H. STRUCTURAL BIOLOGY CENTER
H-b. Molecular Biomechanism Laboratory - Nobuo Shimamoto Group

RESEARCH ACTIVITIES

(1) The branched mechanism of transcription initiation in E. coli

Motoki Susa¹, Shouji Yagi¹ and Nobuo Shimamoto¹ (¹Structural Biology Center, National Institute of Genetics and and Department of Genetics, School of Life Science, The Graduate University for Advanced Studies)

--For several decades, the mechanism of transcription initiation has been assumed to be a sequence of three essential steps: formation of a complex between RNA polymerase and a promoter (closed complex), formation of another complex with partially melted DNA duplex to form phosphodiester bonds (open complex and chemical reaction), and escape of RNA polymerase from the promoter associated with the progress of RNA elongation (promoter clearance). There is a process called “abortive initiation", an iterative synthesis and release of oligo-RNA molecules. This process is often excluded from the mainstream of the mechanism, because its role as well as its occurrence in vivo has been unknown. However, this process has been observed in vitro with all prokaryotic and eukaryotic RNA polymerases so far isolated. The products of the process, abortive transcripts, are typically 2 to 15 nucleotides in length. On the assumption that these short transcripts are “unsuccessful precursors" of the full-length transcript, abortive synthesis has been considered to precede promoter clearance.
--We have been clarifying for several years that this sequential mechanism is not the case and the initiation follows branched pathways, one of which contains the moribund complex, being defined as a complex that produces only abortive and no full-length transcripts. Followings are its characteristics. 1. The moribund complex, as well as the productive complex that synthesizes full-length product, are formed from the same homogeneous fraction of enzyme molecules, and dissociation of the molecules from the promoter DNA cancels any difference between them. 2. Structural differences between these complexes have been demonstrated. 3. At some promoters, a moribund complex is converted into a dead-end complex that still retains a short transcript but has no elongation activity. Therefore, the initiation pathway is branched into the conventional productive pathway and the abortive pathway that can lead towards a dead end. 4. The fates of a moribund complex are either inactivation as a dead-end complex, dissociation from the DNA, or direct conversion into a productive complex, and the rates of these reactions vary with the promoter. 5. There are factors that affect the fate of the moribund complex in a manner that depends on the promoter.
--To examine the existence and significance of the branched pathway in vivo, we selected GreA and GreB for clues. At the λP_RAL promoter these factors enhance conversion of the moribund complex into the productive one, in the presence of high concentrations of initiating nucleoside triphosphate in vitro. If the branched mechanism exists in vivo, absence of the Gre factors should result in reduction of productive transcription from promoters at which the moribund complex is susceptible to these factors. We constructed a double-disruptant of E.coli, ΔgreAΔgreB, and then arbitrarily selected 10 genes from among those whose levels of transcripts in the mutant strain were found to be lower than those in the parental greA⁺greB⁺ strain. Finally, the promoter for three of these genes, atpC (uncC), cspA, and rpsA, passed a further conventional test which confirmed that they displayed a branched initiation pathway in a reconstituted transcription system composed of purified components. The results obtained prove that the branched initiation pathway exists in vivo and is utilized in regulation of transcription initiation from some promoters, through modulation of the fraction of polymerase-promoter complexes entering each branch of the pathway.
--In this year, we examined various physiological conditions that affect on the levels of the Gre factors. The determination of the level of GreB was observed to be constitutive. The level of GreA remained the same through the growth phase, and did not respond much to the richness of the culture media. However, it decreased into half in aerobic conditions, indicating that some genes are regulated by GreA. Therefore, the regulatory circuit responding to the levels of proteins involving GreA, namely the branched pathway mechanism, is working in cells.

(2) Applicability of thermodynamics to equilibria in biology

Nobuo Shimamoto¹ and Jyun-ichi Tomizawa¹ (¹Structural Biology Center, National Institute of Genetics and Department of Genetics, School of Life Science, The Graduate University for Advanced Studies)

--Most DNA-binding proteins are biologically functional as a specific complex, one containing a special short DNA segment. Such a complex is usually assumed as a state tenable for thermodynamic analysis of binding equilibrium. Thus, forward and backward reactions should balance at equilibrium in every pathway, and the affinity should be independent of the length of DNA. However, we have found that the balance at equilibrium is broken for some proteins by their sliding along DNA during association but not dissociation and that their affinities for their specific sites dependent on the length of DNA harboring the sites. This seeming disagreement is explained by an indeliberate use of the state of specific complex in thermodynamics. In the presence of sliding, the state does not satisfy the second law (the ergodic condition) and thus is disqualified for thermodynamic analysis. A general treatment of binding equilibrium, while maintaining the specific complex as a distinct state, is proposed on the base of the master equation or chemical kinetics.

(3) Systematic search for promoters encoded in the genomic DNA sequences of E. coli

Nobuo Shimamoto¹, Hideki Nakayama¹ and Hironori Aromaki² (¹Structural Biology Center, National Institute of Genetics and Department of Genetics, School of Life Science, The Graduate University for Advanced Studies. ²Daiichi College of Pharmaceutical Sciences)

--Irrespective of a pile of compiled sequences identified as promoters for E. coli vegetative RNA polymerase holoenzyme, σ⁷⁰ holoenzyme, there is no successful methods to predict strength of a promoter. To construct such prediction method, we started to design a functional SELEX to select promoter sequences. The complexity of random oligo-DNA available in a lab scale, 4^12~14 is too small to cover the all-possible promoters. Therefore we limited the candidate sequences to those involved in the genomic sequence. In order to construct and select a library, we transferred and amplified parts of the genomic sequence with PCR with a single primer. As a drawback of the use of single primer, the constructed library contains DNA fragments generated from in vitro recombination. A theoretical method to diminish the effect of such recombinants has been developed.

PUBLICATIONS

Papers
1. Sakata-Sogawa, K. and Shimamoto, N. (2004). RNA polymerase can track a DNA groove during promoter search. Proc. Nat. Sci. U.S.A. 101, 14731-14735.

Reviews
2. 嶋本伸雄（2004）レーザートラップのナノ操作標準技術としての確立.医学の歩み,医歯薬出版.
3. 中山秀喜（2004）第2章プロテインチップの応用技術「抗体チップとDNAチップの比較」Inバイオチップの最新技術と応用（松永是監修）シーエムシー出版.

Books
4. 嶋本伸雄（2004）バイオテクノロジーとナノテクノロジーの融合,早稲田大学産業技術創成研究所.

ORAL PRESENTATIONS

1. Shimamoto, N. Molecular Memory in Transcription Initiation and Its Regulatory roles. NCBS International Symposium "Molecules, Machines and Networks", Bangalore, India January, 2004
2. Shimamoto, N. Protein sliding along DNA: Its existence, biological significance and physical implications for the relationship between microscopic and macroscopic sciences NIG-NCBS International Workshop on Single-molecule Biophysics, Bangalore, India January, 2004
3. ○中山秀喜、嶋本伸雄、単一プライマーを用いたゲノムライブラリー作成法、日本生物工学会平成16年度大会、名古屋、9月
4. Shimamoto, N. and Tomizawa J. Chemical and Biological consequences of one-dimensional diffusion of proteins along DNA. Asian Conference of Transcription 8, Bangkok, November, 2004

POSTER PRESENTATIONS

1. ○中山秀喜、嶋本伸雄、単一プライマーでつくるゲノムライブラリーとそれを用いたプロモーター検索、第27回日本分子生物学会年会、神戸、12月

EDUCATION

Dean of School of Life Science, Graduate School of Advansed Studies

Lectures in other universities and institutes
1. Shimamoto, N. “RNA polymerase and a secret of nano-biomachines", Center For DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India January, 2004.
2. Shimamoto, N. “The firm start-point of nanobiology and biological nanotechnology: Understanding the characteristics of biological nanomachines" (in Japanese,「ナノバイオの揺るぎない出発点生体のナノマシンの特色」), Graduate School of Engeneering, The university of Tokyo, June 2004.
3. Shimamoto, N. “Chemical and Biological consequences of one-dimensional diffusion of proteins along DNA School of Chemistry", Seoul National University, Seoul, Korea, September 2004.
4. Shimamoto, N. “RNA polymerase and a secret of nano-biomachines", Department of Life Sciences, Seoul National University, Seoul, Korea, September 2004.
5. Shimamoto, N. “Proteins as biologically functional molecules in Fundamental Course of Molecular and Cellular Biology (KAST)" (in Japanese,基礎から学ぶKAST分子細胞生物学コース:機能分子としてのタンパク質) The Institute of Medical Science, The University of Tokyo October 2004.
6. Shimamoto, N. “Acey-deucy biological nanotechnology: Truths and ghosts in nano-biological science and technology (in Japanese,「玉か石か?:ナノバイオを通して見る科学と技術の虚実」), The Osaka Cabinet of Commerce and Industry (大阪商工会議所), November 2004.

SOCIAL CONTRIBUTIONS AND OTHERS

Organization of International Meeting
1. Shimamoto, N and Shivashankar, G. V. (Organizers),
Nakayama, H and Susa, M. (Organizing Members)
JSPS-DST Asia Academic Seminar:
NIG-NCBS International Workshop on Single-molecule Biophysics, Bangalore, India January, 2004. (25 International Invitees from US, France, Swiss, Israel, Korea, Japan, India and 30 Asian Trainees from India, Japan, China, Korea, Malaysia, Singapore)
2. Member of international Committee of Asian Conference of Transcription (ACT), the delegate of Japan. (Organizing members of ACT8, Bangkok, November, 2004)

特許
1. 嶋本伸雄・中山秀喜・荒牧弘範（発明者）,国立遺伝学研究所長（出願者）,2004年3月3日,ゲノムライブラリー作成方法,および同方法により作成されたゲノムライブラリー,特願2004-59900,機構整理番号,U2003P394
2. 嶋本伸雄・中山秀喜・荒牧弘範（発明者）,国立遺伝学研究所長が代表する日本国（出願者）,2004年3月16日,ゲノムライブラリー作成方法,および同方法により作成されたゲノムライブラリー,PCT/JP2004/003507,機構整理番号,U2003P103
3. 嶋本伸雄・須佐太樹・福島和久（発明者）,横河電機株式会社（出願者）,2004年10月6日,生体高分子検出方法およびバイオチップ並びに抗体固定法及び抗体固定基板,米国およびドイツに出願
4. 嶋本伸雄・須佐太樹・福島和久（発明者）,横河電機株式会社（出願者）,2004年10月6日,生体高分子検出方法およびバイオチップ並びに抗体固定法及び抗体固定基板,中国特許出願番号200410090010.5

学会活動等
1. Member of international Committee of Asian Conference of Transcription(ACT), the delegate of Japan.(Organizing members of ACT8, Bangkok, November, 2004)
2. 嶋本伸雄,財団法人未踏科学技術協会「生命を測る」組織幹事

各種委員
1. 嶋本伸雄,文部科学省科学技術・学術審議会専門委員
2. 嶋本伸雄,NEDO「ナノバイオテクノロジー産業化推進調査」委員,WG