ABSTRACT
DNA ligases join the breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 3'-hydroxyl and 5'-phosphate termini. They fall into two classes that require either ATP or NAD(+) as the source of an AMP group that is covalently attached to a strictly conserved lysine. Conformational flexibility is essential for the function of multi-domain DNA ligases because they must undergo large conformational changes involving domain rearrangements during the course of the reaction. In the absence of the nicked DNA substrate, both open and closed conformations have been observed for the ATP-dependent DNA ligases from Sulfolobus solfataricus and Pyrococcus furiosus. Here, the crystal structure of an ATP-dependent DNA ligase from Archaeoglobus fulgidus has been determined in the DNA-unbound unadenylated state. It resembles the closed conformation of P. furiosus DNA ligase but was even more closed, thus enhancing our understanding of the conformational variability of these enzymes.
Subject(s)
Archaeoglobus fulgidus/metabolism , DNA Ligases/isolation & purification , DNA Ligases/metabolism , DNA, Archaeal/chemistry , Molecular Conformation , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Archaeoglobus fulgidus/genetics , Binding Sites/genetics , Conserved Sequence , DNA Ligase ATP , DNA Ligases/chemistry , DNA Ligases/genetics , DNA, Archaeal/metabolism , Data Collection , Escherichia coli/genetics , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Phosphates/chemistry , Protein Binding/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Statistics as Topic , Temperature , Water/chemistryABSTRACT
The yeast protein Dom34 is a key component of no-go decay, by which mRNAs with translational stalls are endonucleolytically cleaved and subsequently degraded. However, the identity of the endoribonuclease is unknown. Homologs of Dom34, called Pelota, are broadly conserved in eukaryotes and archaea. To gain insights into the structure and function of Dom34/Pelota, we have determined the structure of Pelota from Thermoplasma acidophilum (Ta Pelota) and investigated the ribonuclease activity of Dom34/Pelota. The structure of Ta Pelota is tripartite, and its domain 1 has the RNA-binding Sm fold. We have discovered that Ta Pelota has a ribonuclease activity and that its domain 1 is sufficient for the catalytic activity. We also demonstrate that domain 1 of Dom34 has an endoribonuclease activity against defined RNA substrates containing a stem loop, which supports a direct catalytic role of yeast Dom34 in no-go mRNA decay.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , RNA Stability , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Thermoplasma/enzymology , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Endoribonucleases , Humans , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Termination Factors/chemistry , Protein Structure, Quaternary , Protein Structure, Secondary , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Ribonucleases/metabolism , Solutions , Structural Homology, Protein , Substrate SpecificityABSTRACT
IS200 transposases, originally identified in Salmonella typhimurium LT2, are present in many bacteria and archaea and are distinct from other groups of transposases. To facilitate further structural comparisons among IS200-like transposases, structural analysis has been initiated of a putative transposase from Thermoplasma acidophilum encoded by the Ta0474 gene. Its 137-residue polypeptide shows high levels of sequence similarity to other members of the IS200 transposase family. The protein was overexpressed in intact form in Escherichia coli and crystallized at 297 K using a reservoir solution consisting of 100 mM Na HEPES pH 7.5 and 20%(v/v) ethanol. X-ray diffraction data were collected to 1.78 A. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 65.00, b = 34.07, c = 121.58 A, alpha = 90, beta = 100.20, gamma = 90 degrees. Four monomers, representing two copies of a dimeric molecule, are present in the asymmetric unit, giving a crystal volume per protein weight (V(M)) of 2.02 A(3) Da(-1) and a solvent content of 39.2%.
