Akira Ishihama

Functional characterization in Vitro of all two-component signal transduction systems from Escherichia coli

Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Self-phosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.

Twelve Species of the Nucleoid-associated Protein from Escherichia coli SEQUENCE RECOGNITION SPECIFICITY AND DNA BINDING AFFINITY

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid. We expressed and purified 12 species of the DNA-binding protein, i.e. CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Qβ), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor oftd− phenotype A). The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays. Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities. Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA. From the apparent dissociation constant (K d ) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nm (HU binding to the non-curved DNA) to 250 nm (Hfq binding to the non-curved DNA), under the assay conditions employed.

Bipartite functional map of the E. coli RNA polymerase α subunit: Involvement of the C-terminal region in transcription activation by cAMP-CRP

The alpha subunit of Escherichia coli RNA polymerase plays a major role in the subunit assembly. Carboxyterminal deletion derivatives lacking 73 or 94 amino acid residues were assembled in vitro into enzyme molecules. Core enzymes consisting of these C-terminal-truncated alpha subunits were as active in RNA synthesis as native core enzyme. By the addition of sigma 70 subunit, these mutant enzymes initiated transcription from certain promoters. The mutant RNA polymerases, however, did not show cAMP-CRP activated transcription. These results demonstrate that the N-terminal region of the alpha subunit is involved in the formation of active enzyme molecule, while the C-terminal region plays an essential role in response to transcription activation by cAMP-CRP.

Single-Molecule Imaging of RNA Polymerase-DNA Interactions in Real Time

Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of lambda-phage DNA suspended in solution by optical traps. The interactions of RNA polymerase molecules were not homogeneous along DNA. They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of approximately 0.66 s-1, which was more than severalfold smaller than the rate at other positions. The association rate constant for the slow dissociation sites was 9.2 x 10(2) bp-1 M-1 s-1. The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions. RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA. The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA. The present method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the process of transcription.

Transcriptional response of Escherichia coli to external copper

Transcriptional response of Escherichia coli upon exposure to external copper was studied using DNA microarray and in vivo and in vitro transcription assays. Transcription of three hitherto‐identified copper‐responsive genes, copA (copper efflux transporter), cueO (multicopper oxidase) and cusC (tripartite copper pump component) became maximum at 5 min after addition of copper sulphate, and thereafter decreased to the preshift levels within 30 min. Microarray analysis at 5 min after addition of copper indicated that a total of at least 29 genes including these three known genes were markedly and specifically affected (28 upregulated and one downregulated). Transcription of the divergent operons, cusCFB and cusRS, was found to be activated by CusR, which bound to a CusR box between the cusC and cusR promoters. Except for this site, the CusR box was not identified in the entire E. coli genome. On the other hand, transcription of copA and cueO was found to be activated by another copper‐responsive factor CueR, which bound to a conserved inverted repeat sequence, CueR box. A total of 197 CueR boxes were identified on the E. coli genome, including the CueR box associated with the moa operon for molybdenum cofactor synthesis. At least 10 copper‐induced genes were found to be under the control of CpxAR two‐component system, indicating that copper is one of the signals for activation of the CpxAR system. In addition, transcription of yedWV, a putative two‐component system, was activated by copper in CusR‐dependent manner. Taken together we conclude that the copper‐responsive genes are organized into a hierarchy of the regulation network, forming at least four regulons, i.e. CueR, CusR, CpxR and YedW regulons. These copper‐responsive regulons appear to sense and respond to different concentrations of external copper.

Two types of localization of the DNA-binding proteins within the Escherichia coli nucleoid.

The genome DNA of Escherichia coli is folded into the nucleosome‐like structure, often called a nucleoid, by the binding of several DNA‐binding proteins. We previously determined the specificity and affinity of DNA‐binding for 12 species of the E. coli DNA‐binding protein, and their intracellular concentrations at various growth phases. The intracellular localization of these proteins in E. coli could be predicted from these data, but no attempt has been made thus far to directly observe the intracellular distribution of the DNA‐binding proteins.

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.

Modulation of the nucleoid, the transcription apparatus, and the translation machinery in bacteria for stationary phase survival.

Upon sensing an impending saturation level of their population density, Escherichia coli cells enter into the stationary phase. We have identified structural and functional modulations of the nucleoid, the transcription apparatus and the translation machinery occurring during the transition from exponential growth to stationary phase. The major DNA‐binding proteins, Fis, HU and Hfq, in the exponential‐phase nucleoid are replaced by a single stationary‐phase protein Dps, thereby compacting the nucleoid and ultimately leading to silencing of the DNA functions. The transcription apparatus is modified by replacing the major promoter recognition subunit, σ70, with σS. A stationary‐phase protein, Rsd (Regulator of Sigma D), with the binding activity of σ70 is involved in the efficient replacement of σ and/or the storage of unused σ70. Changes in cytoplasmic composition also differentially influence the activity of Eσ70 and EσS holoenzymes. Together, these effects may result in the preferential transcription of stationary‐phase specific genes. The translation machinery is also modulated in stationary phase, by the formation of translationally incompetent 100S ribosomes. A small stationary‐phase protein, RMF (Ribosome Modulation Factor), is involved in the dimerization of 70S ribosome monomers.

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.

Prokaryotic genome regulation: multifactor promoters, multitarget regulators and hierarchic networks

The vast majority of experimental data have been accumulated on the transcription regulation of individual genes within a single model prokaryote, Escherichia coli, which form the well-established on-off switch model of transcription by DNA-binding regulatory proteins. After the development of modern high-throughput experimental systems such as microarray analysis of whole genome transcription and the Genomic SELEX search for the whole set of regulation targets by transcription factors, a number of E. coli promoters are now recognized to be under the control of multiple transcription factors, as in the case of eukaryotes. The number of regulation targets of a single transcription factor has also been found to be more than hitherto recognized, ranging up to hundreds of promoters, genes or operons for several global regulators. The multifactor promoters and the multitarget transcription factors can be assembled into complex networks of transcription regulation, forming hierarchical networks.