Mature 5S RNA has five stems connected and terminated by loops; the primary transcript has a 3' single stranded tail which is removed by processing. The internal domain consisting of loop D, stem IV and loop E is dispensable for processing. We have made numerous substitutions in Drosophila 5S RNA to map the influence of sequence and structure on processing, and interpreted the distribution of processing elements dispersed through stems I, II, III and loops B and C in two ways. First, sequence changes which stimulate processing cooperate when combined; we suggest that a processing protein makes multiple contacts with 5S RNA (long range cooperation). Second, poorly processed sequence variants cluster in the center of stems I and II, flanked by positions where changes which weaken base pairing improve processing. We suggest that a polypeptide arm reaches in from loop A yields stem I and from loop A yields stem II to make required central contracts (the breathing model). We propose to investigate long range cooperation and helix breathing at stem/loop junctions by separating the proteins involved in 5S RNA processing. Once the processing proteins are isolated, we will assess their sites of interaction with wild type and variant 5S RNA by footprinting. Protein contacts in stems I, II, III and loops B and C would be consistent with long range cooperation; such contacts should be weakened or absent from sequence variants with reduced processing. We will probe wild type and variant 5S RNA structure to evaluate athe breathing model. Substitutions which improve processing should increase probe access to central stem contracts in the event of breathing at stem/loop junctions. Finally, a randomization/processing size selection experiment will enable us to analyze many combinations of sequence variants for processing which would be impractical by site-specific mutagenesis. These investigations will contribute to our understanding of nucleic acid- protein interactions by using a small stable RNA as a processing substrate.

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Yuan, Yiping; Shen, Xiaotong; Pan, Wei (2012) Maximum Likelihood Estimation Over Directed Acyclic Gaussian Graphs. Stat Anal Data Min 5:
Knapp, John M; Zhu, Jie S; Tantillo, Dean J et al. (2012) Multicomponent assembly of highly substituted indoles by dual palladium-catalyzed coupling reactions. Angew Chem Int Ed Engl 51:10588-91
Fearnley, Stephen Philip; Thongsornkleeb, Charnsak (2010) Oxazolone cycloadducts as heterocyclic scaffolds for alkaloid construction: synthesis of (+/-)-2-epi-pumiliotoxin C. J Org Chem 75:933-6
Desamero, Ruel Z B; Kang, Jeonghee; Dol, Chrystel et al. (2009) Spectroscopic characterization of the SH2- and active site-directed peptide sequences of a bivalent Src kinase inhibitor. Appl Spectrosc 63:767-74
Levinger, Louis; Hopkinson, Angela; Desetty, Rohini et al. (2009) Effect of changes in the flexible arm on tRNase Z processing kinetics. J Biol Chem 284:15685-91
Zhang, Chun-Yang; Johnson, Lawrence W (2009) Single quantum-dot-based aptameric nanosensor for cocaine. Anal Chem 81:3051-5
Juszczak, Laura J; Desamero, Ruel Z B (2009) Extension of the tryptophan chi2,1 dihedral angle-W3 band frequency relationship to a full rotation: correlations and caveats. Biochemistry 48:2777-87
Macneil, Margaret A; Purrier, Sheryl; Rushmore, R Jarrett (2009) The composition of the inner nuclear layer of the cat retina. Vis Neurosci 26:365-74
Arsov, I; Li, X; Matthews, G et al. (2008) BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development. Cell Death Differ 15:1385-95
Hopkinson, Angela; Levinger, Louis (2008) Effects of conserved D/T loop substitutions in the pre-tRNA substrate on tRNase Z catalysis. RNA Biol 5:104-11

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