ncRNAP · Structural and functional studies of the interaction between non coding RNAs and the RNA polymerase

Structural and functional studies of the interaction between non coding RNAs and the RNA polymerase.

grant No. PERG08-GA-2010-276861, ncRNAP The last decade a large number of non-coding RNAs (ncRNAs) has been identified and their function as modulators of gene expression at various cellular levels in all kingdoms of life has been revealed. 6S is an abundant, widespread bacterial ncRNA with a highly conserved secondary structure. 6S RNA accumulates to high levels during the stationary phase and is specifically bound to the housekeeping form of the sigma70-RNAP. The project’s objectives was the biophysical characterization of the isolated 6S RNA and the determination of structure in complex with RNAP. This information gives insights not only on the transcriptional regulation by small RNAs, which is a recently discovered mode of regulation but also to the recognition and interactions between structured RNAs and proteins. Moreover, because small non-coding RNAs have been recently discovered to make complexes with eukaryotic RNA polymerases, the structural information obtained from this project will also apply to the transcriptional regulation of eukaryotes. Lastly, given that bacterial RNAP is a proven target for broad-spectrum antibacterial therapy, the structural information may also be eventually useful for chemical intervention at the level of the transcriptional regulation to discover lead compounds for drugs design. Initially we focused on the attempt to produce separately each of the sigma70-RNAP and the 6S RNA. For the production of the endogenous sigma70-RNAP we followed established protocols. The 6S RNA was successfully isolated from E.coli cells and purified by a combination of electroelution and affinity chromatography producing high purity sample in mg quantities. The 6S sample resisted all the crystallization trials, however it was subjected to detailed biophysical characterization in solution produced a theoretical 3D model. Our attempts to synthesize the complex by the separately isolated components in mg quantities remained unsuccessful. We next focused on the isolation of the in vivo formed complex using recombinant material. We were able to establish protocols for the efficient isolation and purification of the intact nucleoprotein complex however the crystallization trials were unsuccessful. The sample was analyzed with cryo electron microscopy, non-dissociating mass spectroscopy and small angle X-ray scattering. A low resolution model of the complex is being developed. In conclusion, during this project we have achieved the structural characterization of the non coding 6S RNA and we have proposed a model for its 3D-structure. We have established a new method for electroelution of RNA molecules and we are currently exploring an overexpression method for the production of mg quantities of non coding RNAs which are necessary for structural studies. We have also established protocols for the large scale isolation of high quality sigma70-RNAP/6S RNA complex. During the project the fellow has succeeded to clarify many of the questions of the project, has established solid collaborations with national and international research groups and has accomplished the required qualifications to start her own independent group. Thus, creating the basis for obtaining a permanent academic position.

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