NUCLEIC ACID STRUCTURE AND MECHANISMS OF GENE EXPRESSION

We are investigating the formation of RNA structure, RNA folding pathways during chain elongation, and the mechanisms(s) of polymerase pausing during RNA synthesis. Our model system employs the 220 nucleotide long, highly structured, and completely sequenced QBeta replicase template, MDV-1 RNA or its DNA copy, MDV-1 DNA. We have devised simple procedures to prepare large amounts of MDV-1 RNA and DNA templates with a variety of site-specific sequence changes. These templates are being employed to investigate the effects of sequence changes on the pattern of RNA folding and polymerase pausing during RNA directed RNA synthesis by QBeta replicase and DNA directed RNA synthesis by E coli and T1 RNA polymerases. We have shown that the folding of the RNA during chain elongation is not solely responsible for the patterns of polymerase-pausing observed and that the template employed or the entire polymerase/template/product complex may be involved. We are continuing our investigations into the confirmation of MDV-1 RNA and plan to study the progressive denaturation of MDV-1 (+) and (-) RNA conformation in the presence and absence of QBeta replicase. Analogous to certain types of DNA transposition and early reverse-transcription of retroviral RNA, QBeta replicase can undergo intra-strand recombination during chain elongation. This seems to occur in regions of inverted repeat sequence which are flanked by direct repeats. We have designed a model system to investigate the sequence and structural requirements involved in the mechanism. Finally, we have been investigating the elements of very high G-C sequence which flank the "TATA homology" of the major late promotor of Adenovirus-5. We have site-directed mutations into this region, characterized them by DNA sequence analysis, and are rebuilding by ligation whole virus DNAs which contain the mutated promotor elements. We plan to investigate not only the levels of transcription from these mutant major late promotor viral DNAs but also the effect of these mutations upon the entire program of viral gene regulation.