REGULATORY RNA IN HELA AND OTHER CELLS

我们的具体目标是：（1）研究RNA引发的 转录在真核细胞和病毒中具有普遍意义。 流感病毒感染HeLa细胞启动子转录的进一步研究 细胞提取物和它们未感染的对应物将进行， 特别强调RNA特定亚群的起源 最近在HeLa亚细胞提取物中发现了聚合酶II产物。 这些可能代表了细胞转录本的第一个例子， 通过添加能够充当 流感病毒转录系统中的引物。 两种控制 实验将与上述研究平行进行：（a） 流感病毒RNA在新型HeLa细胞中启动转录的机制 细胞提取系统最近在这个实验室工作， （B）一系列合作研究， 细胞RNA引发转录的另一个真实案例--正在发生 在锥虫系统-将继续提供另一个例子， 这样的系统。 (2)第二个目标是研究HeLa细胞中的调节RNA 通过关注两种发挥这种作用的RNA--（a）5S RNA， 调节自身的转录我们最近研究出 将RNA结构和功能相关联的测定系统;该测定将 应用于5S rRNA及其转录产物之间的特异性复合物 控制因子IIIA复合物。 (b)RNA加工的RNa亚基 酶，核糖核酸酶P，集中在特定的方式，这种RNA（在 HeLa细胞和其他地方）可以促进整个集合的特异性切割 不同的tRNA前体。 使用的方法包括RNA和DNA模板的体外转录; 通过亲和和杂交方法纯化RNA群体; 二维凝胶电泳RNA结构表征; RNA分子的特定UV诱导衍生物的形成，其中 在生物活性位点引入单一、特异性交联 局部三级结构;以及常规的RNA和DNA指纹图谱， 测序方法 因此，RNA信号在基因调控中的作用将 进一步分析，方法和途径的延伸首先起作用 将扩展到细胞群体，这两种细胞都是正常的 并转化了

Our specific aims are (1) to study the possibility that RNA-primed transcription is of general significance in eukaryotic cells and viruses. Further studies on primed transcription in influenza virus infected HeLA cell extracts and their uninfected counterpartts will be conducted, with particular emphasis on the origin of a particular sub-population of RNA polymerase II products recently discovered in HeLa subcellular extracts. These may represent the first example of cellular transcripts whose synthesis is stimulated by addition of capped RNA's capable of serving as primers in the influenza virus transcription system. Two sorts of control experiments will be carried out in parallel to the above studies: (a) the mechanics of influenza viral RNA primed transcription in the novel HeLa cell extract system recently worked out in this laboratory will continue to be ascertained; and (b) a series of collaborative studies on what may be another authentic case of cellular RNA-primed transcription--that occurring in the trypanosome system--will be continued to provide another example of such a system. (2) A second aim is to study regulatory RNA in HeLa cells by focusing on two RNA's which carry out such a role--(a) 5S RNA, which regulates its own transcription and for which we have recently worked out an assay system correlating RNA structure and function; this assay will be applied to specific complexes between 5S rRNA and its transcriptional control complex with factor IIIA. (b) The RNa subunit of an RNA processing enzyme, RNase P, concentrating on the specific way in which this RNA (in HeLa cells and elsewhere) can promote the specific cleavage of a whole set of differing tRNA precursors. Methods to be used include in vitro transcription of RNA and DNA templates; purification of RNA populations by affinity and hybridization methods; two-dimensional gel electrophoretic RNA structural characterization; formation of specific UV-induced derivatives of RNA molecules in which a single, specific crosslink is introduced at a site of biologically active local tertiary structure; and conventional RNA and DNA fingerprinting and sequencing methods. The role of RNA signals in gene regulation will thus be further analyzed, and the extension of methods and pathways first worked out in viral systems will be extended to cellular populations, both normal and transformed.