RNA's central role in biological function and regulation has become increasingly apparent. A vast number of non-coding RNAs have been discovered, and there is extensive cellular regulation by RNAs such as riboswitches and microRNAs, by alternative splicing of pre-mRNAs, by association of RNA binding proteins with families of mRNAs encoding functionally related proteins, and, likely, by the involvement of ncRNAs in epigenetics. The importance of RNA machines in cellular function and regulation is also profound, with roles in telomere maintenance, pre-mRNA splicing, protein synthesis, and membrane targeting. And researchers are increasingly turning to RNA for cellular engineering and as a therapeutic target. In-depth studies of RNAs that allow dissection of functional properties and uncovering of fundamental RNA properties are more important than ever. Over the past two decades, group I introns have served as unparalleled systems for developing broad experimental approaches for RNA study and for deep investigation of RNA structure and function. Comparing and contrasting the properties of RNA and protein enzymes has allowed generalization of fundamental features of biological catalysts and has, conversely, highlighted critical functional distinctions between these structured, biological macromolecules. Over the last funding period single molecule methods, new structure-function tools, and new methods to probe RNA dynamics have emerged, and our group has made foundational contributions to these breakthroughs and has exploited their power. Also emerging were the first x-ray structures of group I introns, opening a new era of RNA structure-function studies. Our proposed studies capitalize on these highly developed tools, the recent structural information, and the extremely tractable group I RNA system to ask RNA structure-function questions that lie at the frontier of our understanding and to answer these questions at unprecedented depth. We will continue to pioneer approaches that will be broadly applicable to other, more complex RNA and RNA/protein systems. We will dissect the transition states for P1 helix docking to the Tetrahymena and Azoarcus ribozyme cores, while developing innovative experimental/computational approaches to dissect conformational transition paths. We will use comparative structure-function analyses to understand how the Azoarcus group I ribozyme is able to utilize 6 kcal/mol more tertiary stabilization energy than the Tetrahymena ribozyme. We will incisively probe competing mechanistic proposals for group I ribozyme active site interactions, unifying information from structural and functional studies. Finally, we will take on the grand challenge of understanding how a functional RNA is assembled, in particular probing the roles and mode of action of remote elements that facilitate function within the catalytic core.

Public Health Relevance

RNA is the central component in gene expression, transmitting information from DNA's stored genetic code. But RNA molecules are also dynamic and structured entities, and interactions with them are critical in development and in the regulation of all life forms so that aberrant behavior of RNA can cause disease, and, conversely, the control of RNA-mediated processes in pathogens provides potential routes to new therapies. By unraveling the fundamental properties and behaviors of RNA molecules, we and others, ultimately, will be empowered to manipulate and control RNA to re-engineer cells to efficiently carry out useful transformations and to effectively implement therapies with RNA targets.

National Institute of Health (NIH)
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Macromolecular Structure and Function E Study Section (MSFE)
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Barski, Oleg
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Stanford University
Schools of Medicine
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Shi, Xuesong; Bisaria, Namita; Benz-Moy, Tara L et al. (2014) Roles of long-range tertiary interactions in limiting dynamics of the Tetrahymena group I ribozyme. J Am Chem Soc 136:6643-8
Gleitsman, Kristin R; Herschlag, Daniel H (2014) A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction. RNA 20:1732-46
Shi, Xuesong; Herschlag, Daniel; Harbury, Pehr A B (2013) Structural ensemble and microscopic elasticity of freely diffusing DNA by direct measurement of fluctuations. Proc Natl Acad Sci U S A 110:E1444-51
Shi, Xuesong; Solomatin, Sergey V; Herschlag, Daniel (2012) A role for a single-stranded junction in RNA binding and specificity by the Tetrahymena group I ribozyme. J Am Chem Soc 134:1910-3
Sengupta, Raghuvir N; Herschlag, Daniel; Piccirilli, Joseph A (2012) Thermodynamic evidence for negative charge stabilization by a catalytic metal ion within an RNA active site. ACS Chem Biol 7:294-9
Greenfeld, Max; Solomatin, Sergey V; Herschlag, Daniel (2011) Removal of covalent heterogeneity reveals simple folding behavior for P4-P6 RNA. J Biol Chem 286:19872-9
Forconi, Marcello; Porecha, Rishi H; Piccirilli, Joseph A et al. (2011) Tightening of active site interactions en route to the transition state revealed by single-atom substitution in the guanosine-binding site of the Tetrahymena group I ribozyme. J Am Chem Soc 133:7791-800
Benz-Moy, Tara L; Herschlag, Daniel (2011) Structure-function analysis from the outside in: long-range tertiary contacts in RNA exhibit distinct catalytic roles. Biochemistry 50:8733-55
Solomatin, Sergey V; Greenfeld, Max; Chu, Steven et al. (2010) Multiple native states reveal persistent ruggedness of an RNA folding landscape. Nature 463:681-4
Wan, Yaqi; Suh, Hyejean; Russell, Rick et al. (2010) Multiple unfolding events during native folding of the Tetrahymena group I ribozyme. J Mol Biol 400:1067-77

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