–329) of the Escherichia coli RNA polymerase α subunit carries the contact site I for positive transcription factors. For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C‐terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene. Reconstituted holoenzyme containing the mutant a subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP–CRP. DNA sequence determination of several ‘Lac‐’ mutant rpoA genes revealed that all had mutations clustered within a short segment near the C‐terminus of α between amino acid residues 265 and 270. A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s).

IDENTIFICATION OF THE CPDA GENE ENCODING CYCLIC 3',5'-ADENOSINE MONOPHOSPHATE PHOSPHODIESTERASE IN ESCHERICHIA COLI

We have identified a gene, cpdA, located at 66.2 min of the chromosome of Escherichia coli that encodes cyclic 3′,5′-adenosine monophosphate phosphodiesterase (cAMP phosphodiesterase, EC3.1.4.17). The expression of β-galactosidase, which is a product of the lacZ gene, was repressed in cells that harbored multiple copies of the plasmid carrying the cpdA gene. Northern blotting showed that the transcription of the lacZ gene was inhibited in these cells. Multiple copies of the cpdA gene decreased the intracellular concentration of cAMP, which is a positive regulator for transcription of the lacZ gene. We found that the purified CpdA protein repressed in vitro transcription from the lacP1 promoter by decreasing cAMP. In addition, we showed that the CpdA protein hydrolyzed cAMP to 5′-adenosine monophosphate and that its activity was activated by iron. Our results suggested that regulation of intracellular concentration of cAMP is dependent not only on synthesis of cAMP but also on hydrolysis of cAMP by cAMP phosphodiesterase.

Ambidextrous transcriptional activation by SoxS: requirement for the C-terminal domain of the RNA polymerase alpha subunit in a subset of Escherichia coli superoxide-inducible genes

Purified MalE–SoxS fusion protein specifically stimulated in vitro transcription of the Escherichia colizwffprfumCmicFnfo, and sodA genes, indicating that activation of the superoxide regulon requires only SoxS. As in vivo, a 21 bp sequence adjacent to the zwf promoter was able to activate transcription of an heterologous promoter in vitro. Activation of zwf and fpr transcription required the C‐terminal domain (CTD) of the RNA polymerase alpha subunit, while stimulation of fumCmicFnfo, and sodA transcription was independent of CTD truncation. Thus, like the catabolite gene activator protein (CAP), SoxS is an ‘ambidextrous’ activator, activation only requiring the α CTD in a subset of regulated promoters. Indeed, the −35 hexamers of the zwf and fpr promoters lie downstream of the respective MalE–SoxS binding sites, while the binding sites of fumCmicFnfo, and sodA overlap their −35 promoter hexamers.

Active recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD.

The sequence elements determining the binding of the σ54‐containing RNA polymerase (σ54‐RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the −12/−24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C‐terminal domain of the α subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in σ70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of σ54‐RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of σ54‐RNAP for the −12/−24 site.