This proposal consists of three projects which all involve studying the structure of isolated domains of larger RNA molecules and understanding how that structure is related to biological function. Recently developed methods for the in vitro transcription of synthetic DNA with T7 RNA polymerase will be refined and used to prepare RNA fragments of defined sequence and approximately 20 nucleotides in length. The structure of these fragments and carefully designed sequence variants will be studied using well established chemical modification methods and by nuclear magnetic resonance techniques. Attempts will be made to prepare diffraction quality crystals. A study of the interaction of bacteriophage R17 coat protein with the replicase translational operator region of R17 RNA will be continued with the focus on the protein. A combined biochemical and molecular biological approach will be used to identify amino acids involved in RNA binding and protein-protein association. The role of this interaction in translational repression and phage assembly will be investigated using both in vitro and in vivo experiments. Recent experiments demonstrating a new, very small RNA enzyme will be extended. By constructing variants of the active sequences, both the structure and the mechanism of the reaction will be studied.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Molecular and Cellular Biophysics Study Section (BBCA)
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University of Colorado at Boulder
Schools of Arts and Sciences
United States
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Nelson, Jennifer A; Shepotinovskaya, Irina; Uhlenbeck, Olke C (2005) Hammerheads derived from sTRSV show enhanced cleavage and ligation rate constants. Biochemistry 44:14577-85
Blount, Kenneth F; Grover, Neena L; Mokler, Victor et al. (2002) Steric interference modification of the hammerhead ribozyme. Chem Biol 9:1009-16
Dertinger, D; Uhlenbeck, O C (2001) Evaluation of methylphosphonates as analogs for detecting phosphate contacts in RNA-protein complexes. RNA 7:622-31
O'Rear, J L; Wang, S; Feig, A L et al. (2001) Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations. RNA 7:537-45
Dertinger, D; Dale, T; Uhlenbeck, O C (2001) Modifying the specificity of an RNA backbone contact. J Mol Biol 314:649-54
Dertinger, D; Behlen, L S; Uhlenbeck, O C (2000) Using phosphorothioate-substituted RNA to investigate the thermodynamic role of phosphates in a sequence specific RNA-protein complex. Biochemistry 39:55-63
Feig, A L; Panek, M; Horrocks Jr, W D et al. (1999) Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy. Chem Biol 6:801-10
Scott, E C; Uhlenbeck, O C (1999) A re-investigation of the thio effect at the hammerhead cleavage site. Nucleic Acids Res 27:479-84
Johansson, H E; Dertinger, D; LeCuyer, K A et al. (1998) A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein. Proc Natl Acad Sci U S A 95:9244-9
Tsu, C A; Uhlenbeck, O C (1998) Kinetic analysis of the RNA-dependent adenosinetriphosphatase activity of DbpA, an Escherichia coli DEAD protein specific for 23S ribosomal RNA. Biochemistry 37:16989-96

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