The broad objective of this project is to understand how RNA and protein molecules assemble to form native ribonucleoprotein complexes and collaborate to perform RNA-based catalysis. In the first system, we will explore assembly of the bl5 ribonucleoprotein, consisting of the bl5 group I intron RNA and its single obligatory protein cofactor, CBP2. In particular, we will use this system to understand the structure of the RNA collapsed state, identified in the P.l.'s lab, that forms prior to subsequent assembly with the CBP2 protein cofactor. It is becoming clear that related collapsed states play fundamental, productive, roles in many RNA folding and ribonucleoprotein assembly reactions. The second system is the bl3 group I intron ribonucleoprotein. Splicing by the bl3 intron requires two facilitating proteins. The intron-encoded bl3 maturase binds a monomer; whereas, two Mrs1 dimers bind, cooperatively, to yield a six-component complex. The bl3 ribonucleoprotein will be developed as a model for understanding ribonucleoproteins that require binding by multiple protein cofactors to achieve a biologically functional state. Both simple systems are sufficiently sophisticated to illustrate principles generalizable to biomedically important ribonucleoproteins like the ribosome and pre-mRNA complexes.
Specific aims are: (1) To determine the physical basis by which the collapsed state functions to prevent mis-assembly of the bl5 ribonucleoprotein. (2) To study the structure of the bl5 RNA under conditions likely to predominate in yeast mitochondria. (3) To understand the molecular basis for the predominance of near-native structures in the bl5 collapsed state. (4) To develop a comprehensive kinetic and thermodynamic framework for assembly of the six-component bl3 group I intron RNP catalyst. (5) To begin to explore the molecular mechanism by which Mrs1 facilitates catalysis of the bl3 group I intron. (6) To develop a high resolution model for the maturase-bl3 RNA complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056222-08
Application #
7036535
Study Section
Biochemistry Study Section (BIO)
Program Officer
Rhoades, Marcus M
Project Start
1998-05-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
8
Fiscal Year
2006
Total Cost
$246,350
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Duncan, Caia D S; Weeks, Kevin M (2010) The Mrs1 splicing factor binds the bI3 group I intron at each of two tetraloop-receptor motifs. PLoS One 5:e8983
Mortimer, Stefanie A; Weeks, Kevin M (2009) Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution. Nat Protoc 4:1413-21
McGinnis, Jennifer L; Duncan, Caia D S; Weeks, Kevin M (2009) High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly. Methods Enzymol 468:67-89
Jones, Christie N; Wilkinson, Kevin A; Hung, Kimberly T et al. (2008) Lack of secondary structure characterizes the 5'ends of mammalian mitochondrial mRNAs. RNA 14:862-71
Duncan, Caia D S; Weeks, Kevin M (2008) SHAPE analysis of long-range interactions reveals extensive and thermodynamically preferred misfolding in a fragile group I intron RNA. Biochemistry 47:8504-13
Bokinsky, Gregory; Nivon, Lucas G; Liu, Shixin et al. (2006) Two distinct binding modes of a protein cofactor with its target RNA. J Mol Biol 361:771-84
Longo, Antonella; Leonard, Christopher W; Bassi, Gurminder S et al. (2005) Evolution from DNA to RNA recognition by the bI3 LAGLIDADG maturase. Nat Struct Mol Biol 12:779-87
Buchmueller, Karen L; Weeks, Kevin M (2004) Tris-borate is a poor counterion for RNA: a cautionary tale for RNA folding studies. Nucleic Acids Res 32:e184
Garcia, Ivelitza; Weeks, Kevin M (2004) Structural basis for the self-chaperoning function of an RNA collapsed state. Biochemistry 43:15179-86
Garcia, Ivelitza; Weeks, Kevin M (2003) Small structural costs for evolution from RNA to RNP-based catalysis. J Mol Biol 331:57-73

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