Substrate properties of C′-methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me-UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me-UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force-field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N-type conformation.     

High-throughput screening of RNA polymerase inhibitors using a fluorescent UTP analog

RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (gamma-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of gamma-AmNS-PPi, which has higher intrinsic fluorescence than (gamma-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 microM. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC(50) below 100 microM. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has >99% sequence identity with Mycobacterium tuberculosis RNAP subunits.     

真核生物RNA聚合酶组装及其生物学意义

RNA聚合酶负责RNA生物合成,是维持机体细胞生长和器官发育的重要调控机器.真核生物主要通过3种多亚基RNA聚合酶(RNAPⅠ、RNAPⅡ和RNAPⅢ)进行基因转录.RNA聚合酶Ⅱ由10个核心亚基组成,分子质量约为520 ku.RNA聚合酶结构已经解析,但其组装过程却还不清楚.RNA聚合酶各亚基无法完成体外自我组装,说明细胞内其装配过程需要组装因子帮助.RNA聚合酶组装是一个复杂的生物学过程,近年来组装因子的鉴定和发现使RNA聚合酶组装研究成为热点.在酿酒酵母中发现的组装因子有Rba50、Bud27和GPN蛋白家族等.其中GPN蛋白家族是重要的GTP酶(GTPase)家族,从古细菌到酵母以及高等真核生物中都存在,且高度保守.近期研究发现,Rba50在动植物的同源蛋白(RPAP1和IYO)与细胞分化和发育有关.GPN等组装因子编码基因的突变与细胞发育及恶性肿瘤发生发展密切关联.本文对真核生物RNA聚合酶组装最新进展做出综述,以期为RNA聚合酶组装机制的最终阐明及其与疾病发生的关联研究提供基础.