This project concerns the investigation of the mechanism of transcription termination in Escherichia coli in the highly purified in vitro transcription system. This system involves the E. coli RNA polymerase 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 ogripo 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. Its 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 are currently addressing 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) The disruption of RNA:DNA hybrid from its 5-end can explain destabilizing effect of RNA hairpin in transcription termination. (ii) The stability of elongation complex depends on sequence of RNA:DNA hybrid at the 3-end of the transcript, e. g. the weaker hybrid makes the elongation complex less stable.
