This project concerns the investigation of the mechanism of transcription termination in Escherichia coli and the yeast S. cerevisiae in the highly purified in vitro transcription system. This system involves the E. coli RNA polymerase [hexahistidine-tagged at the carboy-terminus of beta' subunit (rpoC)] and the yeast RNA polymerase II (Pol II, hexahistidine-tagged at N-terminus of RPB3 subunit) core enzyme immobilized in a solid phase to which synthetic DNA and RNA components were added to reconstruct the authentic elongation complex. The elongation complex represents a form of RNA polymerase traveling along the gene and synthesizing the RNA transcript. Normally, the elongation complex is highly stable and grips the DNA and the RNA very tightly. However, when RNA polymerase reaches the sequence called transcription terminator, the """"""""grip"""""""" is relieved and the complex falls apart momentarily. The terminator consists of two elements: the stable hairpin-like structure formed in the RNA behind RNA polymerase, and the oligo-uridine track located next to the hairpin in the RNA. The mechanism, that causes RNA polymerase dissociation at terminator is unknown. It's believed, that the strength with which the end of the RNA is hybridized to the DNA in the moving polymerase is a major factor of the elongation complex stability. When the hybrid is weak or short the complex spontaneously dissociates. One of the ideas how the hairpin might work is that it reduces the RNA:DNA hybrid in the polymerase beyond its normal length. We addressed the role of the RNA hairpin in destabilization of elongation complex by performing the statistical measurements of the dissociation kinetics of elongation complexes at different variants of transcription terminator. The project involved four consecutive steps. First, the elongation complex was reconstituted followed by the analyses of its stability using the salt-sensitivity assay. The conclusions we have reached can be summaried as follows: (i) We show that folding of the hairpin disrupts the three upstream base pairs of the 8-bp RNA:DNA hybrid, a major stability determinant in the elongation complex. During termination, the hairpin does not directly compete for base pairing with the 8-bp hybrid. Thus, melting of the hybrid results from spatial restrictions in RNA polymerase that couple the hairpin formation with the disruption of the hybrid immediately downstream from the stem. Shortening the weak rU:dA hybrid from 8 nt to 5 nt causes dissociation of the complex. (ii) We demonstrate that a similar mechanism, involving melting of 8-bp RNA:DNA hybrid by the hairpin, disrupts elongation complexes of yeast RNA polymerase II in vitro. Thus, melting of the RNA:DNA hybrid arises as a general mechanism inducing RNA polymerase release in prokaryotes and eukaryotes.
