Use of the rnabinding protein u1a as a crystal doudna lab. The rnaifold software provides two algorithms to solve the inverse folding problem. Crystal structures of transcription factor nusg in light of. The rna 6000 nano system has a broad dynamic range and can quantitate between 25 500 ngml of rna with a covariance of 10%. Rnastructure is a complete package for rna and dna secondary structure prediction and analysis. These socalled crystallization modules include the u1a rna binding protein and fab bl36, which bind to their cognate hairpins, and the l7ae family of proteins that bind to the kink turn motifs 15,17,18. Protein engineering as a tool for crystallography sciencedirect. Preparation of rnaprotein complexes for xray crystallography and nmr. Molecular biologyrna protocols protocol online your. These socalled crystallization modules include the u1a rna binding protein. The role of rna in biology is varied and rich, with rna playing diverse roles in both healthy and diseased cells. Sederivatized rnas for xray crystallography springerlink. The structure of the nterminal rrm of u1a residues 2117 has been determined by nmr spectroscopy32 and xray crystallography residues 392,33 and the complex of the nterminal rrm of u1a residues 297 with sl2 rna 21 bases u1arna has been determined by xray crystallography,34 albeit with two surface mutations. Proteinassisted rna fragment docking rnax for modeling rna.
This system coexpresses u1apgfp with the rna of interest tagged with u1a aptamers, and has been proven to effectively track rna in vivo. When bound to pie rna, u1a proteins also bind to the enzyme responsible for. It includes algorithms for secondary structure prediction, including facility to predict base pairing probabilities. Raman crystallography is the application of raman spectroscopy to single crystals. The human u1a rnabinding domain rbd1 adopts one of the most common protein folds, the rnarecognition motif, and is a paradigm for understanding rnaprotein interactions. Pairfold can be used, for example, to predict interactions between a probe and target rna molecule, or between pairs of strands in biomolecular nanostructures. This series of talks is about rna structure, function, and recognition. Anaelu is composed of three interconnected applications, corresponding to three crystallographic tasks. A software package that integrates three rna analysis tools based on the abstract shapes approach. Although xray diffraction played a role in its solution by james d. Our results provide a new tool set for the crystallization of rna and rna. When bound to pie rna, u1a proteins also bind to the enzyme responsible for formation of the mature 3.
The crystallographic structure of the synthetic oligoribonucleotide, uua6a, has been solved at 2. This position is critical for rna binding as the phenylalanine stacks to a certain nucleotide in the complex formed by u1a and rna shiels et al. The development release allows access to the latest features for feedback. List of rna structure prediction software wikipedia. This work was supported by the intramural research program of the nih. Expanding crystallization tools for nucleic acid complexes. Cryoem structure of saccharomyces cerevisiae u1 snrnp. Together with foldx protein design tool suite we were able to engineer. The second group comprises multiple structures of u1a rrm in complex with the. The role of rna structure in the interaction of u1a protein with u1.
The selfcleaving ribozyme of the hepatitis delta virus hdv is the only catalytic rna known to be required for the viability of a human pathogen. Polya tail regulation requires cooperative binding of two human u1a proteins to an rna regulatory region called the polyadenylation inhibition element pie. Crystal structure reveals details of nonstandard rna. Tertiary structure detection software tools rna data. However, rna binding as, for example, in the spliceosomal u1a oubridge et al. Athena, artemis, hephaestus this software package is based on the ifeffit library of numerical and xas algorithms. Use of the spliceosomal protein u1a to facilitate crystallization and structure determination of complex rnas. Here, we describe the application of raman crystallography to functional rna molecules. Use of the u1a protein to facilitate crystallization and structure. Attempts to crystallize the wild type u1a protein and its cognate rna hairpin result in crystals diffracting no.
The function of many biologically important rnas is conferred by their threedimensional folds, and xray crystallography remains the most powerful tool to determine their structure. The pdb archive contains information about experimentallydetermined structures of proteins, nucleic acids, and complex assemblies. Cryoem structure of saccharomyces cerevisiae u1 snrnp offers. If acrrm bound rna in the way observed in related structures, the cterminal. Oct 19, 2017 d sequence alignment between human and yeast u1a rrm1 demonstrates differences between residues in rnp1 and rnp2 that are known to bind rna in human u1a red triangles and the long loop 2 in. These socalled crystallization modules include the u1a rna binding protein and fab bl36 16, which bind to their cognate hairpins. The role of rna sequence versus structure in rnaprotein interactions remains a. The availability of sequenced and annotated genomes greatly facilitates rnai experiments in an increasing number of organisms that were previously not genetically tractable. Md simulations of u1arna systems the md trajectories computed for this study are summarizedintable1. Md simulations were performed with explicit water and counterions. Thermophiles are natural sources of such molecules. The human u1a rna binding domain rbd1 adopts one of the most common protein folds, the rna recognition motif, and is a paradigm for understanding rna protein interactions. Anaelu software package anaelu analytical emulator laue utility has been created for aiding the interpretation of twodimensional xray diffraction patterns produced by textured bulk and nanostructured samples. In this method, the rna of interest is attached to the 5.
To solve the crystal structure of fabspinach1 rna complex, we. Rna interference reagent design software tools omictools. Crystallographic studies of dna and rna sciencedirect. The structure determination of complex rna molecules such as ribozymes, riboswitches and aptamers by xray crystallography hinges on the preparation of wellordered crystals. The rna used in crystallization experiments comprised an 11bp duplex in the center of which was the t. Notably, the u1 rna hairpin sequence is conserved in viridiplantae and animalia. Even mrna, a relatively linear molecule, can contain distinctive three dimensional structures. A design and visualization tool for rna secondary structure.
Oscail is a windows based software for molecular modelling and crystallography. Macromolecular crystallographic software links ccp4 cns phenix eden crystallography coot o macros for o uppsala software factory pymol home page sharp mosflm other useful crystallography links crystallography on os x xray absorption edges the protein data bank pdb molecular movies data base nucleic acid databank international. The software can also design interacting rna molecules using rnacofold of the viennarna package. The tagged rna system has recently emerged as a feasible noninvasive solution for such purpose. Crystal structure of a hepatitis delta virus ribozyme nature. If the rna is universally abundant, homologs may sometimes be purified directly from a thermophile e. Methods in molecular biology methods and protocols, vol 941. The ability of u1a to bind rna in different structural contexts raises the. The design of rna interference rnai reagents is an essential step for performing lossoffunction studies in many experimental systems. The instrument software automatically compares the peak areas from unknown rna samples to the combined area of the six rna 6000 ladder rna peaks to determine the concentration of the unknown samples. The first structure of an rna m5c methyltransferase, fmu. The rna was cocrystallized with a mixture of archeoglobus fulgidus l7ae and human u1a. Crystallographic software university of california. Carine crystallography is a crystallographic software which has been developed since 1989 and which is used by thousands of persons around the world for teaching, research and edition is now available in a totally rethought version.
C the u1a model was subjected to rna docking, taking into account the. If you want to visualize tertiary structures, you will need to install the software ucsf chimera and to link it to assemble2. Jul 03, 2000 moreover, the rna fragment, as seen in the u1a. This indicates that the mutation f56w should not prohibit rna binding to u1a rbd law, 2005, shiels et al. Molecular biologyrna protocols protocol online your lab. Aug 22, 2017 highresolution crystal structure reveals a new pathway for rna during a nontraditional form of transcriptionthe process by which rna is produced from a dna template. A utility for collecting statistics from aligned and structurally annotated rna sequences. Rna molecules of interest can be constructed to contain a motif that binds to its cognate rna binding protein and the complex is advanced to crystallization trials. The nmr structure of the 38 kda u1a protein pie rna. The duplex section terminated with a 10nt loop that is the binding site for the u1a protein. Affinity maturation of a portable fabrna module for chaperone. For smaller rnas rna purification system has been developed that is capable of producing sufficient rna for crystallography screening batey and kieft, 2007.
Crystallographic studies of dna and rna molecules share many considerations that we outline here. Structure of a rare nonstandard sequence kturn bound by. Pdf determining structures of rna aptamers and riboswitches. The application of crystallography to rna is illustrated with the structure determination of the cug repeat that is linked to type i myotonic dystrophy. In this chapter, we describe in detail the u1a based tagged rna system. As a member of the wwpdb, the rcsb pdb curates and annotates pdb data according to agreed upon standards. Affinity maturation of a portable fabrna module for. Fmu contains three subdomains which share structural homology to dna m5c methyltransferases and two rna. Structural basis of the rnabinding specificity of human u1a protein. It is important to design multiple constructs to vary the length of the stem connecting the inserted element to the core of the rna, which will alter their relative orientations fig. Highresolution crystal structure reveals a new pathway for rna during a nontraditional form of transcriptionthe process by which rna is produced.
Crystallization, crystallizability, spliceosomal protein, rna biding domain. This windows software provides an integrated high quality system for building and modelling molecules mopac and iconc are included and the system drives pc gamess and tinker. Affinity and specificity of protein u1arna complex. Expanding crystallization tools for nucleic acid complexes using. Embl, gene expression program, 1 meyerhofstrasse, heidelberg, d69117. The crystallographic refinement permitted the identification of 91 solvent molecules, with a final agreement factor of %. The modules have yielded promising results for rna crystallization and structure determination. Pyle explains that many rna molecules have elaborate structures that are essential for their functions. Pairfold predicts the minimum free energy secondary structure formed by two input dna or rna molecules. U1 spliceosomal rna is the small nuclear rna snrna component of u1 snrnp small nuclear ribonucleoprotein, an rna protein complex that combines with other snrnps, unmodified premrna, and various other proteins to assemble a spliceosome, a large rna protein molecular complex upon which splicing of premrna occurs. You will find here the stable release and the development release for assemble2. Theyincludewildtypeu1arna complex, free wildtype u1a in closed and open conformations, and free sl2 rna, as well as those of three u1a mutants, f56a, f56l and f56w. Crystal structure of human acinus rna recognition motif. A general strategy to solve the phase problem in rna.
Center for molecular biology of rna at university of california, santa cruz. As in protein crystallography, crystal quality may be improved by using nucleic acids with higher thermal stability. Collection of tissue for rna analysis laboratory of experimental pathology how to collect and store tissue sample for rna. Xray diffraction from fibrous dna tipped off watson, crick to double helix it is one of the most famous molecular structures ever determined, the now iconic dna double helix. The nmr structure of the 38 kda u1a protein pie rna complex. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis.
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