Crystallization of RNA

Holbrook, Stephen R.; Holbrook, Elizabeth L.; Walukiewicz, Hanna E.

doi:10.1007/pl00000851

Cited by 19 publications

(14 citation statements)

References 34 publications

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“…Well-ordered single crystals are required for structural studies by x-ray diffraction methods, and obtaining them is often the most difficult step of the structure determination process [82]. Most RNAs exist naturally as protein–RNA complexes, and the crystallization of these complexes has several advantages over the crystallization of RNA alone.…”

Section: Rna Structure Determinationmentioning

confidence: 99%

Computational approaches to 3D modeling of RNA

Laing

Schlick²

2010

J. Phys.: Condens. Matter

108

126

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Many exciting discoveries have recently revealed the versatility of RNA and its importance in a variety of functions within the cell. Since the structural features of RNA are of major importance to their biological function, there is much interest in predicting RNA structure, either in free form or in interaction with various ligands, including proteins, metabolites and other molecules. In recent years, an increasing number of researchers have developed novel RNA algorithms for predicting RNA secondary and tertiary structures. In this review, we describe current experimental and computational advances and discuss recent ideas that are transforming the traditional view of RNA folding. To evaluate the performance of the most recent RNA 3D folding algorithms, we provide a comparative study in order to test the performance of available 3D structure prediction algorithms for an RNA data set of 43 structures of various lengths and motifs. We find that the algorithms vary widely in terms of prediction quality across different RNA lengths and topologies; most predictions have very large root mean square deviations from the experimental structure. We conclude by outlining some suggestions for future RNA folding research.

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Section: Rna Structure Determinationmentioning

confidence: 99%

Computational approaches to 3D modeling of RNA

Laing

Schlick²

2010

J. Phys.: Condens. Matter

108

126

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“…Purification of in vitro transcribed RNA by denaturing methods followed by annealing has been used extensively to generate RNA samples suitable for crystallization (Holbrook et al 2001;Golden and Kundrot 2003;Ke and Doudna 2004;Mooers 2009). We find that in the case of the B. stearothermophilus (Bst) RNA, refolding of the denatured RNA by slowly cooling over several minutes in the presence of 25 mM Mg 2+ and 200 mM K + generates a highly homogeneous sample that elutes as a sharp, symmetric peak from an HR GF column ( Fig.…”

Section: Preparation Of Rna Samples To Minimize Refolding Artifactsmentioning

confidence: 99%

Solution structure of RNase P RNA

Kazantsev¹,

Rambo²,

Karimpour³

et al. 2011

RNA

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The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 29-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.

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“…TheDNAtemplatecanbeobtainedfrom differentsources:(i)Fromtwochemically synthesized strands, (ii) by amplification fromatinyamountofdsDNAbyPCR,or (iii)fromalinearizedplasmid [21]. Thefirstsixnucleotidesatpositions+1 to+6havethegreatestimpactontranscriptioninitiationandthesubsequentstructural change of T7 to the elongation complex; hence, these nucleotides determine the yieldofthetargetRNAtoalargeamount.…”

Section: 2generaltranscription Requirementsmentioning

confidence: 99%

In vitro Transcription and Purification of RNAs of Different Size

Gallo¹,

Furler²,

Sigel³

2005

Chimia

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In vitro runoff transcription from a double-stranded DNA template by T7 RNA polymerase is an elegant way to obtain highly pure and uniform RNA oligonucleotides of lengths ranging from about 15 to several thousand nucleotides. Here we describe the different strategies applied and optimized in our laboratory to enzymatically synthesize RNAs as necessary when working at the interface of bioinorganic chemistry, coordination chemistry, RNA biochemistry and structural biology.

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Crystallization of RNA

Cited by 19 publications

References 34 publications

Computational approaches to 3D modeling of RNA

Computational approaches to 3D modeling of RNA

Solution structure of RNase P RNA

In vitro Transcription and Purification of RNAs of Different Size

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