Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 2. Processivity, ATP consumption, and RNA binding.
Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 2. Processivity, ATP consumption, and RNA binding.
复制标题
DOI：
10.1021/bi963180r
复制
发表时间：
1997-07
期刊：
影响因子：
2.9
通讯作者：
K. Walstrom;J. Dozono;P. V. von Hippel
中科院分区：
文献类型：
--
作者：
K. Walstrom;J. Dozono;P. V. von Hippel
用长度为255和391个核苷酸残基的天然RNA分子研究了大肠杆菌转录终止因子rho的RNA结合和RNA-DNA解旋酶活性。杆菌通过使一种或多种DNA寡聚物与位于RNA分子3 ′-末端或其附近的互补序列退火，以形成长度范围为20 - 100 bp的确定的RNA-DNA杂合序列，从这些RNA分子制备解旋酶底物。通过比较与rho结合的RNA分子的分数与在一轮解旋酶反应期间从RNA中去除的结合的DNA寡聚体的分数，我们已经表明rho在37 ° C下在含有50 mM KCl的缓冲液中易位至proc。解旋酶反应和ATP酶的测量进行了平行的存在下，含有RNA-DNA杂交的各种长度的RNA分子，我们表明，无论是易位率的rho六聚体沿着的RNA链和ATP的消耗率是相似的，DNA杂交的RNA转录。通过结合易位和ATP酶速率的测量，我们估计rho在37 ℃下在50 mM KCl中易位RNA链的1个核苷酸残基时消耗约1-2个ATP分子。rho的ATP酶活性在一轮解旋酶反应后保持不变，表明rho似乎以相同的速率水解ATP，无论它是沿着RNA移位，分离RNA-DNA杂交体，还是结合在RNA底物的3 '端。我们还表明，在我们的标准解旋酶反应条件下，rho与RNA底物协同结合（每条RNA链约2-4个rho六聚体）。然而，协同结合对于解旋酶活性不是必需的，因为通过用互补DNA寡核苷酸阻断解旋酶底物的rho结合位点的任一末端的约100 nt，这种结合化学计量可以减少到每255个核苷酸残基RNA链约1.5个rho六聚体，而解旋酶性质没有变化。这些结果的影响模型的rho解旋酶的功能和rho在终止的作用进行了讨论。
The RNA-binding and RNA-DNA helicase activities of the Escherichia coli transcription termination factor rho have been investigated using natural RNA molecules that are 255 and 391 nucleotide residues in length and that contain the trp t' rho-dependent termination sequence of E. coli. Helicase substrates were prepared from these RNA molecules by annealing one or more DNA oligomers to complementary sequences located at or near the 3'-ends of the RNA molecules to form defined RNA-DNA hybrid sequences ranging in length from 20 to 100 bp. By comparing the fraction of the RNA molecules bound to rho with the fraction of bound DNA oligomers removed from the RNA during one round of the helicase reaction, we have shown that rho translocates processively at 37 degrees C in buffer containing 50 mM KCl. Helicase reactions and ATPase measurements were performed in parallel in the presence of RNA molecules containing RNA-DNA hybrids of various lengths, and we show that both the rate of translocation of the rho hexamer along the RNA chain and the rate of ATP consumption are similar, whether or not DNA is hybridized to the RNA transcript. By combining measurements of translocation and ATPase rates, we estimate that rho consumes approximately 1-2 ATP molecules in translocating over 1 nucleotide residue of the RNA chain at 37 degrees C in 50 mM KCl. The ATPase activity of rho remains the same after one round of the helicase reaction, indicating that rho appears to hydrolyze ATP at the same rate, whether it is translocating along the RNA, separating RNA-DNA hybrids, or bound at the 3'-end of the RNA substrate. We also show that rho binds cooperatively ( approximately 2-4 rho hexamers per RNA chain) to the RNA substrates under our standard helicase reaction conditions. However, cooperative binding is not essential for helicase activity, since this binding stoichiometry can be reduced to approximately 1.5 rho hexamers per 255-nucleotide residue RNA chain by blocking approximately 100 nt of either end of the rho binding site of the helicase substrate with complementary DNA oligonucleotides, with no change in helicase properties. The implications of these results for models of rho helicase function and for the role of rho in termination are discussed.