Biochemical Studies of Ribonuclease P of Tetrahymena Thermophila

153 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1998.Photochemical crosslinking experiments with a photoaffinity-labeled substrate have been performed with the RNase P RNA subunit and holoenzymes from various sources. Experimental studies clearly reveal that the prokaryotic RNA subunit is involved in substrate binding, but that the eukaryotic RNA subunit alone is unable to bind substrate. Crosslinking experiments with the holoenzyme from various sources suggest that protein components of the eukaryotic holoenzyme contribute to the active site architecture and may mediate substrate binding functions. Thus, one role of the protein subunit in the eukaryotic RNase P may be simply to recognize and bind precursor tRNAs and position them into the holoenzyme active site for correct cleavage. To further understand the differences in substrate-binding capability of RNase P RNA subunits from various sources, chimeric molecules were constructed and analyzed. These molecules are comprised of the human (HeLa) subunit backbone with small structural regions replaced with structures from the catalytic Bacillus subtilis RNA. This data suggests that the inability to generate a specific region of tertiary structure in the RNA may be important in the ability to form a structure competent for binding RNA substrates.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD