? Dynamics in the ribosome that accompany translation are not well characterized. One example of a dynamic process in the 30S subunit is the switching of helix 27 from one base-pairing pattern to another, a proposed trigger of peptide synthesis. This so-called """"""""switch helix"""""""" was implicated by Lodmell and Dahlberg (1997) in decoding of mRNA and selection of cognate transfer RNA at the A-site. However, all crystal structures of the 30S subunit solved to date depict one conformation of helix 27, creating a controversey over whther helix 27 actually changes conformation in vivo. The experiments descibed in this proposal establish a kinetic and thermodynamic framework for helix 27 dynamics. By measuring 13C and 15N spin-lattice, spin-spin, and rotating-frame spin-lattice relaxation rates for nuclei in different switch helix constructs, local dynamics of this sequence on a ps-ms timescale will be determined. Residual dipolar coupling studies will reveal complementary global motions of the switch helix conformers. I will also use various fluorescence resonance energy transfer (FRET) techniques, both ensenble and single molecule, to observe motions of this helix on a sub-second to ms timescale that facilitate conformational switching. These methods will give us information about the motions of helix 27 on a variety of timescales, and help us to determine whether the inherent dynamics of this helix imply a functional role in translation. ? ?
