SEQUENCE-SPECIFIC DNA-PROTEIN INTERACTIONS

我们研究的主要长期目标是了解详细的 蛋白质识别特定蛋白质并与其相互作用的分子机制 DNA序列。这个问题是通过一个详细的 II型限制和修饰的结构和功能研究 系统。EcoRI系统是最具特点的。基因和氨基酸 测定了两种酶的酸序列，并进行了X-射线分析 结晶学分析开始提供关于 核酸内切酶-DNA复合体的结构。互补性接触 将探索酶和它们的底物序列之间的关系。 EcoRI内切酶的突变体将被分离和定位。更改后的 酶将被提纯并分析功能参数，如 DNA水解动力学、二聚体结构稳定性、平衡 结合常数和与DNA底物的接触。其他类型II 将对系统进行检查，以确定 来自EcoRI系统的信息。具体地说，是一种异构体 已在球形红假单胞菌中鉴定出EcoRI。虽然R. 球状杆菌和大肠杆菌有相当远的亲缘关系，初步认为 实验表明，这些内切酶具有共同的起源。RSRI 限制性内切酶和修饰系统将克隆到大肠杆菌中。脱氧核糖核酸 序列分析将被用来推断该蛋白的氨基酸序列。 酵素。这些酶将被提纯，功能参数将被 与EcoRI的同行相比。来自这两家公司的对比数据 系统将根据三维结构进行分析 X射线结晶学衍生的野生型酶-DNA复合体 分析。识别DNA中已定义的序列是一个必不可少的过程 在DNA复制、重组和基因组表达过程中 细胞新陈代谢和发育。对涉及的机制的阐明 利用相对简单的系统的特定序列的相互作用应该 说明这些复杂的过程。 II型限制性内切酶修饰所利用的基因调控方式 对于任何系统，系统都没有被阐明。在EcoRI的情况下 系统，已经证明了激活甲基酶是必需的 核酸内切酶的表达。这种激活的机制还没有 已被确认身份。β-半乳糖苷酶融合质粒将用于 监测甲基酶的激活情况。体内转录将通过分析 Northern杂交分析和S1定位。

The major long-term objective of our research is to understand the detailed molecular mechanisms by which proteins recognize and interact with specific DNA sequences. This problem is being approached through a detailed structural and functional study of type II restriction and modification systems. The EcoRI system is the best characterized. The gene and amino acid sequences of both enzymes have been determined, and X-ray crystallographic analysis is beginning to yield detailed information about the structure of the endonuclease-DNA complex. Complementary contacts between the enzymes and their substrate sequence will be explored. Mutants of the EcoRI endonuclease will be isolated and mapped. Altered enzymes will be purified and analyzed for functional parameters such as kinetics of DNA hydrolysis, stability of dimer structure, equilibrium binding constants and contacts with the DNA substrate. Other type II systems will be examined in order to determine the generality of information derived from the EcoRI system. In particular, an isoschizomer of EcoRI has been identified in Rhodopseudomonas sphaeroides. Although R. sphaeroides and E. coli are rather distantly related, preliminary experiments indicate that the endonucleases have a common origin. The RsrI restriction and modification system will be cloned into E. coli. DNA sequence analysis will be used to deduce the amino acid sequences of the enzymes. The enzymes will be purified and functional parameters will be compared to the EcoRI counterparts. Comparative data from both of these systems will be analyzed in relation to the three-dimensional structure of the wild type enzyme-DNA complex derived by X-ray crystallographic analyses. Recognition of defined sequences in DNA is an essential process in DNA replication, recombination and the expression of the genome during cellular metabolism and development. Elucidation of mechanisms involved in sequence-specific interactions utilizing relatively simple systems should illuminate these complex processes. The mode of gene regulation utilized by type II restriction-modification systems has not been elucidated for any system. In the case of the EcoRI system, it has been shown that the methylase is required to activate ezpression of the endonuclease. The mechanism of this activation has not been identified. Beta-galactosidase fusion plasmids will be used to monitor methylase activation. In vivo transcription will be analyzed by Northern blot analysis and S1 mapping.