PHOTOCHEMICAL & SPECTROSCOPIC PROBES OF RNA POLYMERASE

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The proposed work is to map the path of the leading end of nascent ribonucleic acid (RNA) across the surface of the bacterial enzyme RNA polymerase and on to the ribosome. In situ preparation of RNA tagged with azide photoaffinity labels, and subsequent photolysis and analysis of labeled products, will show which subunits of the enzyme are contacted by the growing nucleotide chain and also the RNA chain length at which RNA separates from DNA template. Spectroscopic probes attached to the RNA and other sites will provide complementary information about distances between sites on the macromolecular complex, and restrictions on the freedom of movement of nascent RNA. Bifunctional chelating agents, analogs of ethylenediaminetetraacetic acid which can be covalently attached to biological molecules, will be used as multipurpose spectroscopic probes. In particular, luminescent lanthanide ions in bifunctional chelating agents attached to nascent RNA will be used as donors for energy-transfer studies. Also, diffusion-enhanced energy transfer from chelated lanthanides in solution will reveal the accessibility of the leading end of nascent RNA, and of chromophoric probe molecules, in macromolecular complexes. These experiments will provide new knowledge of the molecular mechanisms of DNA transcription and transcription-translation coupling; better understanding of these basic life processes may ultimately benefit human health.