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The diverse functional roles of RNA are determined by its underlying structure. Accurate and comprehensive knowledge of RNA structure would inform a broader understanding of RNA biology and facilitate exploiting RNA as a biotechnological tool and therapeutic target. Determining the pattern of base pairing, or secondary structure, of RNA is a first step in these endeavors. Advances in experimental, computational, and comparative analysis approaches for analyzing secondary structure have yielded accurate structures for many small RNAs, but only a few large (>500 nts) RNAs. In addition, most current methods for determining a secondary structure require considerable effort, analytical expertise, and technical ingenuity. In this review, we outline an efficient strategy for developing accurate secondary structure models for RNAs of arbitrary length. This approach melds structural information obtained using SHAPE chemistry with structure prediction using nearest-neighbor rules and the dynamic programming algorithm implemented in the RNAstructure program. Prediction accuracies reach ≥95% for RNAs on the kilobase scale. This approach facilitates both development of new models and refinement of existing RNA structure models, which we illustrate using the Gag-Pol frameshift element in an HIV-1 M-group genome. Most promisingly, integrated experimental and computational refinement brings closer the ultimate goal of efficiently and accurately establishing the secondary structure for any RNA sequence.

G-proteins cycle between an inactive GDP-bound state and active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage-display to identify a series of peptides that bind Gα subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069-1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP·AlF 4 − -and GTPγS-bound states of Gα i subunits. KB-1753 blocks interaction of Gα transducin with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated Gα in vitro. The crystal structure of KB-1753 bound to Gα i1 ·GDP·AlF 4 − reveals binding to a conserved hydrophobic groove between switch II and α3 helices, and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for Gα i subunits.Heterotrimeric G-proteins serve as critical relays that transmit cues from extracellular stimuli as diverse as neurotransmitters, hormones, photons, and odorants/tastants to intracellular signaling cascades responsible for eliciting specific cellular effects (1,2). In the traditional model of G-protein signaling, cell surface G-protein coupled receptors (GPCRs), upon activation by the aforementioned stimuli, catalyze the exchange of GDP for GTP on the Gα *To whom correspondence should be addressed: UNC Pharmacology, 1106 M.E. Jones Bldg., Chapel Hill, NC 27599-7365. Telephone: 919-843-9363. Fax: 919-966-5640. E-mail: dsiderov@med.unc.edu. Current addresses: Entegrion, 5312 Farrington Rd., Chapel Hill, NC 27517 (J.K.R); Becton Dickinson, 21 Davis Dr., RTP, NC 27709 (R.B.); Amgen Inc., 1201 Amgen Court W., Seattle, WA 98119 (Z.F.). † This work was supported by NIH R01 GM074268 (D.P.S.) and EY12859 (V.Y.A.). C.A.J. was supported by an NIH postdoctoral fellowship (1 F32 GM076944). V.Y.A. is the recipient of the Senior Scientific Investigator Award from Research to Prevent Blindness Inc. Coordinates of the KB-1753/Gα i1 ·GDP·AlF 4 − complex were deposited in the Protein Data Bank (accession code 2G83). 1 Abbreviations: AlF 4 − , aluminum tetrafluoride; CFP, cyan fluorescent protein; cGMP, cyclic guanosine monophosphate; FRET, fluorescence resonance energy transfer; GAP, GTPase-accelerating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GMP, guanosine monophosphate; GPCR, G protein-coupled receptor; GTP, guanosine triphosphate; PDE, phosphodiesterase; RGS, regulator of G-protein signaling; ROS, rod outer segment; SPR, surface plasmon resonance; YFP, yellow fluorescent protein. subunit. This results in adoption of the active, GTP-bou...

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