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Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

New Insights into the Mechanism of Cleavage of Sarcin Ricin Loop (SRL) RNA

The hydroxyl radical (⋅OH) is a highly reactive oxygen species that is known to damage nucleic acids. Hydroxyl radical cleavage has been widely used as an important tool in understanding the structure of DNA (1) and RNA (2). The mechanism of strand cleavage has been proposed to occur via the initial abstraction of a (deoxy)ribose hydrogen atom by the hydroxyl radical. Deuterium kinetic isotope effect experiments from our lab have shown that the solvent accessibility of a particular hydrogen atom determines the extent of DNA strand cleavage by the hydroxyl radical (3). Of late, the hydroxyl radical has also been used as a high-resolution probe of RNA folding and structure. However, the mechanism of RNA cleavage by ⋅OH remains to be elucidated. Recent work in the Tullius lab has been focused on deuterium kinetic isotope effect experiments on hydroxyl radical cleavage of the 27-nucleotide Sarcin Ricin Loop (SRL) domain of ribosomal RNA from E. coli, a structurally well-defined model RNA system. Our approach is to replace hydrogen atoms on the ribose sugar with deuterium atoms. In vitro transcription is used to incorporate 4'- and 5'-deuterated nucleotides into the SRL RNA molecule. Kinetic isotope experiments have been performed to elucidate which hydrogen atoms are involved in the mechanism of RNA cleavage by the hydroxyl radical. Our results provide valuable insights into the use of a high-resolution chemical probe for understanding RNA structure and folding.

References and Footnotes
  1. Tullius, T. D., and Dombroski, B. A., Science 230, 679-681 (1981)
  2. Latham, J., Cech, T., Science 245, 276-282 (1989).
  3. Balasubramanian, B., Pogozelski, W. K., and Tullius, T. D., Proc. Natl. Acad. Sci. USA 95, 9738-9743 (1998).
  4. Correll, C.C., Beneken, J., Plantinga, M.J., Lubbers, M., and Chan, Y.L., Nuc. Acids. Res. 31, 6806-6818 (2003).

Swapan S. Jain*
Orlando Suero
and
Thomas D. Tullius

Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, MA 02215 USA

Phone: (001) 617-353-8810 Fax:617-353-4814 Email: swapan@bu.edu