DYNAMICS OF T7 RNA POLYMERASE TRANSCRIPTION
T7 RNA 聚合酶转录动力学
基本信息
- 批准号:6684087
- 负责人:
- 金额:$ 29.04万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1995
- 资助国家:美国
- 起止时间:1995-09-30 至 2004-11-30
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
DESCRIPTION: As structural information on the cell's macromolecular machinery
accumulates, our attention turns towards understanding the dynamics of this
machinery. For a multi-step reaction, such as transcription, we want to know
how the macromolecular complex moves from one step to another along the
reaction pathway. When alternate pathways exist (i.e., pause or not; terminate
or continue; displace the RNA or form a hybrid for priming replication) we want
to understand what determines the choice between these pathways and how the
decision is made. Finally, we want to understand how these reaction mechanisms
may be sensitive to regulation. Despite limited structural similarity between
the multi-and single subunit RNAPs, the transcription reactions mediated by
these enzymes are remarkably similar, even in details such as the length of the
RNA at which promoter release occurs or the fine structure of terminators,
suggesting that these structurally dissimilar molecules have converged upon
similar solutions for executing a transcription reaction. We propose to use the
structurally well characterized single-subunit 17RNAP as a model to understand
the conformational dynamics of transcription. We will use nucleases and FeBABE
conjugated RNAPs to probe the changes in RNAP:RNA/DNA
interactions that occur as the polymerase moves from initiation to elongation.
The changes in elongation complex conformation which accompany pausing, NTP
binding, or formation of extended hybrids will be similarly characterized.
Engineered disulphide cross-links will be used measure the effects of
conformational restriction on RNAP function. The roles of sequence and
supercoiling in deterrr aboutining whether the RNA is displaced, or whether
extended or persistent hybrids form, will be defined. The sequence-specific
interactions required for site-specific pausing will be identified. The
conformational state of the active site, which is regulated via binding of T7
lysozyme, will be monitored with carboxypeptidase as the RNAP pauses. The
mechanisms of promoter unwinding and RNA displacement will be characterized by
mutagenizing the RNAP elements proposed to be important for these processes,
and by mapping the DNA/RNA interactions made by these elements. Finally, the
mechanism by which T7RNAP primes T7 DNA replication will be studied to reveal
how an RNAP engaged in elongation transfers a priming transcript to a DNA
polymerase.
描述:作为细胞大分子机制的结构信息
积累,我们的注意力转向了解这一动态,
机械.对于多步反应,例如转录,我们想知道
大分子复合物如何从一个步骤移动到另一个沿着
反应途径当存在替代途径时(即,停不停
或继续;取代RNA或形成杂交以引发复制),
了解是什么决定了这些途径之间的选择,
已经决定了。最后,我们想了解这些反应机制
可能对监管很敏感。尽管有限的结构相似性之间
多亚基和单亚基RNAP,由
这些酶是非常相似的,甚至在细节上,如长度的酶。
启动子释放发生的RNA或终止子的精细结构,
这表明这些结构不同的分子聚集在
用于执行转录反应的类似解决方案。我们建议使用
结构良好表征的单亚基17 RNAP作为模型,以了解
转录的构象动力学我们将使用核酸酶和FeBABE
结合RNAP以探测RNAP:RNA/DNA中的变化
当聚合酶从起始移动到延伸时发生的相互作用。
伴随停顿的延伸复合体构象的变化,NTP
延伸杂交体的结合或形成将被类似地表征。
工程二硫化物交联将被用来衡量的影响,
RNAP功能的构象限制。序列的作用和
超螺旋在决定RNA是否被置换,或者
扩展或持久的杂交形式,将被定义。序列特异
将确定站点特定暂停所需的交互。的
活性位点的构象状态,其通过T7的结合来调节
当RNAP暂停时,用羧肽酶监测溶菌酶。的
启动子解旋和RNA置换的机制的特征在于:
诱变RNAP元素被认为对这些过程是重要的,
以及绘制这些元件所产生的DNA/RNA相互作用。最后
T7 RNAP引发T7 DNA复制的机制将被研究,以揭示
参与延伸的RNAP如何将引发转录物转移到DNA
聚合酶。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('RUI J. SOUSA', 18)}}的其他基金
Technology for Synthesis of Chemically Diverse RNAs
化学多样性 RNA 的合成技术
- 批准号:
9199672 - 财政年份:2016
- 资助金额:
$ 29.04万 - 项目类别:
Development of imaging reagents to monitor GTP levels and GTP/GDP ratios in vivo
开发成像试剂来监测体内 GTP 水平和 GTP/GDP 比率
- 批准号:
8129628 - 财政年份:2010
- 资助金额:
$ 29.04万 - 项目类别:
Development of imaging reagents to monitor GTP levels and GTP/GDP ratios in vivo
开发成像试剂来监测体内 GTP 水平和 GTP/GDP 比率
- 批准号:
7941549 - 财政年份:2010
- 资助金额:
$ 29.04万 - 项目类别:
Biology and Mechanism of the Single Subunit RNA Polymerases
单亚基RNA聚合酶的生物学和机制
- 批准号:
7920708 - 财政年份:2009
- 资助金额:
$ 29.04万 - 项目类别:
STRUCTURE/FUNCTION STUDIES OF T7 RNA POLYMERASE
T7 RNA 聚合酶的结构/功能研究
- 批准号:
2191581 - 财政年份:1995
- 资助金额:
$ 29.04万 - 项目类别:
Conformational Dynamics of T7 RNAP Transcription
T7 RNAP 转录的构象动力学
- 批准号:
7318332 - 财政年份:1995
- 资助金额:
$ 29.04万 - 项目类别:
Conformational Dynamics of T7 RNAP Transcription
T7 RNAP 转录的构象动力学
- 批准号:
6987143 - 财政年份:1995
- 资助金额:
$ 29.04万 - 项目类别:
Biology and Mechanism of the Single Subunit RNA Polymerases
单亚基RNA聚合酶的生物学和机制
- 批准号:
7743367 - 财政年份:1995
- 资助金额:
$ 29.04万 - 项目类别:
STRUCTURE/FUNCTION STUDIES OF T7 RNA POLYMERASE
T7 RNA 聚合酶的结构/功能研究
- 批准号:
2900837 - 财政年份:1995
- 资助金额:
$ 29.04万 - 项目类别:
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