RNA processing is intimately related to its structure, as RNA interactions organize the catalytic center of processing complexes and create or mask sites for regulatory proteins. The goal of the proposed research is to understand how the long-range folding of RNA influences gene expression using the Tetrahymena self-splicing group I intron as a model system. This system is advantageous for studying RNA structure because the catalytic activity of the intron directly reflects its conformation. Alternative secondary structures attenuate self-splicing by trapping the pre-ribosomal RNA in an inactive conformation during transcription in vitro. In vivo, kinetic barriers to splicing are overcome by factors not yet understood. RNA interactions that lead to the formation of inactive or active pre-rRNA will be identified by chemical modification and phosphorothioate interference. The effect of mutations, temperature, magnesium concentration, and the transcription process itself on the competition between inactive and active RNA structures will be determined. Direct strand-exchange between alternative helices in inactive and active pre-rRNA will be tested by disrupting stacking of adjacent helices and by trapping unpaired sequences with complementary DNA oligonucleotides. These experiments will establish a general framework for RNA folding kinetics that is expected to be relevant to other systems such as the spliceosome. Factors that facilitate RNA folding in vivo will be investigated using bacterial genetics. The Tetrahymena intron is spliced as rapidly from the homologous position of 23S rRNA in E. coli as it is in Tetrahymena. Ribosomal RNA sequences will be randomly mutated and selected for inhibition of splicing. Proteins that facilitate folding of splicing-competent pre-rRNA will be identified by complementation of splicing-defective pre-rRNA. This work will be directly relevant to pre-rRNA processing, splice site selection, and intron evolution.
| Zheng, Amy; Panja, Subrata; Woodson, Sarah A (2016) Arginine Patch Predicts the RNA Annealing Activity of Hfq from Gram-Negative and Gram-Positive Bacteria. J Mol Biol 428:2259-2264 |
| Santiago-Frangos, Andrew; Kavita, Kumari; Schu, Daniel J et al. (2016) C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA. Proc Natl Acad Sci U S A 113:E6089-E6096 |
| Panja, Subrata; Paul, Rakesh; Greenberg, Marc M et al. (2015) Light-Triggered RNA Annealing by an RNA Chaperone. Angew Chem Int Ed Engl 54:7281-4 |
| Panja, Subrata; Woodson, Sarah A (2015) Fluorescence reporters for Hfq oligomerization and RNA annealing. Methods Mol Biol 1259:369-83 |
| Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J et al. (2015) Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing. J Mol Biol 427:3491-3500 |
| Peng, Yi; Curtis, Joseph E; Fang, Xianyang et al. (2014) Structural model of an mRNA in complex with the bacterial chaperone Hfq. Proc Natl Acad Sci U S A 111:17134-9 |
| Peng, Yi; Soper, Toby J; Woodson, Sarah A (2014) Positional effects of AAN motifs in rpoS regulation by sRNAs and Hfq. J Mol Biol 426:275-85 |
| Panja, Subrata; Schu, Daniel J; Woodson, Sarah A (2013) Conserved arginines on the rim of Hfq catalyze base pair formation and exchange. Nucleic Acids Res 41:7536-46 |
| Panja, Subrata; Woodson, Sarah A (2012) Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing. J Mol Biol 417:406-12 |
| Peng, Yi; Soper, Toby J; Woodson, Sarah A (2012) RNase footprinting of protein binding sites on an mRNA target of small RNAs. Methods Mol Biol 905:213-24 |
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