It was a surprise of the last decade the large number of small, noncoding RNAs, which have been identified to play regulatory roles in the modulation of gene expression in all kingdoms of life. The bacterial 6S RNA is a transcription regulator of the σ70-RNA polymerase holoenzyme during the stationary phase. It has been shown that 6S binds with high affinity and specificity onto the σ70-RNAP and presumably blocks the sites of interaction of RNAP with DNA promoters. It has also been shown that upon outgrowth from the stationary phase 6S serves as template for the RNAP and the product RNAs, which are synthesized, destabilize the complex and liberate the polymerase from 6S RNA. The objective of this proposal is the structure determination of the whole complex between 6S and σ70-RNAP, of truncated forms and sub-assemblies by X-ray crystallography. The project will involve the preparation of RNA-protein complexes in amounts, purity and homogeneity suitable for studies with X-ray crystallography. The obtained structural information will help to understand the nature of 6S regulation of transcription in details. It will also help to elucidate the features which determine the σ70-RNAP specificity for 6S RNA interactions and may allow us to understand the molecular mechanism of RNAP liberation upon product RNAs synthesis.
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