Nobuo Shimamoto

Morphological pathway of flagellar assembly in Salmonella typhimurium

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.

Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase.

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

Applications of electrostatic stretch-and-positioning of DNA

The authors have previously reported that the electrostatic orientation and the dielectrophoresis (DEP) of DNA occur under a approximately=1 MHz, >1*10/sup 6/ V/m field, by which the DNA strands are stretched straight along field lines and positioned onto electrode edges. In the present work they discuss some applications of this stretch-and-positioning method to genetic engineering. It is shown that the DNA size distribution, as well as the activities of nuclease, can be determined by the measurement of the apparent length of stretched DNA. Several methods are developed to immobilize stretched DNA onto a substrate, including immobilization onto a conducting substrate for observations with scanning tunneling microscopy and anchoring onto a substrate only at the two ends of DNA using a special electrode configuration, and/or molecular binding between avidin and biotin. The DNA can be held without contact to the substrate in the latter method, so that it does not cause steric hindrances to the DNA-binding enzymes. A novel fluid integrated circuit (FIC) device is proposed in which stretched DNAs are cut by laser beam for successive sequencing. A method for obtaining unidirectionally oriented DNAs is developed.<<ETX>>

One-dimensional Diffusion of Proteins along DNA ITS BIOLOGICAL AND CHEMICAL SIGNIFICANCE REVEALED BY SINGLE-MOLECULE MEASUREMENTS

Evidence for sliding of proteins along DNA has been provided by many kinetic studies, but single-molecule-based measurements have uncovered distinct problems, the solutions of which may lead us to an understanding of new mechanisms for gene regulation. Furthermore, they reveal a deep problem lying between chemistry and physics regarding the seemingly simple binding between DNA and protein. Single-molecule dynamics provides a tool to solve this problem without prejudgments or unsound assumptions.

Molecular surgery of DNA based on electrostatic micromanipulation

A novel method for the space-resolved dissection (molecular surgery) of DNA using electrostatic molecular manipulation is proposed and demonstrated. In conventional biochemistry, DNA cutting enzymes and DNA are mixed in water, so the cutting reactions occur only by stochastic chances. In contrast, the present method is based upon a physical manipulation and enables the deterministic cutting of DNA at arbitrary position on a DNA molecule. In order to realize this space-resolved cutting, the target DNA is stretched straight by electrostatic orientation, and anchored onto a solid surface by dielectrophoresis, using a high intensity (/spl ges/1 MV/m) high frequency (/spl ap/1 MHz) field generated in microfabricated electrodes. The molecular surgery presented in this paper is expected to realize space-resolved molecular operations, not only limited to dissections, but also chemical modifications, or even insertion of genes may become possible in the future.

Regions of the Escherichia coli primary sigma factor σ70 that are involved in interaction with RNA polymerase core enzyme

The σ factors of bacterial RNA polymerase are required for recognition of promoters in transcription initiation. Most σ factors share several regions with significant homology in their amino acid sequences (regions 1–4). Some primary σ factors carry a large nonconserved segment between regions 1 and 2. The binding of an σ factor to the core enzyme alters the structure and properties of the σ factor, but little is known about the binding mechanism and subsequent reactions. In this report, we employed the protein footprinting method to investigate the alteration of the structure and function of Escherichia coli σ70 by binding to core enzyme and promoter DNA.

A pathway branching in transcription initiation in Escherichia coli.

In transcription initiation, all RNA polymerase molecules bound to a promoter have been conventionally supposed to proceed into elongation of transcript. However, for Escherichia coli RNA polymerase, evidence has been accumulated for a view that only its fraction can proceed into elongation and the rest is retained at a promoter in non‐productive form: a pathway branching in transcription initiation. Proteins such as GreA and GreB affect these fractions at several promoters in vitro. To reveal the ubiquitous existence of the branched mechanism in E. coli, we searched for candidate genes whose transcription decreased by disruption of greA and greB using a DNA array. Among the arbitrarily selected 11 genes from over 100, the atpC, cspA and rpsA passed the test by Northern blotting. The Gre factors activated transcription initiation from their promoters in vitro, and the results demonstrated that the branched mechanism is exploited in vivo regulation. Consistently, decrease in the level of the GreA in an anaerobic stationary condition accompanied a decrease in the levels of transcripts of these genes.

Physical interference between Escherichia coli RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation

Transcription initiation is accompanied with iterative synthesis and release of short transcripts. The molar ratio of enzyme to template was found to be critical for the amounts and distribution of the abortive products synthesized by Escherichia coli RNA polymerase from several promoters. At a high ratio abortive synthesis of 4-8 nt were enhanced at thelambda P R promoter. Removing excess RNA polymerase just before initiation, achieved by washing immobilized transcription complexes, prevented this enhancement. At this high ratio synthesis of an unexpected 6 nt transcript was enhanced when the enzyme stalled at position +32, but not when it stalled at position +73. This transcript had misincorporations at its fifth and sixth positions, probably due to slippage. Hydroxyl radical footprinting of the complex stalled at +32 in the presence of excess enzyme showed that more than one molecule of RNA polymerase was tandemly bound to the same DNA. These results suggest that: (i) when RNA polymerase molecules are tandemly transcribing the same DNA, transient collisions enhance abortive synthesis by the trailing molecule; (ii) when the leading polymerase stalled in the initially transcribed region blocks progression of the trailing polymerase, the latter can commit misincorporations, probably due to slippage synthesis.

Conformational switching of Escherichia coli RNA polymerase-promoter binary complex is facilitated by elongation factor GreA and GreB

The initiation arrest at a modified λPR promoter is caused by irreversible divergence of the reaction pathway into productive and arrested branches. Escherichia coli GreA and GreB induce cleavage of the nascent transcript and relieve arrest in elongation. They also reduce abortive synthesis at several promoters and relieve initiation arrest. Their mechanism of action during initiation, and its relationship to the branched initiation pathway are unknown.

Imaging of DNA molecules by scanning near-field microscope

Abstract We utilized a novel scanning near-field optical microscope (SNOM) for imaging DNA molecules. The microscope system was constructed with a commercial inverse microscope and a newly developed scanning unit. In the system, a bent optical fiber probe is used to operate in a dynamic mode atomic-force microscope (AFM). A λDNA solution of 5 μM (base) with 5 μM and 500 nM YOYO-1 was prepared and cast on a γ-APTES treated cover slip. The λDNA was aggregated in line and immobilized on the cover slip. The percentage of fluorescence intensity of λDNA with 5 μM YOYO-1 showed integers at almost each point on the DNA. As the fluorescence intensity correlated with the areas of a cross-section of the DNA topography, it appeared that YOYO-1 intercalated in the DNA homogeneously. The fluorescence images of λDNA with 500 nM YOYO-1, however, were irregular and did not correlate with the area of the topographic cross-section, suggesting that YOYO-1 did not intercalate in the λDNA uniformly in this concentration and intercalated cooperatively.