Specific RNA recognition by proteins is a fundamental problem in studying macromolecule interactions. We selected the association of a nucleocapsid protein (N) and its complex with RNA as a model system. The association of N with the genomic RNA is absolutely required for both replication/transcription and particle assembly. The only RNA that is recognized by the specific polymerase is the one that is encapsidated by N. The long term goal of this proposal is to study the detailed interactions of N with the RNA template. We have previously developed a system in which the N protein was expressed in E.coli. This system produces a soluble complex of N oligomer that encapsidates a 90 base RNA. This complex is also crystallized, but the crystals only diffracted to a 7.5Angstrom units resolution. We therefore seek support for our exploratory efforts to improve crystal quality. The following specific aims are proposed to explore various experimental conditions in order to produce high resolution crystals:
Aim 1. High resolution crystals of RNA-N complex. To prepare crystals that can diffract X-rays to higher resolution, several conditions need to be experimented with, such as pH, salt concentrations, detergents and/or additives, and temperature. We will systematically screen these conditions to improve crystal quality. In addition, the removal of the exterior domain of the N-RNA complex by trypsin digestion may give us an alternative complex that may crystallize better.
Aim 2. The incorporation of homogeneous RNA in the complex. The newly synthesized N recognizes only its genome specific RNA. This recognition could be mediated by specific sequences in the leader or trailer region, or the specific secondary structure formed by these regions in the genome. Experiments will be designed to incorporate these RNA sequences into the complex in the E.coli expression system. Sequences coding for the leader and trailer RNAs will be co-transcribed in E.coli with the N mRNAs. Their encapsidation by the N in this system will be assessed. The result of this experiment may provide a more homogeneous RNA-N complex since a unique RNA sequence may be encapsidated instead of random RNA sequences as it is now. The results from these experiments will lead to the crystallization of a unique protein-RNA complex that produces high resolution diffraction data. The ultimate structure of this protein-RNA complex will help us understand how protein recognizes specific RNA sequences or structures.
