Poster abstracts

Poster number 24 submitted by Elizabeth Dethoff

NMR Studies of the Structural Dynamics of Wild-Type TAR RNA

Elizabeth Dethoff (Chemistry; Universtiy of Michigan), Catherine Musselman (Chemistry; Universtiy of Michigan), Hashim Al-Hashimi (Chemistry and Biophysics; Universtiy of Michigan)

Abstract:
The transactivation response element (TAR) RNA has multiple roles in HIV replication, including transcriptional elongation. Once bound to TAR, the viral protein Tat recruits the transcription elongation factor, P-TEFb, which eventually leads to phosphorylation of the RNA polymerase and thus transcriptional elongation.(1) Both an internal bulge and apical loop in TAR are believed to be essential for proper binding of Tat and P-TEFb. To date, NMR studies that have investigated the structural dynamics of TAR have used a TAR construct in which the six-residue hairpin loop was replaced with a UUCG tetraloop. Here, we report initial steps towards complete NMR characterization of the structural dynamics of wt-TAR and its Tat binding properties. Our results indicate that mutation of the apical loop has minimal effects on the stem-bulge-stem element of TAR. This is in contrast to a previous biochemical study indicating formation of a U31•A22-U40 base-pair.(2) The wild-type loop undergoes complex internal motions spanning nanosecond to millisecond timescales. The NMR data will be compared with a 65 ns molecular dynamics simulation of wt-TAR in explicit solvent.

References:
1 Price, D. H. (2000). P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol 20, 2629-34.

2 Huthoff, H., Girard, F., Wijmenga, S. S. & Berkhout, B. (2004). Evidence for a base triple in the free

Keywords: TAR, NMR, dynamics