This revised application focuses on 3 projects addressing structural and functional aspects of folding, recognition and catalysis by RNA regulatory domains. Project 1 focuses on the structural characterization of the core domains of metabolite-sensing mRNAs discovered in the Ronald Breaker laboratory, which adopt complex junctional topologies capable of ligand-induced functional modulation. Our structural research will initially focus on the free and bound core domains of guanine/adenine-sensing and thiamine pyrophosphate-sensing mRNAs, for which we have collected promising NMR and crystallographic data. Our structural studies combined with functional efforts in the Breaker laboratory should highlight the principles of molecular recognition and metabolite encapsulation by mRNA, and define the allosteric mRNA transitions associated with the modulation of gene expression levels and metabolic homeostasis. Project 2 on ribozymes catalyzing chemical reactions, focuses on the structural characterization of RNA motifs with Diels-Alderase catalytic activities discovered in the Andres Jaschke laboratory. We are attempting to structurally characterize the catalytic RNA scaffold in the context of bound substrates, transition state analogs and products, with promising crystallographic and NMR data collected on the product complex. Our structural characterization of the Diels-Alderase ribozyme, together with mutational and energetics studies in the Jaschke laboratory, should identify principles for generation of novel ribozymes with controllable catalytic activities, tunable specificites and enantiomeric capabilities. Project 3 focuses on protein-RNA recognition events that mediate the degradation of metazoan histone mRNA, a process tightly coupled to cell cycle progression. Histone mRNAs contain a unique bipartite stem-loop scaffold followed by an ACCA sequence, whose stem, loop and flanking sequences are targeted along opposite faces, both separately and simutaenously, by a stem-loop binding protein and a histone mRNA 3' end-specific exonuclease. Our proposed structural and energetics characterization of binary and ternary protein-RNA recognition events involved in histone mRNA 3'-end recognition and cleavage should provide insights into the mechanisms that cells use to achieve precise cell cycle-regulated mRNA degradation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073618-19
Application #
7337311
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
1988-04-01
Project End
2009-12-31
Budget Start
2008-01-01
Budget End
2009-12-31
Support Year
19
Fiscal Year
2008
Total Cost
$338,485
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Serganov, Alexander; Patel, Dinshaw J (2012) Molecular recognition and function of riboswitches. Curr Opin Struct Biol 22:279-86
Huang, Lili; Ishibe-Murakami, Satoko; Patel, Dinshaw J et al. (2011) Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch. Proc Natl Acad Sci U S A 108:14801-6
Teplova, Marianna; Wohlbold, Lara; Khin, Nyan W et al. (2011) Structure-function studies of nucleocytoplasmic transport of retroviral genomic RNA by mRNA export factor TAP. Nat Struct Mol Biol 18:990-8
Pikovskaya, Olga; Polonskaia, Anna; Patel, Dinshaw J et al. (2011) Structural principles of nucleoside selectivity in a 2'-deoxyguanosine riboswitch. Nat Chem Biol 7:748-55
Huang, Lili; Serganov, Alexander; Patel, Dinshaw J (2010) Structural insights into ligand recognition by a sensing domain of the cooperative glycine riboswitch. Mol Cell 40:774-86
Pikovskaya, Olga; Serganov, Artem A; Polonskaia, Ann et al. (2009) Preparation and crystallization of riboswitch-ligand complexes. Methods Mol Biol 540:115-28
Serganov, Alexander (2009) The long and the short of riboswitches. Curr Opin Struct Biol 19:251-9
Serganov, Alexander; Patel, Dinshaw J (2009) Amino acid recognition and gene regulation by riboswitches. Biochim Biophys Acta 1789:592-611
Serganov, Alexander; Huang, Lili; Patel, Dinshaw J (2009) Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch. Nature 458:233-7
Serganov, Alexander; Patel, Dinshaw J (2008) Towards deciphering the principles underlying an mRNA recognition code. Curr Opin Struct Biol 18:120-9

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