Structural investigation of nuclear RNP particles containing pre-mRNA by different fluorescence techniques

Ethidium bromide (EB) adsorption isotherms on 30S nuclear RNP particles isolated from liver nuclei has revealed 6% of double-stranded regions in pre-mRNA (dsRNA). It has been established by measurements of the EB fluorescence polarization that the bulk of dsRNA regions in RNP is rigidly attached to RNP. They are longer than 45 degree A. The increase of NaCl concentration from 0.1 up to 0.4 M causes a significant loosening of dsRNA-protein bonds. As a result the dsRNA segments become more flexible. Measurements of energy transfer from fluorescamine (covalently bound to the protein) to EB (adsorbed on dsRNA) have yielded information about dsRNA location. The fact that absorbtion of exciting light by fluorescamine causes pronounced increase of EB fluorescence is consistent with the idea that helical regions of RNA are located outside the RNP particles.