PROKARYOTIC CHROMATIN AND TRANSCRIPTIONAL REGULATION

原核染色质和转录调控

基本信息

批准号：
2684873
负责人：
ERNEST PETER GEIDUSCHEK
金额：
$ 23.35万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-04-01 至 2001-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2684873
关键词：
DNA binding protein DNA directed RNA polymerase bacterial virus chromatin conformation fluorimetry genetic manipulation genetic transcription nucleic acid sequence prokaryote transcription factor virus genetics virus protein

项目摘要

The research program deals with mechanisms for regulating the activities of genes. Its broad implications are for understanding gene regulation in developing systems and infection processes, and for precise interventions concerning protein-nucleic acid interactions and gene activity. The specific system under analysis involves the bacteriophage SPO1 encoded chromatin-forming protein TF1, which is a homolog of the ubiquitous prokaryotic type II DNA-binding proteins (DBPII). The DBPII family is remarkable for including proteins that bind to DNA nonspecifically, sequence-specifically and, in the case of TF1, sequence- as well as modified nucleotide-specifically. Experiments dealing with the regulation of phage SPO1 late genes are also proposed. The following lines of work deal with the DNA-binding and -bending protein TF1: (1) We shall determine how the principal contributions to the total affinity of binding are distributed along the large DNA site that binds a single TF1 dimer. (2) The ideal DNA-binding site for TF1 will be defined by a reiterative selection-amplification procedure. (3) TF1 binds preferentially to specific DNA sites in hydroxymethyluracil (hmUra)-containing DNA but not in T-containing DNA. (HmUra substitutes entirely for thymine (T) in phage SPO1 DNA. It occurs in animal and human DNA as a potentially mutagenic oxidation product of T and as an oxidation-plus-deamination product of 5-methylcytosine.) Binding of TF1 to hybrid DNA containing hmUra and T in different combinations is to be analyzed. (4) TF1 will be mapped to its DNA-binding site by site- specific photocrosslinking, using reagents developed in our laboratory. (5) Site-directed mutagenesis experiments seeking to alter TF1 so as to define and manipulate its DNA-binding properties are proposed. (6) TF1 forms higher order DNA complexes, compacting the latter. Experiments to analyze the fine structure of these complexes are proposed. (7) Our work involves a collaboration to determine the 3-dimensional structure of TF1 and TF1-DNA complexes by NMR methods. An in vitro analysis of transcription of SPO1 late genes is proposed. The current focus of interest in these experiments is an analysis of another phage system (T4) in which replication proteins act as transcription factors, utilizing enhancer-like mechanisms with novel properties of protein-protein communication by tracking along DNA. The rationale of a search for the involvement of similar principles in SPO1 late gene regulation is presented and a relevant set of experiments is proposed.

该研究计划涉及调节活动的机制基因。它的广泛含义是理解基因调节在开发系统和感染过程中，并精确有关蛋白质核酸相互作用和基因的干预措施活动。分析的特定系统涉及噬菌体 SPO1编码的染色质蛋白TF1，这是无处不在的核核II型DNA结合蛋白（DBPII）。 DBPII 家族在包括结合DNA的蛋白质方面显着非特定于序列特定于序列，在TF1的情况下，序列 - 以及特定于修饰的核苷酸。实验正在处理还提出了噬菌体Spo1后期基因的调节。以下工作线涉及DNA结合和弯曲蛋白质TF1：（1）我们将确定主要贡献结合的总亲和力沿着大型DNA位点分布结合单个TF1二聚体。（2）TF1的理想DNA结合位点将通过重申的选择放大程序来定义。（3） TF1优先与羟甲基拉西中的特定DNA位点结合（HMURA） - 含DNA，但不含在T的DNA中。（Hmura替代品完全用于噬菌体SPO1 DNA中的胸腺嘧啶（T）。它发生在动物中，人DNA作为T的潜在诱变氧化产物，作为 5-甲基胞嘧啶的氧化加脱氨基产物。）TF1的结合以不同组合的含有Hmura和T的混合DNA为分析。（4）TF1将通过位点映射到其DNA结合位点 - 使用实验室中开发的试剂，特定的光叠链接链接。（5）试图改变TF1的地点指导的诱变实验提出了定义并操纵其DNA结合特性。（6）TF1 形成高阶DNA复合物，将后者压实。实验提出了分析这些复合物的精细结构。（7）我们的工作涉及协作以确定TF1的3维结构 NMR方法和TF1-DNA复合物。提出了SPO1后期基因转录的体外分析。这些实验中目前感兴趣的重点是分析另一个噬菌体系统（T4），其中复制蛋白充当转录因子，利用具有新颖的增强剂样机制通过沿着DNA跟踪蛋白质蛋白通信的特性。这寻求参与SPO1中类似原则的理由提出了晚期基因调节，并且一组相关的实验是建议的。