Taq聚合酶的结构,聚合酶活性位点上的DNA
1Department of Molecular Biophysics, Yale University, New Haven, Connecticut 06520-811, USA.
Nature
|July 18, 1996
概括
研究人员确定了Taq聚合酶在活性部位与DNA结合的结构. 这揭示了酶如何结合不的DNA,与之前的克伦诺碎片结构不同.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 分子生物学分子生物学
背景情况:
- 塔克聚合酶与大肠杆菌DNA聚合酶I (Pol I) 具有同质性,具有聚合酶和5'核酶域.
- 在Taq聚合酶中的3'-5'外核酶域在结构上发生了改变,与Pol I的Klenow片段相比,它缺乏编辑功能.
- 之前的结构研究缺乏一个复合的Taq聚合酶与多重DNA在聚合酶活性部位.
研究的目的:
- 为了阐明Taq聚合酶与聚合酶活性位点的形双重DNA结合的共同晶体结构.
- 了解DNA结合和Taq聚合酶在其活性位点上的识别的结构基础.
主要方法:
- Taq聚合酶与形双重DNA的联合晶体学.
- 进行X射线衍射分析以确定三维结构.
主要成果:
- 共同晶体结构揭示了Taq聚合酶与活性位点裂内的形双重DNA结合.
- DNA没有明显的曲,并且采用B-DNA和A-DNA之间的结构形式.
- 一个宽的小沟促进了蛋白质与DNA末端的相互作用,结合位与外核酶位重叠.
结论:
- 确定的结构提供了对Taq聚合酶在活性位点结合DNA的机制的洞察.
- 结构上的差异解释了Taq聚合酶中3'-5'外核酶活性缺失.
- 这项工作促进了对热稳定酶中的DNA聚合酶-DNA相互作用的理解.
相关概念视频
PCR
Overview
DNA Replication
DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied. After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication uses a large number of...
Replication in Prokaryotes
DNA replication uses a large number of...
The Replisome
DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
Translesion DNA Polymerases
Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
Bacterial RNA Polymerase
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
Nucleic Acid Structure
The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms a 5′ to 3′ phosphodiester linkage.
DNA Structure
DNA has a double-helix structure. The...
DNA Structure
DNA has a double-helix structure. The...


