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.
