T7 RNA-POLYMERASE DOES NOT INTERACT WITH THE 5'-PHOSPHATE OF THE INITIATING NUCLEOTIDE
T7 RNA-POLYMERASE DOES NOT INTERACT WITH THE 5'-PHOSPHATE OF THE INITIATING NUCLEOTIDE
复制标题
DOI：
10.1021/bi00433a002
复制
发表时间：
1989-04-04
期刊：
影响因子：
2.9
通讯作者：
COLEMAN, JE
中科院分区：
文献类型：
--
作者：
MARTIN, CT;COLEMAN, JE
T7 RNA 聚合酶的启动子尺寸较小，有利于研究转录动力学，从而允许使用具有精心定义的序列的合成寡核苷酸模板。我们之前曾使用这种方法来测量五碱基径流转录本 GGACU 生产的 Michaelis-Menten 稳态动力学。特别地，在所有四种三磷酸核苷酸的饱和水平下，酶和模板之间的相互作用的Km显示为大约0.02μM。我们现在表明，在径流转录本 GACU 的类似模板编码上启动的相应 Km 和 Vmax 与早期研究相同（Km = 0.02 μM；kcat = 40-50 min-1）。这个新模板允许测量初始核苷酸 GTP 与酶或酶-DNA 复合物关联的 Km。结果表明，在进行性延伸的后期阶段，添加延伸性 GTP 时，KmgTP (0.60 mM) 略高于早期的 Km 近似值。正如预期的那样，GTP 类似物 ITP (KmITP) 的（起始）Km 增加（约 2 倍），可能是由于 Watson-Crick 碱基配对减弱的结果。然而，GTP 类似物 GMP 和鸟苷的 Km 值比较表明，对 5''-三磷酸分别被单磷酸或羟基取代几乎没有影响。这一结果表明，单个活性位点已经进化适应从等待核苷酸的5''端接受起始时的5''-三磷酸和延伸过程中的5''-单磷酸酯(RNA)。这种双重功能是在与等待核苷酸的 5'' 末端失去任何正向或负向结合相互作用的情况下实现的。
The study of transcription kinetics by T7 RNA polymerase is facilitated by the small size of its promoter, allowing the use of synthetic oligonucleotide templates with carefully defined sequences. We have previously used this approach to measure Michaelis-Menten steady-state kinetics for production of the five-base runoff transcript GGACU. In particular, Km for the interaction between enzyme and template under saturating levels of all four nucleotide triphosphates was shown to be approximately 0.02 .mu.M. We now show that the corresponding Km and Vmax for initiation on a similar template coding for the runoff transcript GACU are the same as for the earlier study (Km = 0.02 .mu.M; kcat = 40-50 min-1). This new template allows the measurement Km for association of the initial nucleotide GTP with enzyme or with the enzyme-DNA complex. The results show that KmgTP (0.60 mM) is somewhat higher than earlier approximations of Km for addition of elongating GTP during the later phase of processive elongation. As expected, the (initiating) Km for the GTP analogue ITP (KmITP) is increased (by about 2-fold), presumably as a result of weakened Watson-Crick base pairing. However, comparison of Km values for the GTP analogues GMP and guanosine shows little effect on substitution of the 5''-triphosphate by monophosphate or by a hydroxyl, respectively. This result suggests that a single active site has been evolutionarily adapted to accept from the 5'' end of a waiting nucleotide both a 5''-triphosphate at initiation and a 5''-monophosphate ester (RNA) during elongation. This dual functionality is achieved at the loss of any binding interaction, positive or negative with the 5'' end of the waiting nucleotide.