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The chemical ratchet, a mechanism indifferent to detailed balance, is evidenced by a single-molecule experiment involving the binding of a bacterial repressor to its operator.

When a reaction is accompanied by a change with the speed close to or slower than the reaction rate, a circulating reaction flow can exist among the reaction states in the macroscopic stationary state. If the accompanying change were at equilibrium in the timescale of the relevant reaction, the transition-state theory would hold to eliminate the flow.

The study of transcription and its regulation is an interdisciplinary field that is closely connected with genetics, structural biology, and reaction theory. Among these, although less attention has been paid to reaction theory, it is becoming increasingly useful for research on transcription. Rate equations are commonly used to describe reactions involved in transcription, but they tend to be used unaware of the timescales of relevant physical processes. In this review, we discuss the limitation of rate equation for describing three-dimensional diffusion and one-dimensional diffusion along DNA. We then introduce the chemical ratchet mechanism recently proposed for explaining the antenna effect, an enhancement of the binding affinity to a specific site on longer DNA, which deviates from a thermodynamic rule. We show that chemical ratchet cannot be described with a single set of rate equations but alternative sets of rate equations that temporally switch no faster than the binding reaction.

RNA polymerase can cleave a phosphodiester bond at the 3′ end of a nascent RNA in the presence of pyrophosphate producing NTP. Pyrophosphorolysis has been characterized during elongation steps of transcription where its rate is significantly slower than the forward rate of NMP addition. In contrast, we report here that pyrophosphorolysis can occur in a millisecond time scale during the transition of Escherichia coli RNA polymerase from initiation to elongation at the psbA2 promoter. This rapid pyrophosphorolysis occurs during productive RNA synthesis as opposed to abortive RNA synthesis. Dissociation of σ(70) or RNA extension beyond nine nucleotides dramatically reduces the rate of pyrophosphorolysis. We argue that the rapid pyrophosphorolysis allows iterative cycles of cleavage and re-synthesis of the 3′ phosphodiester bond by the productive complexes in the early stage of transcription. This iterative process may provide an opportunity for the σ(70) to dissociate from the RNA exit channel of the enzyme, enabling RNA to extend through the channel.

Several DNA-binding proteins show the affinities for their specific DNA sites that positively depend on the length of DNA harboring the sites, i. e. antenna effect. DNA looping can cause the effect for proteins with two or more DNA binding sites, i. e. the looping mechanism. One-dimensional diffusion also has been suggested to cause the effect for proteins with single DNA sites, the diffusion mechanism, which could violate detailed balance. We addressed which mechanism is possible for E. coli TrpR showing 104-fold antenna effect with a single DNA binding site. When a trpO-harboring DNA fragment was connected to a nonspecific DNA with biotin-avidin connection, the otherwise sevenfold antenna effect disappeared. This result denies the looping mechanism with an unknown second DNA binding site. The 3.5-fold repression by TrpR in vivo disappeared when a tight LexA binding site was introduced at various sites near the trpO, suggesting that the binding of LexA blocks one-dimensional diffusion causing the antenna effect. These results are consistent with the chemical ratchet recently proposed for TrpR-trpO binding to solve the deviation from detailed balance, and evidence that the antenna effect due to one-dimensional diffusion exists in cells.

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on the sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences, when enriched with thymine bases, contain the signal for inducing abortive transcription, whereas certain repetitive sequence elements embedded in promoter regions constitute the signal for inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we suggest that repetitive sequence elements within the promoter DNA modulate the nonlocal base pair stability of its double-stranded form. This stability profoundly influences the reaction coordinates of the productive initiation via pausing.

Transcriptional regulations have been widely studied as one of the main bridges between biology and other basic sciences as well as medicine. The traffic across it has been mostly unidirectional: chemistry and physics provided a lot of tools for biology, although the supply is now saturating. The traffic in opposite direction, the supply of subjects to develop chemistry and physics, has been only a little. However, if there are any, the supply will be at least from transcription, because the notion of chemical reaction is the strongest. This topic is aimed to increase the opposite traffic by introducing the forefront of physicochemical studies of transcription.

A repressor composed of homodimeric subunits, as is often found in bacteria, possesses two effector-binding sites per molecule, enabling sophisticated regulation by the cooperative binding of two effector molecules. Positive cooperativity generates a narrower region of effector concentration for switching, but little is known about the role of negative cooperativity. d-camphor, an inducer for Pseudomonas putida cytochrome P450cam hydroxylase operon (camDCAB), binds to the homodimeric cam repressor (CamR). Here, we report solid evidence that the complex of CamR and an operator DNA is not dissociated by the first binding of d-camphor but, at a higher concentration, is dissociated by the second binding. d-camphor thus binds to the CamR in two steps with negative cooperativity, yielding two distinct dissociation constants of Kd1 = 0.064 ± 0.030 and Kd2 = 14 ± 0.3 μm, as well as the Hill coefficient of 0.56 ± 0.05 (

If Mg2+ion is replaced by Mn2+ion, RNA polymerase tends to misincorporate noncognate nucleotide, which is thought to be one of the reasons for the toxicity of Mn2+ion. Therefore, most cells have Mn2+ion at low intracellular concentrations, but cyanobacteria need the ion at a millimolar concentration to maintain photosynthetic machinery. To analyse the mechanism for resistance against the abundant Mn2+ion, we compared the properties of cyanobacterial andE. coliRNA polymerases. The cyanobacterial enzyme showed a lower level of abortive transcription and less misincorporation than theE. colienzyme. Moreover, the cyanobacterial enzyme showed a slower rate of the whole elongation by an order of magnitude, paused more frequently, and cleaved its transcript faster in the absence of NTPs. In conclusion, cyanobacterial RNA polymerase maintains the fidelity of transcription against Mn2+ion by deliberate incorporation of a nucleotide at the cost of the elongation rate. The cyanobacterial and theE. colienzymes showed different sensitivities to Mg2+ion, and the physiological role of the difference is also discussed.

Diamond could be an excellent support for nanodevices utilizing biomolecules if it is covered with a polymer layer immobilizing a variety of biomolecules. We report a wet method to form a 3-aminopropyltriethoxysilane (APTES) multilayer with a controlled hardness, roughness, and capacity for immobilizing protein. The method is feasible in typical biochemical laboratories where biomolecules are prepared. Atomic force microscopy (AFM) revealed that the surface geometries and nanoscopic hardness of the multilayers on an oxygen-terminated single-crystalline diamond surface depended on the dielectric constant of the solvent; the smaller the constant, the harder the layer. The hard multilayers had holes and APTES aggregates on the surfaces, while less hard ones had homogeneous surfaces with rare holes and little aggregates. The secondary deposition of APTES in a solvent with a large dielectric constant on a hard multilayer removed the holes, and further treatment of the multilayer in acidic ethanol solution diminished the aggregates. Such a surface can immobilize streptavidin with enough specificity against nonspecific adsorption using a combination of polyethylene glycol reagents. The results of a scratching test and nanoindentation test with AFM provided consistent results, suggesting some universality of the scratching test independent of the tip structure of the cantilever. The mechanism of formation of multilayers on the diamond surface and their binding to it is discussed.

The fate of the giant jellyfish Nemopilema nomurai after death was examined in the southwest Sea of Japan. The density of dead jellyfish was greater than that of live animals. The dead animals are heavier than the Japan Sea Proper Water which occurs deeper than 200 m, suggesting that dead jellyfish sink to the sea floor. The sea floor survey, conducted with a towed video tape recorder (VTR) monitoring system between mid September and mid October, observed a total of 138 jellyfish during 28 of 29 operations. The density of carrion ranged between 0.2 and 5.1 individuals/1,000 m2 (mean ± SE = 1.1 ± 0.2). Ophiuroids occurred abundantly at 23 jellyfish carcasses and a sea anemone was observed attached to five carcasses. The VTR surveys confirmed that carrion sinks to the sea floor not only during the winter, the normal end of life for medusae, but also during the fall. A trap survey baited with medusae was also employed, and four different species were sampled with the traps: the snow crab (Chionoecetes opilio), a shrimp (Pandalopsis japonica), the ivory shell (Buccinum striatissimum) and an ophiuroid (Ophiura sarsii). Much of the trap bait remained (49–68% weight-mean = 60.3%) during the 23 h soak-time, and the reduction in weight was greater than that observed by bacterial decomposition, suggesting benthic animals consume dead organisms. The present study indicates that dead N. nomurai sink to the sea floor continuously and were subsequently consumed by benthic scavengers.

Ribosomal proteins S10 and S2 were each fused with GFP to track the fates of these proteins in the stationary growth phase and the following decay period in Escherichia coli. The fused proteins localized mainly in the cytoplasm, and their amounts were proportional to the colony-forming unit. S10-GFP strains that lacked genes responsible for regulating 100S ribosomes and S2-GFP strain that was unable to form 100S both showed shortened stationary phases. This result indicates that these strains exhibit earlier death in the absence of 100S formation (S2-GFP, S10-GFP∆rmf and S10-GFP∆hpf) and breakdown (S10-GFP∆yfiA). Therefore, in addition to the mere presence of 100S, the correct timing of 100S formation and breakdown is required to maintain viability. We propose a model in which 100S acts as a tentative repository of ribosomes that are protected from degradation and provide a source of amino acids in later growth period.

Summary 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.

第Ⅲ部 一般論文 第１章, 嶋本 伸雄［総合研究大学院大学 生命科学研究科長］

Many proteins select special DNA sequences to form functional complexes. In one possible mechanism, protein molecules would scan DNA sequences by tracking a groove without complete dissociation. Upon dragging single molecules of DNA over a surface carrying fixed Escherichia coli RNA polymerase holoenzyme, we detected rotation of individual DNA molecules, providing direct evidence that a DNA-binding protein can track a DNA groove. These results confirm our previous observations of longitudinal movement of RNA polymerase along fixed, extended DNA and, moreover, imply that groove tracking facilitates scanning of DNA sequences.

Construction of plasmids has been occupying a significant fraction of laboratory work in most fields of experimental biology. Tremendous effort was made to improve the traditional method for constructing plasmids, in which DNA fragments digested with restriction enzymes were ligated. However, the traditional method remained to be a standard protocol more than 40 years. At last, several recent inventions are rapidly and completely replacing the traditional method, because they are far quicker with less cost, and requiring less material. We here introduce three such methods that cover up most of the cases. Moreover, they are complementary with each other. Our lab protocols are provided for “no strain, no pain” construction of plasmids.

Background 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. Results The Gre factors mitigated initiation arrest only when they were added to the binary complex of the holoenzyme bound to the λPR promoter, prior to RNA synthesis. They exerted little effect when they were added to ternary initiation complexes. They accelerated the exchange of the binary complex with its free components by 6–9-fold. When they are present, a high concentration of the initiating nucleotide increased yield of the full-length transcript, whereas a low concentration did not. Conclusions All the results presented above can be explained by a model where the productive and arrested pathways diverge at the binary complex stage. The Gre factors relieve the initiation arrest by introducing reversibility between subspecies of the binary complex that are precursors of the two pathways. RNA cleavage is unlikely to cause relief of initiation arrest.

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.

新分野ナノバイオロジー, 嶋本伸雄［遺伝学学専攻・国立遺伝学研究所］＊所属は当時のものを記載

The single-stranded DNA (ssDNA) binding protein from Escherichia coli (EcoSSB) plays a central role in DNA replication, recombination and repair. The tertiary structure of EcoSSB was determined at 2.2 A resolution. This is rather higher resolution than previously reported. Crystals were grown from the homogeneous intact protein but the EcoSSB tetramer in the crystals contains truncated subunits lacking a part of the C-terminal. The structure determined includes biologically important flexible loops and C-terminal regions, and revealed the existence of concavities. These concavities include the residues important for ssDNA binding. An ssDNA can be fitted on the concavities and further stabilized through interactions with the loops forming flexible lids. It seems likely to play a central role in the binding of ssDNA.

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.

The crystal structure of the Escherichia coli replication-terminator protein (Tus) bound to terminus-site (Ter) DNA has been determined at 2.7 A resolution. The Tus protein folds into a previously undescribed architecture divided into two domains by a central basic cleft. This cleft accommodates locally deformed B-form Ter DNA and makes extensive contacts with the major groove, mainly through two interdomain beta-strands. The unusual structural features of this complex may explain how the replication fork is halted in only one direction.

The time-course of synthesis of long and short (abortive) transcripts by Escherichia coli RNA polymerase was investigated under single round conditions in vitro. The synthesis of long RNA initiated at the lambda PR (with an altered leader sequence) or at the lacUV5 promoter was completed within five minutes, but abortive transcripts were continuously synthesized for at least 20 minutes. The results indicate the presence of transcription complexes that are capable only of abortive synthesis, and not of productive elongation. Consistently, only one in four of the RNA polymerase molecules that initially associated with the lambda PR promoter synthesized long RNA. The enzyme reisolated from productive complexes synthesized both long and abortive transcripts, behaving just like the original enzyme. This suggests that RNA polymerase is homogeneous with respect to its ability to synthesize the two types of product. Overall, the results indicate that many transcription complexes can be irreversibly trapped in vitro in an abortive cycle.

This paper proposes, and experimentally demonstrates, a novel method of 'molecular surgery' of DNA, where a enzyme-labeled particle or probe is pressed against DNA, which is immobilized on the substrate with known conformation and orientation, to make chemical modifications at arbitrary positions of the DNA molecule. In the method presented in this paper, DNA is subjected to high-intensity high-frequency electric field (≥1MV/m, ≈1MHz) created in micro-fabricated electrodes, so that it is 1) stretched to a straight conformation parallel to the field by the electrostatic orientation effect, and 2) positioned onto a substrate by the dielectrophoresis. Once DNA is thus stretch-and-positioned, one has access to arbitrary position of any one of aligned molecules. A latex particle, 2.8μm in diameter, is labeled with DNA-cutting enzyme, DNaseII, and is laser-manipulated and contacted with DNA. It is observed that DNA is instantaneously cut at the contact point. This result is considered to be the first demonstration of space-resolved biochemical modification.

© 2012 Shimamoto et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nanoindentation as a Tool to Clarify the Mechanism Causing Variable Stiffness of a Silane Layer on Diamond

The feeding habit of red sea bream, Pagrus major, was examined by stomach content analysis with special reference to its seasonal changes in Tomogashima Channel waters, an important habitat in the eastern Seto Inland Sea. The fish fed through its life on many kinds of organisms, but intensively on a few kinds of them during the feeding season from spring to autumn. It was noted here that juveniles fed on zooplankton such as Acartia omorii and Oikopleura sp. from June to July, and youngs on the pasiphaeid shrimps Leptochela from August onward. These shrimps were in fact the main prey for the fish of age 1 and over. The stomach contents were poor and changed after November, because of the fall in water temperature and the increase in its population density with overwintering assemblage. Consequently, until March, or the end of the overwintering season, fish growth was retarded with a continuous decrease in the condition factor. The change in feeding habit with the winter assemblage was most remarkable for age 0 fish. The winter season would appear to be a critical period for the survival of the fish especially at this age.

A repressor composed of homodimeric subunits, as is often found in bacteria, possesses two effector-binding sites per molecule, enabling sophisticated regulation by the cooperative binding of two effector molecules. Positive cooperativity generates a narrower region of effector concentration for switching, but little is known about the role of negative cooperativity. d-camphor, an inducer for Pseudomonas putida cytochrome P450cam hydroxylase operon (camDCAB), binds to the homodimeric cam repressor (CamR). Here, we report solid evidence that the complex of CamR and an operator DNA is not dissociated by the first binding of d-camphor but, at a higher concentration, is dissociated by the second binding. d-camphor thus binds to the CamR in two steps with negative cooperativity, yielding two distinct dissociation constants of K(d1 ) =( ) 0.064 ± 0.030 and K(d2 ) =( ) 14 ± 0.3 μm, as well as the Hill coefficient of 0.56 ± 0.05 (1). The first binding guarantees the high specificity of the inducer by the high affinity, although the second binding turns on the gene expression at a 200-fold higher concentration, a more suitable switching point for the catabolism of d-camphor.

In growing bacterial cells, the global reorganization of transcription is associated with alterations of RNA polymerase composition and the superhelical density of the DNA. However, the existence of any regulatory device coordinating these changes remains elusive. Here we show that in an exponentially growing Escherichia coli rpoZ mutant lacking the polymerase ω subunit, the impact of the Eσ38 holoenzyme on transcription is enhanced in parallel with overall DNA relaxation. Conversely, overproduction of σ70 in an rpoZ mutant increases both overall DNA supercoiling and the transcription of genes utilizing high negative superhelicity. We further show that transcription driven by the Eσ38 and Eσ70 holoenzymes from cognate promoters induces distinct superhelical densities of plasmid DNA in vivo. We thus demonstrate a tight coupling between polymerase holoenzyme composition and the supercoiling regimen of genomic transcription. Accordingly, we identify functional clusters of genes with distinct σ factor and supercoiling preferences arranging alternative transcription programs sustaining bacterial exponential growth. We propose that structural coupling between DNA topology and holoenzyme composition provides a basic regulatory device for coordinating genome-wide transcription during bacterial growth and adaptation., IMPORTANCE Understanding the mechanisms of coordinated gene expression is pivotal for developing knowledge-based approaches to manipulating bacterial physiology, which is a problem of central importance for applications of biotechnology and medicine. This study explores the relationships between variations in the composition of the transcription machinery and chromosomal DNA topology and suggests a tight interdependence of these two variables as the major coordinating principle of gene regulation. The proposed structural coupling between the transcription machinery and DNA topology has evolutionary implications and suggests a new methodology for studying concerted alterations of gene expression during normal and pathogenic growth both in bacteria and in higher organisms.

We have been developing a low invasive cell manipulation technology based on inserting an ultra-thin needle--“nanoneedle”--into a living cell by using an atomic force microscope (AFM). The nanoneedle, made from a silicon AFM tip by focused-ion-beam etching, has a diameter of several hundred nanometers and a length of about 10 microns. Successful insertion of the nanoneedle into the cell can be confirmed by the appearance of a steep relaxation of repulsive force in the force-distance curve as monitored by the AFM system. This technology, termed “cell surgery”, can be applied for the detection of intracellular proteins in a living cell or for highly efficient gene transfer. The present study shows that the durability of a tapered nanoneedle is superior to that of a cylindrical nanoneedle, and that a proper aspect ratio for the tapered nanoneedle must be chosen to maintain sufficient insertion efficiency for a particular target cell: tapered nanoneedles of an aspect ratio over 20 showed high insertion efficiency for various kinds of mammalian cells. We then used diamond for the material of the nanoneedle because its specific properties, such as high stiffness, heat conductivity, and electrical conductivity capacitated by boron doping, were deemed useful for the analysis and manipulation of intracellular phenomena. We compared the capability of the diamond nanoneedle in cell manipulation with that of the silicon nanoneedle. Evaluation of the effect of the former on transcription efficiency and localization analysis of p53 expression revealed the low invasiveness for cell manipulation as was also the case for the silicon nanoneedle. We also succeeded in achieving highly efficient plasmid DNA delivery into a mouse fibroblast C3H10T1/2 using the diamond nanoneedle. The diamond nanoneedle is expected to contribute to the versatility of “cell surgery” technology.