Swart J R, Griep M A
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
Biochemistry. 1995 Dec 12;34(49):16097-106. doi: 10.1021/bi00049a025.
The kinetics of primer RNA initiation and elongation by Escherichia coli primase were measured. The single-stranded DNA template that was used to develop the system, d(CAGA-(CA)5CTGCAAAGC), contained: (1) the preferred initiating trinucleotide d(CTG); (2) five residues 3' to the d(CTG), the minimum required for efficient primer synthesis; and (3) a single guanine placed near the 5'-end to facilitate study of cytidine triphosphate analog incorporation at a unique site. The assay monitored radiolabeled nucleotide incorporation into the RNA primers. The various primers were separated by length using denaturing polyacrylamide gel electrophoresis. Different types of primers were observed when synthesis was monitored using gamma- versus alpha-radiolabeled nucleotides as the probe. When [gamma-32P]-ATP incorporation was the probe, only primers initiated with ATP from the unique template thymine were observed. The sequences of these primers were complementary to that of the template. No primers shorter than a 12-mer accumulated, demonstrating that formation of the first phosphodiester bond was much slower than that of the next 10 phosphodiester bonds. The longest primer observed when monitoring [gamma-32P]ATP incorporation was 16 nucleotides long, the correct length for a primer completely template-directed and initiated at the unique thymine. Misinsertion of a noncognate nucleotide at the template's guanine indicated very poor nucleotide discrimination by this enzyme. When [alpha-32P]UTP was the probe for primer synthesis, all primers synthesized were observed whether or not they were initiated with ATP. Under these conditions, "overlong" primers and the above-described template length-dependent primers were observed. The template length-dependent primers accumulated faster than the overlong primers, but, at long incubation times, the overlong primers became the dominant species. The overlong primers were not fully related to the template length-dependent primers since they were not initiated complementary to the template d(CTG). Nevertheless, the overlong primers did appear to arise as a consequence of the template length-dependent species since their length was double and they arose in the time course after the length-dependent species.
对大肠杆菌引发酶引发的引物RNA起始和延伸动力学进行了测定。用于构建该系统的单链DNA模板d(CAGA-(CA)5CTGCAAAGC)包含：(1) 优选的起始三核苷酸d(CTG)；(2) d(CTG) 3'端的五个残基，这是高效引物合成所需的最少数量；(3) 一个位于5'端附近的鸟嘌呤，以利于在一个独特位点研究胞苷三磷酸类似物的掺入。该测定监测放射性标记的核苷酸掺入RNA引物的情况。使用变性聚丙烯酰胺凝胶电泳按长度分离各种引物。当使用γ-与α-放射性标记的核苷酸作为探针监测合成时，观察到了不同类型的引物。当以[γ-32P]-ATP掺入作为探针时，仅观察到从独特模板胸腺嘧啶起始且以ATP起始的引物。这些引物的序列与模板互补。没有积累到短于12聚体的引物，这表明第一个磷酸二酯键的形成比接下来的10个磷酸二酯键的形成慢得多。监测[γ-32P]ATP掺入时观察到的最长引物为16个核苷酸长，这是完全由模板指导并在独特胸腺嘧啶处起始的引物的正确长度。在模板的鸟嘌呤处错插入一个非同源核苷酸表明该酶的核苷酸辨别能力很差。当以[α-32P]UTP作为引物合成的探针时，无论是否以ATP起始，都观察到了所有合成的引物。在这些条件下，观察到了“超长”引物和上述模板长度依赖性引物。模板长度依赖性引物的积累速度比超长引物快，但在长时间孵育时，超长引物成为主要种类。超长引物与模板长度依赖性引物并不完全相关，因为它们不是与模板d(CTG)互补起始的。然而，超长引物似乎确实是模板长度依赖性种类的结果，因为它们的长度是其两倍，并且在长度依赖性种类之后的时间进程中出现。
