There are a number of genes whose expression is thought to be controlled, at least in part, during transcriptional elongation. The most medically relevant include the human c-myc, c-myb, and c-fos oncogenes and the human immunodeficiency virus. The long-term goal of this research project is to understand the mechanism by which RNA polymerase II elongates RNA chains, and how this process is influenced and/or regulated by transcription factors. RNA polymerase becomes blocked in elongation during traversal of certain DNA sequences, termed intrinsic arrest sites. These are probably multipartite structures utilizing both template and transcript sequences to establish the transcriptional blockade. Thus, these sites provide an excellent model system to examine the concerted effects of both transcript and template sequence on the configuration of the ternary complex. Using a promoter-initiated single-round transcription assay, the nucleic acid determinants that signal and/or stabilize the arrested configuration are being investigated. These studies include different arrest sites which probably use slightly different mechanisms to trigger arrest. Using the single-round transcription assay it is possible to purify essentially homogenous populations of complexes arrested at bona fide arrest sites, or stalled at a defined location or within sequences that partially resemble arrest sites. These studies have demonstrated that one of the signals that triggers arrest is a U-rich transcript 3' end. Different models that could account for recognition of this structure by polymerase will be tested using poly-U competition and heteroduplex stabilization analyses. In the presence of elongation factor SII, ternary complexes become a ribonuclease, truncating their nascent transcript in a 3'-->5' direction. Stalled complexes cleave primarily in a dinucleotide increment, whereas arrested complexes display a large (7-17 nt) cleavage increment. In both cases, nucleotide sequence probably determines cleavage rates and site specificity. DNA mutagenesis and heteroduplex stabilization approaches are being used to ask whether the DNA, the RNA, or both contribute to the properties of the cleavage reaction. To define further the structural changes that accompany arrest, and to test certain models of arrest, the constraints imposed on the template and transcript by homogeneous populations of stalled and arrested complexes will be examined using single and double strand-specific nuclease protection assays and complexes generated on templates encoding a ribozyme. At present, efficient transcription over long stretches of template requires exposing ternary complexes transiently to the nonionic detergent Sarkosyl. However, these complexes are devoid of the known elongation factors. Preliminary studies suggest that using competitor DNA instead of Sarkosyl generates complexes that have stably loaded SII. Studies to further characterize DNA-competed complexes will employ both in vitro transcription and Western blot analyses. To understand transcriptional elongation will require a complete inventory of factors that influence elongation. A crude nuclear fraction termed TFIIX contains a unique cleavage activity. This activity will be further characterized and the protein(s) responsible purified.
