For most genes in eukaryotic cells, splicing of the primary transcript is undertaken by a megadalton molecular machine, the spliceosome, consisting of over 100 proteins and five small nuclear RNAs (snRNAs). However, this splicing reaction is also carried out by the Group II intron RNA enzymes (ribozymes) without the need of proteins. Such ribozymes share striking structural and mechanistic similarities to the spliceosome, which makes them an attractive model for studying the basic biochemistry of splicing. It is now well established that domains 1 through 3 (D123), domain 5 (D5) and exon substrates form the minimal active site essential for catalysis by group II introns. The only known structure of the active site component is D5 in isolation. Yet D5 alone cannot mimic the active catalytic configuration. Examining the structure of D5 in the context of D123 and substrates is therefore critical for advancing our knowledge of atomic basis for catalysis. To this end we have reconstructed a group II intron from Pylaiella littoralis into a novel tripartite ribozyme system that has some important difference from previous model systems and that makes it an attractive target for structural studies. We hypothesize that the internal bulge of D5 and substrate form a key part of the catalytic site. We will apply powerful biophysical and biochemical tools to obtain detailed structural insight into the minimal catalytic core of this ribozyme. Our overall specific aims are: 1. Determine the molecular basis for the catalytic deficiency of a D5 bulge deletion mutant-D5A25delta. 2. Map the binding interface between D5 bulge mutant and D123 in the presence and absence of substrates using NMR, mutagenesis, and UV crosslinking 3. To obtain an atomic view of the minimal active site and to identify the features that are important for binding and catalysis, determine the structure of D5 and D5 bulge mutant bound to D123 (and or fragments of D123) and substrate using NMR, and 4. Validate the structure using biochemical assays. Significance: We anticipate that completion of these studies will provide an atomic framework for interpreting the extensive biochemical and genetic data on group II introns to extend our understanding of catalysis, these might provide the first evidence for the details of RNA interactions in cell splicing, and suggest novel approaches to developing group II introns as potential ribozyme therapeutics. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM077326-03
Application #
7741619
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2007-08-01
Project End
2012-05-31
Budget Start
2008-09-01
Budget End
2009-05-31
Support Year
3
Fiscal Year
2008
Total Cost
$247,500
Indirect Cost
Name
University of Maryland College Park
Department
Chemistry
Type
Schools of Earth Sciences/Natur
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
Luo, Yiling; Sintim, Herman O; Dayie, T Kwaku (2014) Synthesis of a biotinylated photocleavable nucleotide monophosphate for the preparation of natively folded RNAs. Methods Enzymol 549:115-31
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Luo, Yiling; Zhou, Jie; Watt, Sarah K et al. (2012) Differential binding of 2'-biotinylated analogs of c-di-GMP with c-di-GMP riboswitches and binding proteins. Mol Biosyst 8:772-8
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Arthur, Patrick K; Alvarado, Luigi J; Dayie, T Kwaku (2011) Expression, purification and analysis of the activity of enzymes from the pentose phosphate pathway. Protein Expr Purif 76:229-37
Thakur, Chandar S; Dayie, T Kwaku (2011) Asymmetry of (13)C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy. J Biomol NMR 51:505-17
Luo, Yiling; Eldho, Nadukkudy V; Sintim, Herman O et al. (2011) RNAs synthesized using photocleavable biotinylated nucleotides have dramatically improved catalytic efficiency. Nucleic Acids Res 39:8559-71
Thakur, Chandar S; Sama, Jacob N; Jackson, Melantha E et al. (2010) Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle. J Biomol NMR 48:179-92

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