The goal of this project is to define the mechanisms by which formation of secondary structures (RNA hairpins) in nascent transcripts causes pausing and termination during transcription. RNA secondary structures are important regulatory signals in prokaryotes where transcription pausing and termination are major components of genetic regulatory mechanisms; pausing and premature termination also occur in eukaryotes where their regulatory significance is only now emerging. The proposed studies will provide a detailed understanding of how RNA hairpins form and how they interact in the transcription complex. Understanding this model systems will be an important contribution to knowledge about transcription regulation, the basic process that governs all gene expression. These studies also will refine techniques and concepts that will be employed in future work on eukaryotic transcription complexes. A combination of biochemical, genetic, and recombinant DNA techniques will be used to study RNA hairpin-transcription complex interactions. Systematic variation of model RNA hairpins will define the sequence and structural requirements for induction of transcription pausing and termination by RNA hairpin formation. A major goal will be to determine if the formation of RNA hairpins causes pausing and termination only by altering nucleic acid conformations in the transcription complex or if specific interactions between proteins in the transcription complex and portions of the RNA hairpin are involved. RNA hairpin-specifying regions will be constructed from synthetic oligonucleotides and inserted into special plasmid vectors in front of the T7 A1 promoter to facilitate study of their pausing and termination phenotypes. Transcription complexes containing defined nascent transcript RNA hairpins will be isolated from in vitro transcription reactions and subjected to extensive structural analyses using enzymatic and chemical probes of RNA and DNA structure. The beta subunit of RNA polymerase will be analyzed by site-directed mutagenesis of its gene to determine which amino acid residues may participate in RNA hairpin interactions.
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