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In JoVE (1)
Other Publications (12)
- Acta Crystallographica. Section D, Biological Crystallography
- Journal of Molecular Biology
- Protein Science : a Publication of the Protein Society
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of Molecular Biology
- Protein Science : a Publication of the Protein Society
- Nucleic Acids Research
- PLoS Computational Biology
- The Journal of Physical Chemistry. B
- BMC Bioinformatics
- Journal of Applied Crystallography
Articles by Cameron Mura in JoVE
Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples
Jennifer Patterson1, Cameron Mura1
1Department of Chemistry, University of Virginia
Other articles by Cameron Mura on PubMed
Structure of a Nudix Protein from Pyrobaculum Aerophilum Reveals a Dimer with Two Intersubunit Beta-sheets
Acta Crystallographica. Section D, Biological Crystallography. Apr, 2002 | Pubmed ID: 11914479
Nudix proteins, formerly called MutT homolog proteins, are a large family of proteins that play an important role in reducing the accumulation of potentially toxic compounds inside the cell. They hydrolyze a wide variety of substrates that are mainly composed of a nucleoside diphosphate linked to some other moiety X and thus are called Nudix hydrolases. Here, the crystal structure of a Nudix hydrolase from the hyperthermophilic archaeon Pyrobaculum aerophilum is reported. The structure was determined by the single-wavelength anomalous scattering method with data collected at the peak anomalous wavelength of an iridium-derivatized crystal. It reveals an extensive dimer interface, with each subunit contributing two strands to the beta-sheet of the other subunit. Individual subunits consist of a mixed highly twisted and curved beta-sheet of 11 beta-strands and two alpha-helices, forming an alpha-beta-alpha sandwich. The conserved Nudix box signature motif, which contains the essential catalytic residues, is located at the first alpha-helix and the beta-strand and loop preceding it. The unusually short connections between secondary-structural elements, together with the dimer form of the structure, are likely to contribute to the thermostability of the P. aerophilum Nudix protein.
Structure and Function of an Archaeal Homolog of Survival Protein E (SurEalpha): an Acid Phosphatase with Purine Nucleotide Specificity
Journal of Molecular Biology. Mar, 2003 | Pubmed ID: 12595266
The survival protein E (SurE) family was discovered by its correlation to stationary phase survival of Escherichia coli and various repair proteins involved in sustaining this and other stress-response phenotypes. In order to better understand this ancient and well-conserved protein family, we have determined the 2.0A resolution crystal structure of SurEalpha from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (Pae). This first structure of an archaeal SurE reveals significant similarities to and differences from the only other known SurE structure, that from the eubacterium Thermatoga maritima (Tma). Both SurE monomers adopt similar folds; however, unlike the Tma SurE dimer, crystalline Pae SurEalpha is predominantly non-domain swapped. Comparative structural analyses of Tma and Pae SurE suggest conformationally variant regions, such as a hinge loop that may be involved in domain swapping. The putative SurE active site is highly conserved, and implies a model for SurE bound to a potential substrate, guanosine-5'-monophosphate (GMP). Pae SurEalpha has optimal acid phosphatase activity at temperatures above 90 degrees C, and is less specific than Tma SurE in terms of metal ion requirements. Substrate specificity also differs between Pae and Tma SurE, with a more specific recognition of purine nucleotides by the archaeal enzyme. Analyses of the sequences, phylogenetic distribution, and genomic organization of the SurE family reveal examples of genomes encoding multiple surE genes, and suggest that SurE homologs constitute a broad family of enzymes with phosphatase-like activities.
Protein Science : a Publication of the Protein Society. Apr, 2003 | Pubmed ID: 12649441
Intron splicing is a prime example of the many types of RNA processing catalyzed by small nuclear ribonucleoprotein (snRNP) complexes. Sm proteins form the cores of most snRNPs, and thus to learn principles of snRNP assembly we characterized the oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs) from Pyrobaculum aerophilum (Pae) and Methanobacterium thermautotrophicum (Mth). Ultracentrifugation shows that Mth SmAP1 is exclusively heptameric in solution, whereas Pae SmAP1 forms either disulfide-bonded 14-mers or sub-heptameric states (depending on the redox potential). By electron microscopy, we show that Pae and Mth SmAP1 polymerize into bundles of well ordered fibers that probably form by head-to-tail stacking of heptamers. The crystallographic results reported here corroborate these findings by showing heptamers and 14-mers of both Mth and Pae SmAP1 in four new crystal forms. The 1.9 A-resolution structure of Mth SmAP1 bound to uridine-5'-monophosphate (UMP) reveals conserved ligand-binding sites. The likely RNA binding site in Mth agrees with that determined for Archaeoglobus fulgidus (Afu) SmAP1. Finally, we found that both Pae and Mth SmAP1 gel-shift negatively supercoiled DNA. These results distinguish SmAPs from eukaryotic Sm proteins and suggest that SmAPs have a generic single-stranded nucleic acid-binding activity.
Proceedings of the National Academy of Sciences of the United States of America. Apr, 2003 | Pubmed ID: 12668760
To better understand the roles of Sm proteins in forming the cores of many RNA-processing ribonucleoproteins, we determined the crystal structure of an atypical Sm-like archaeal protein (SmAP3) in which the conserved Sm domain is augmented by a previously uncharacterized, mixed alpha/beta C-terminal domain. The structure reveals an unexpected SmAP3 14-mer that is perforated by a cylindrical pore and is bound to 14 cadmium (Cd(2+)) ions. Individual heptamers adopt either "apical" or "equatorial" conformations that chelate Cd(2+) differently. SmAP3 forms supraheptameric oligomers (SmAP3)(n = 7,14,28) in solution, and assembly of the asymmetric 14-mer is modulated by differential divalent cation-binding in apical and equatorial subunits. Phylogenetic and sequence analyses substantiate SmAP3s as a unique subset of SmAPs. These results distinguish SmAP3s from other Sm proteins and provide a model for the structure and properties of Sm proteins >100 residues in length, e.g., several human Sm proteins.
Journal of Molecular Biology. Feb, 2006 | Pubmed ID: 16337963
Hfq is a nucleic acid-binding protein that functions as a global regulator of gene expression by virtue of its interactions with several small, non-coding RNA species. Originally identified as an Escherichia coli host factor required for RNA phage Qbeta replication, Hfq is now known to post-transcriptionally regulate bacterial gene expression by modulating both mRNA stability and translational activity. Recently shown to be a member of the diverse Sm protein family, Hfq adopts the OB-like fold typical of other Sm and Sm-like (Lsm) proteins, and also assembles into toroidal homo-oligomers that bind single-stranded RNA. Similarities between the structures, functions, and evolution of Sm/Lsm proteins and Hfq are continually being discovered, and we now report an additional, unexpected biophysical property that is shared by Hfq and other Sm proteins: E.coli Hfq polymerizes into well-ordered fibres whose morphologies closely resemble those found for Sm-like archaeal proteins (SmAPs). However, the hierarchical assembly of these fibres is dissimilar: whereas SmAPs polymerize into polar tubes (and striated bundles of such tubes) by head-to-tail stacking of individual homo-heptamers, helical Hfq fibres are formed by cylindrical slab-like layers that consist of 36 subunits arranged as a hexamer of Hfq homo-hexamers (i.e. protofilaments in a 6 x 6 arrangement). The different fibrillar ultrastructures formed by Hfq and SmAP are presented and examined herein, with the overall goal of elucidating another similarity amongst the diverse members of the Sm protein family.
Protein Science : a Publication of the Protein Society. Sep, 2007 | Pubmed ID: 17660259
Sm-like proteins are ubiquitous ring-shaped oligomers that exhibit a variety of nucleic acid-binding activities. They have been linked functionally to various cellular events involving RNA, and it is generally believed that their activity is exerted via the passive binding of nucleic acids. Our earlier studies of the Sm-like Escherichia coli protein Hfq provided the first evidence indicating that Hfq is an ATP-binding protein. Using a combination of biochemical and genetic techniques, we have now determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. The results of RNA footprinting and binding analyses suggest that ATP binding by the Hfq-RNA complex results in its significant destabilization. RNA footprinting indicates deprotection of Hfq-bound RNA tracts in the presence of ATP, suggestive of their release by the protein. The results reported herein broaden the scope of potential in vivo roles for Hfq and other Sm-like proteins.
Molecular Dynamics of a KappaB DNA Element: Base Flipping Via Cross-strand Intercalative Stacking in a Microsecond-scale Simulation
Nucleic Acids Research. Sep, 2008 | Pubmed ID: 18653524
The sequence-dependent structural variability and conformational dynamics of DNA play pivotal roles in many biological milieus, such as in the site-specific binding of transcription factors to target regulatory elements. To better understand DNA structure, function, and dynamics in general, and protein...DNA recognition in the 'kappaB' family of genetic regulatory elements in particular, we performed molecular dynamics simulations of a 20-bp DNA encompassing a cognate kappaB site recognized by the proto-oncogenic 'c-Rel' subfamily of NF-kappaB transcription factors. Simulations of the kappaB DNA in explicit water were extended to microsecond duration, providing a broad, atomically detailed glimpse into the structural and dynamical behavior of double helical DNA over many timescales. Of particular note, novel (and structurally plausible) conformations of DNA developed only at the long times sampled in this simulation-including a peculiar state arising at approximately 0.7 micros and characterized by cross-strand intercalative stacking of nucleotides within a longitudinally sheared base pair, followed (at approximately 1 micros) by spontaneous base flipping of a neighboring thymine within the A-rich duplex. Results and predictions from the microsecond-scale simulation include implications for a dynamical NF-kappaB recognition motif, and are amenable to testing and further exploration via specific experimental approaches that are suggested herein.
RapA, the SWI/SNF Subunit of Escherichia Coli RNA Polymerase, Promotes the Release of Nascent RNA from Transcription Complexes
Biochemistry. Aug, 2009 | Pubmed ID: 19580329
RapA, a prokaryotic member of the SWI/SNF protein superfamily, is an integral part of the RNA polymerase transcription complex. RapA's function and catalytic mechanism have been linked to nucleic acid remodeling. In this work, we show that mutations in the interface between RapA's SWI/SNF and double-stranded nucleic acid-binding domains significantly alter ATP hydrolysis in purified RapA. The effects of individual mutations on ATP hydrolysis loosely correlated with RapA's nucleic acid remodeling activity, indicating that the interaction between these domains may be important for the RapA-mediated remodeling of nonproductive transcription complexes. In this study, we introduced a model system for in vitro transcription of a full-length Escherichia coli gene (slyD). To study the function of RapA, we fractionated and identified in vitro transcription reaction intermediates in the presence or absence of RapA. These experiments demonstrated that RapA contributes to the formation of free RNA species during in vitro transcription. This work further refines our models for RapA function in vivo and establishes a new role in RNA management for a representative of the SWI/SNF protein superfamily.
The Journal of Physical Chemistry. B. Jan, 2011 | Pubmed ID: 21192664
By hindering or "silencing" protein translation in vivo, antisense nucleic acid analogues that hybridize to bacterial rRNA could serve as a promising class of antibacterial compounds. Thus, we performed a comparative analysis of the dynamical properties of modified oligonucleotides based upon a sequence (5')r(UGUUACGACU)(3') that is complementary to bacterial ribosomal A-site RNA. In particular, 25 ns explicit solvent molecular dynamics simulations were computed for the following six single-stranded decamers: (1) the above RNA in unmodified form; (2) the 2'-O-methyl-modified RNA; (3) peptide nucleic acid (PNA) analogues of the above sequence, containing either (a) T or (b) U; and (4) two serine-substituted PNAs. Our results show that 2'-O-methylation attenuates RNA backbone dynamics, thereby preventing interconversion between stacked and unstacked conformations. The PNA analogue is rendered less flexible by replacing uracil with thymine; in addition, we found that derivatizing the PNA backbone with serine leads to enhanced base-stacking interactions. Consistent with known solubility properties of these classes of molecules, both RNAs exhibited greater localization of water molecules than did PNA. In terms of counterions, the initially helical conformation of the 2'-O-methyl RNA exhibits the highest Na(+) density among all the simulated decamers, while Na(+) build-up was most negligible for the neutral PNA systems. Further studies of the conformational and physicochemical properties of such modified single-stranded oligomers may facilitate better design of nucleic acid analogues, particularly those capable of serving as specific, high-affinity ribosomal A-site binders.
BMC Bioinformatics. 2011 | Pubmed ID: 21352538
Bioinformatic analyses typically proceed as chains of data-processing tasks. A pipeline, or 'workflow', is a well-defined protocol, with a specific structure defined by the topology of data-flow interdependencies, and a particular functionality arising from the data transformations applied at each step. In computer science, the dataflow programming (DFP) paradigm defines software systems constructed in this manner, as networks of message-passing components. Thus, bioinformatic workflows can be naturally mapped onto DFP concepts.
Journal of Applied Crystallography. Apr, 2011 | Pubmed ID: 22479120
To facilitate flexible and efficient structural bioinformatics analyses, new functionality for three-dimensional structure processing and analysis has been introduced into PyCogent - a popular feature-rich framework for sequence-based bioinformatics, but one which has lacked equally powerful tools for handling stuctural/coordinate-based data. Extensible Python modules have been developed, which provide object-oriented abstractions (based on a hierarchical representation of macromolecules), efficient data structures (e.g.kD-trees), fast implementations of common algorithms (e.g. surface-area calculations), read/write support for Protein Data Bank-related file formats and wrappers for external command-line applications (e.g. Stride). Integration of this code into PyCogent is symbiotic, allowing sequence-based work to benefit from structure-derived data and, reciprocally, enabling structural studies to leverage PyCogent's versatile tools for phylogenetic and evolutionary analyses.