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Espinoza-Herrera S.J.,Case Western Reserve University | Gaur V.,Ohio State University | Gaur V.,International Institute of Molecular and Cell Biology in Warsaw | Suo Z.,Ohio State University | Carey P.R.,Case Western Reserve University
Biochemistry | Year: 2013

Y-Family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. 13C- and 15N-labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg2+ and Mn2+, nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of*C modes in the crystal following soaking out of noncovalently bound C(or*C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following a single d*CTP addition. Adding these two bases caused visible damage to the crystal that was possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn2+ or Mg2+, the primer extension reaction did not occur; instead, a ternary protein·template·d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features caused by ring modes from the template/primer bases being perturbed and from the DNA backbone appear, as well as features from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary complex than in the binary complex. Using standardized Raman intensities followed as a function of time, the C(*C)TP population in the crystal was maximal at ∼20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred. © 2013 American Chemical Society. Source

Bujnicki J.M.,International Institute of Molecular and Cell Biology in Warsaw
Briefings in Bioinformatics | Year: 2011

Noncoding RNAs perform important roles in the cell. As their function is tightly connected with structure, and as experimental methods are time-consuming and expensive, the field of RNA structure prediction is developing rapidly. Here, we present a detailed study on using the ModeRNA software. The tool uses the comparative modeling approach and can be applied when a structural template is available and an alignment of reasonable quality can be performed. We guide the reader through the entire process of modeling Escherichia coli tRNAThr in a conformation corresponding to the complex with an aminoacyl-tRNA synthetase (aaRS). We describe the choice of a template structure, preparation of input files, and explore three possible modeling strategies. In the end, we evaluate the resulting models using six alternative benchmarks. The ModeRNA software can be freely downloaded from http://iimcb.genesilico.pl/moderna/ under the conditions of the General Public License. It runs under LINUX, Windows and Mac OS. It is also available as a server at http://iimcb.genesilico.pl/modernaserver/. The models and the script to reproduce the study from this article are available at http://www.genesilico.pl/moderna/examples/. © The Author 2011. Published by Oxford University Press. Source

Fislage M.,Vrije Universiteit Brussel | Roovers M.,Institute Of Recherches Microbiologiques Jean Marie Wiame | Tuszynska I.,International Institute of Molecular and Cell Biology in Warsaw | Bujnicki J.M.,International Institute of Molecular and Cell Biology in Warsaw | And 3 more authors.
Nucleic Acids Research | Year: 2012

Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNA Phe at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m 2G6 (N 2-methylguanosine) MTase TTCTrmN from Thermus thermophilus and its ortholog PfTrm14 from Pyrococcus furiosus. Structures of PfTrm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. TTCTrmN and PfTrm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNA Phe of T. thermophilus and via site-directed mutagenesis. © 2012 The Author(s). Source

Korneta I.,International Institute of Molecular and Cell Biology in Warsaw | Bujnicki J.M.,International Institute of Molecular and Cell Biology in Warsaw | Bujnicki J.M.,Adam Mickiewicz University
PLoS Computational Biology | Year: 2012

The spliceosome is a molecular machine that performs the excision of introns from eukaryotic pre-mRNAs. This macromolecular complex comprises in human cells five RNAs and over one hundred proteins. In recent years, many spliceosomal proteins have been found to exhibit intrinsic disorder, that is to lack stable native three-dimensional structure in solution. Building on the previous body of proteomic, structural and functional data, we have carried out a systematic bioinformatics analysis of intrinsic disorder in the proteome of the human spliceosome. We discovered that almost a half of the combined sequence of proteins abundant in the spliceosome is predicted to be intrinsically disordered, at least when the individual proteins are considered in isolation. The distribution of intrinsic order and disorder throughout the spliceosome is uneven, and is related to the various functions performed by the intrinsic disorder of the spliceosomal proteins in the complex. In particular, proteins involved in the secondary functions of the spliceosome, such as mRNA recognition, intron/exon definition and spliceosomal assembly and dynamics, are more disordered than proteins directly involved in assisting splicing catalysis. Conserved disordered regions in spliceosomal proteins are evolutionarily younger and less widespread than ordered domains of essential spliceosomal proteins at the core of the spliceosome, suggesting that disordered regions were added to a preexistent ordered functional core. Finally, the spliceosomal proteome contains a much higher amount of intrinsic disorder predicted to lack secondary structure than the proteome of the ribosome, another large RNP machine. This result agrees with the currently recognized different functions of proteins in these two complexes. © 2012 Korneta, Bujnicki. Source

Majewski L.,International Institute of Molecular and Cell Biology in Warsaw | Kuznicki J.,International Institute of Molecular and Cell Biology in Warsaw
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2015

In this review we describe the present knowledge about store operated Ca2+ entry (SOCE) in neurons and the proteins involved in this process: STIM, as well as Orai and TRP channels. We address the issue of whether SOCE is used only to refill Ca2+ in the ER or whether Ca2+ that enters the neuronal cell during SOCE also performs signaling functions. We collected the data indicating that SOCE and its components participate in the important processes in neurons. This has implications for identifying new drug targets for the treatment of brain diseases. Evidence indicates that in neurodegenerative diseases Ca2+ homeostasis and SOCE components become dysregulated. Thus, different targets and strategies might be identified for the potential treatment of these diseases. This article is part of a Special Issue entitled: 13th European symposium on calcium. © 2015. Source

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