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Inter and intramolecular communications in transcription

转录中的分子间和分子内通讯

基本信息

批准号：
8245827
负责人：
TOMASZ HEYDUK
金额：
$ 30.72万
依托单位：
SAINT LOUIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8245827
关键词：
Antibiotic Resistance Antibiotics Archaea Archaeal RNA Bacteria Bacterial RNA Beryllium Binding Biological Process Biology Cell physiology Communication Complex DNA DNA Binding DNA-Directed RNA Polymerase Development Elements Enzymes Eukaryota Event Evolution Exhibits Fluorescence Free Energy Funding Genes Genetic Transcription Goals Health Healthcare Indium Kinetics Knowledge Linker DNA Maps Messenger RNA Molecular Molecular Conformation Mutagenesis Nature Outcome Period Analysis Play Polymerase Positioning Attribute Process Property Proteins RNA Randomized Reaction Regulation Relative (related person)Reporting Research Role Sigma Factor Site-Directed Mutagenesis Structure Techniques Thermodynamics Thymine Time Transcription Initiation Transcription Process combat functional group in vivo insight melting promoter resistant strain

项目摘要

DESCRIPTION (provided by applicant): Transcription is one of the central points of regulation of cellular functions. Understanding molecular mechanisms of transcription and its regulation remains one of the most important goals in biology. DNA-dependent RNA polymerases (RNAP), the enzymes capable of faithfully copying the information encoded in gene sequence into the mRNA product, are of central importance in the process of transcription. Our research is focused on bacterial and archaeal RNAP's. These enzymes are among the simplest multisubunit polymerases, offering the best chance for obtaining detailed molecular understanding of RNAP activity. Our long term goal is to understand the molecular mechanism by which RNA polymerase initiates transcription. Bacterial RNAP is an attractive target for new antibiotics since it is an essential enzyme. Detailed understanding of RNAP properties unique to the bacterial enzyme will aid the development of new antibiotics targeting RNAP. It is clear now that transcription initiation involves a complex interplay between numerous RNAP-promoter contacts. While we are beginning to understand how RNAP utilizes these contacts in transcription initiation reaction, many gaps in knowledge will need to be filled before a comprehensive understanding of transcription initiation is achieved. In this project we will focus on filling in three of such gaps. In aim 1 we will determine the role of conserved nontemplate -7 thymine in promoter melting by bacterial RNA polymerase. In aim 2 we will determine the role of -35 conserved promoter element and the spacer connecting -10 and -35 promoter elements in promoter melting by bacterial RNA polymerase. In aim 3, we will investigate the interplay between RNAP interactions with the core and secondary promoter elements in promoter escape by bacterial RNA polymerase. Furthermore, we will also begin to investigate the mechanism of transcription initiation by archaeal RNA polymerase (aim 4). Archaeal RNA polymerase exhibits eukaryote-like structure but bacteria-like promoter melting (i.e. no auxiliary ATP-dependent activities are necessary for melting). Thus, the studies with archaeal polymerase will provide insights into the evolution of transcription initiation mechanism and will facilitate understanding of general mechanistic features this process. In our studies we will utilize a combination of fluorescence techniques together with mutagenesis, kinetic, thermodynamic and structural analysis of promoter melting reactions. Upon completion of these studies mechanistic insights to a fundamental biological process will be gained. PUBLIC HEALTH RELEVANCE: This project will provide new insights into the mechanism of transcription initiation by bacterial RNA polymerase. Such information will enable development of new antibiotics targeting bacterial RNA polymerase. Development of new antibiotics targeting new targets is essential to combating emerging antibiotic-resistant strains.

描述（由申请人提供）：转录是细胞功能调节的中心点之一。了解转录及其调控的分子机制仍然是生物学最重要的目标之一。 DNA 依赖性 RNA 聚合酶 (RNAP) 能够将基因序列编码的信息忠实地复制到 mRNA 产物中，在转录过程中至关重要。我们的研究重点是细菌和古菌 RNAP。这些酶是最简单的多亚基聚合酶之一，为获得 RNAP 活性的详细分子了解提供了最佳机会。我们的长期目标是了解 RNA 聚合酶启动转录的分子机制。细菌 RNAP 是新型抗生素的一个有吸引力的靶点，因为它是一种必需的酶。详细了解细菌酶特有的 RNAP 特性将有助于开发针对 RNAP 的新型抗生素。现在很清楚，转录起始涉及众多 RNAP 启动子接触之间复杂的相互作用。虽然我们开始了解 RNAP 如何在转录起始反应中利用这些接触，但在全面了解转录起始之前，需要填补许多知识空白。在这个项目中，我们将重点填补其中三个空白。在目标 1 中，我们将确定保守的非模板 -7 胸腺嘧啶在细菌 RNA 聚合酶熔化启动子中的作用。在目标 2 中，我们将确定 -35 保守启动子元件以及连接 -10 和 -35 启动子元件的间隔区在细菌 RNA 聚合酶启动子熔解中的作用。在目标 3 中，我们将研究细菌 RNA 聚合酶启动子逃逸过程中 RNAP 与核心和二级启动子元件的相互作用之间的相互作用。此外，我们还将开始研究古细菌RNA聚合酶的转录起始机制（目标4）。古细菌 RNA 聚合酶表现出类似真核生物的结构，但具有类似细菌的启动子熔化（即熔化不需要辅助的 ATP 依赖性活性）。因此，对古细菌聚合酶的研究将为转录起始机制的进化提供见解，并将有助于理解该过程的一般机制特征。在我们的研究中，我们将结合使用荧光技术以及启动子熔化反应的诱变、动力学、热力学和结构分析。完成这些研究后，将获得对基本生物过程的机械见解。公共健康相关性：该项目将为细菌 RNA 聚合酶转录起始机制提供新的见解。这些信息将有助于开发针对细菌 RNA 聚合酶的新型抗生素。开发针对新靶标的新抗生素对于对抗新出现的抗生素耐药菌株至关重要。

项目成果

期刊论文数量（35）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases.

使用不同的化学蛋白酶通过蛋白质足迹技术绘制环核苷酸诱导的 cyclAMP 受体蛋白构象变化。

DOI：
10.1110/ps.8.3.518
发表时间：
1999
期刊：
Protein science : a publication of the Protein Society
影响因子：
0
作者：
Baichoo,N;Heyduk,T
通讯作者：
Heyduk,T

Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact.

大肠杆菌 RNA 聚合酶的 α 亚基与 DNA 的相互作用：蛋白质-DNA 接触的刚体性质。

DOI：
10.1074/jbc.m107760200
发表时间：
2001
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Heyduk,E;Baichoo,N;Heyduk,T
通讯作者：
Heyduk,T

Escherichia coli RNA polymerase contacts outside the -10 promoter element are not essential for promoter melting.

-10 启动子元件外部的大肠杆菌 RNA 聚合酶接触对于启动子解链并不重要。

DOI：
10.1074/jbc.m507984200
发表时间：
2005
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Niedziela-Majka,Anita;Heyduk,Tomasz
通讯作者：
Heyduk,Tomasz

Interaction of T4 AsiA with its target sites in the RNA polymerase sigma70 subunit leads to distinct and opposite effects on transcription.

T4 AsiA 与其 RNA 聚合酶 sigma70 亚基中的靶位点相互作用，会对转录产生不同且相反的影响。

DOI：
10.1016/s0022-2836(02)01442-0
发表时间：
2003
期刊：
Journal of molecular biology
影响因子：
5.6
作者：
Minakhin,Leonid;Niedziela-Majka,Anita;Kuznedelov,Konstantin;Adelman,Karen;Urbauer,JeffreyL;Heyduk,Tomasz;Severinov,Konstantin
通讯作者：
Severinov,Konstantin

Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human coagulation factor xa. Studies on catalytic efficiency and inhibitor binding.

人凝血因子 xa 蛋白酶结构域中 S1 位点、Na 位点和 Ca2 位点之间的热力学连接。