Real-time fluorescence assay system for gene transcription: Simultaneous observation of protein/DNA binding, localized DNA melting, and mRNA production

被引:19
作者
Dunkak, KS [1 ]
Otto, MR [1 ]
Beechem, JM [1 ]
机构
[1] VANDERBILT UNIV,SCH MED,DEPT PHYSIOL & MOL BIOPHYS,NASHVILLE,TN 37232
关键词
D O I
10.1006/abio.1996.0511
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
This article describes the development of an in vitro multicolor fluorescence assay system for studying protein/DNA complex formation, transcription bubble formation, and mRNA production. These studies were accomplished using three different fluorescent spectroscopic probes: rhodamine-labeled DNA (at the 5' position) to monitor protein/DNA complex formation, DNA internally labeled with the base analog 2-amino-purine in place of adenine to monitor transcription bubble formation, and gamma-fluorophore-labeled UTP nucleotide to measure mRNA transcription rates. Combining these three assay systems allows the simultaneous determination of protein/DNA binding, localized DNA melting transitions, and mRNA production at physiological concentrations of reagents (pM-nM) and millisecond timing resolution. The application of this multicolor fluorescence assay to Escherichia coli RNA polymerase reactions (binding, open complex formation, and mRNA production) demonstrates the importance of kinetically coupled events in gene transcription. (C) 1996 Academic Press, Inc
引用
收藏
页码:234 / 244
页数:11
相关论文
共 32 条
  • [1] ALBERTS B, 1994, MOL BIOL CELL, P2126
  • [2] BEECHEM JM, 1992, METHOD ENZYMOL, V210, P37
  • [3] BEECHEM JM, 1991, TOPICS FLUORESCENCE, V2
  • [4] Kinetic mechanism of DNA binding and DNA-induced dimerization of the Escherichia coli Rep helicase
    Bjornson, KP
    Moore, KJM
    Lohman, TM
    [J]. BIOCHEMISTRY, 1996, 35 (07) : 2268 - 2282
  • [5] PRE-STEADY-STATE KINETIC-ANALYSIS OF SEQUENCE-DEPENDENT NUCLEOTIDE EXCISION BY THE 3'-EXONUCLEASE ACTIVITY OF BACTERIOPHAGE-T4 DNA-POLYMERASE
    BLOOM, LB
    OTTO, MR
    ERITJA, R
    REHAKRANTZ, LJ
    GOODMAN, MF
    BEECHEM, JM
    [J]. BIOCHEMISTRY, 1994, 33 (24) : 7576 - 7586
  • [6] INFLUENCE OF 5'-NEAREST NEIGHBORS ON THE INSERTION KINETICS OF THE FLUORESCENT NUCLEOTIDE ANALOG 2-AMINOPURINE BY KLENOW FRAGMENT
    BLOOM, LB
    OTTO, MR
    BEECHEM, JM
    GOODMAN, MF
    [J]. BIOCHEMISTRY, 1993, 32 (41) : 11247 - 11258
  • [7] BRENNAN CA, 1986, J BIOL CHEM, V261, P7279
  • [8] KINETICS OF OPEN COMPLEX-FORMATION BETWEEN ESCHERICHIA-COLI RNA-POLYMERASE AND THE LAC UV5 PROMOTER - EVIDENCE FOR A SEQUENTIAL MECHANISM INVOLVING 3 STEPS
    BUC, H
    MCCLURE, WR
    [J]. BIOCHEMISTRY, 1985, 24 (11) : 2712 - 2723
  • [9] REAL-TIME FLUORESCENCE DETECTION OF RNA AMPLIFIED BY Q-BETA REPLICASE
    BURG, JL
    CAHILL, PB
    KUTTER, M
    STEFANO, JE
    MAHAN, DE
    [J]. ANALYTICAL BIOCHEMISTRY, 1995, 230 (02) : 263 - 272
  • [10] MULTIPLE RNA-POLYMERASE CONFORMATIONS AND GREA - CONTROL OF THE FIDELITY OF TRANSCRIPTION
    ERIE, DA
    HAJISEYEDJAVADI, O
    YOUNG, MC
    VONHIPPEL, PH
    [J]. SCIENCE, 1993, 262 (5135) : 867 - 873