An intramolecular FRET system monitors fingers subdomain opening in Klentaq1

被引：18

作者：

Allen, William J. ^{[1
,2
]}

Rothwell, Paul J. ^{[1
,2
]}

Waksman, Gabriel ^{[1
,2
,3
]}

机构：

[1] Univ London Birkbeck Coll, Inst Struct Mol Biol, London WC1E 7HX, England

[2] Sch Crystallograph, London WC1E 7HX, England

[3] UCL, Res Dept Struct & Mol Biosci, London WC1E 6BT, England

来源：

PROTEIN SCIENCE | 2008年 / 17卷 / 03期

基金：

英国惠康基金;

关键词：

polymerase; Klentaq1; subdomain movement; FRET; stopped-flow; kinetics;

D O I：

10.1110/ps.073309208

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. However, this FRET system was limited in that it could not directly measure the rate of fingers subdomain opening by FRET after polymerization or in the absence of DNA. Here we report the development of a new system capable of measuring both fingers subdomain closure and reopening by FRET, and show that the rate of fingers subdomain opening is limited only by the rate of polymerization. We anticipate that this system will scale down to the single molecule level, allowing measurement of fingers subdomain movements in the presence of incorrect nucleotide and in the absence of DNA.

引用

页码：401 / 408

页数：8

共 32 条

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