Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purification of p protein to homogeneity from Escherichia coli has shown that its RNA-dependent ATPase activity is physically inseparable from its termination activity. The biochemical properties of pATPase have been studied using poly(C) as the activating RNA. This reaction is stimulated by Mg2+ ions and Mn2+ ions and is prevented by excess EDTA; it is not stimulated by Ca2+ ions. The reaction is not affected by a Zn2+ ion chelator and is inhibited by 1 mM Zn2+. With Mg2+ present, the activity is essentially constant from pH 7 to pH 9.7. pATPase is sensitive to p-hydroxymercuribenzoate and to N-ethylmaleimide. All four ribonucleoside triphosphates are hydrolyzed by p action. ATP has the lowest Km (0.009 mM), while CTP has the highest Vmax. In a mixture containing all four nucleoside triphosphates at a concentration of 0.4 mM, p shows no strong preference for any one of the substrates. The response of p ATPase to a variety of inhibitors of other ATPases and GTPases and of transcription has been studied. Of the compounds tested, aurintricarboxylic acid, an inhibitor of protein-nucleic acid interactions, was found to be a potent inhibitor of p ATPase, while rifampicin and heparin had no effect. pATPase showed partial sensitivity to thiostrepton, fusidic acid, Dio 9, and sodium azide.
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PMID:Characterization of the nucleoside triphosphate phosphohydrolase (ATPase) activity of RNA synthesi termination factor p. I. Enzymatic properties and effects of inhibitors. 13 81

Escherichia coli transcription termination factor rho is an RNA-dependent ATPase, and ATPase activity is required for all its functions. We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer. This stoichiometry has been verified by a number of different techniques, including ultracentrifugation, ultrafiltration, and fluorescence titration studies. We have also shown that ATP can bind to isolated monomers of rho when the hexamer is dissociated with the mild denaturant myristyltrimethylammonium bromide, demonstrating that each promoter of rho carries an ATP binding site. The six binding sites that we observe in the rho hexamer are not equivalent; the hexamer contains three strong (Ka approximately 3 x 10(6) M-1) and three weak (Ka approximately 10(5) M-1) binding sites for ATP. The binding constant of the weak binding site is just the reciprocal of the enzymatic Km for ATP as a substrate; thus these weak sites, as well as the strong sites, can, in principle, take part in the catalytic cycle. The asymmetry induced (or manifested) by ATP binding reduces the symmetry of the rho hexamer from a D3 to a pseudo-D3 state. This "breakage" of symmetry has implications for the molecular mechanism of rho, because an asymmetric structure can lead to directional helicase activity by invoking directionally distinct RNA binding and release reactions (see Geiselmann, J., Yager, T.D., & von Hippel, P.H., 1992c, Protein Sci. 1, 861-873).
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PMID:Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. I. Binding of ATP. 130 71

eIF-4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF-4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5' untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF-4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF-4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF-4A: (i) ATP binding, (ii) RNA-dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site-directed mutagenesis. First we show that recombinant eIF-4A, together with recombinant eIF-4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity.
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PMID:Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. 137 97

PRP16 is an RNA-dependent ATPase required for the second catalytic step of splicing in vitro. A dominant suppressor of a branchpoint mutation in Saccharomyces cerevisiae, the prp16-1 allele, contains a Tyr to Asp change in the nucleotide-binding site consensus sequence. We now find that cells harboring the prp16-1 allele have a general growth defect that is exacerbated at cold temperatures. The mutant is dominant over the wild-type gene when overexpressed. Purified Prp16-1 protein binds to the spliceosome with apparently wild-type affinity; however, it only weakly complements the second-step block in a PRP16-depleted extract. Analysis of purified Prp16-1 revealed that the rate of ATP hydrolysis is greatly reduced. These results can account for the dominant negative growth phenotype and argue that the ATPase activity of PRP16 is essential for its role in splicing. Moreover, since PRP16 is a member of the DEAD/H box families, these findings have important implications for a large class of proteins.
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PMID:A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome. 138 54

Transcription-termination factor rho of Escherichia coli functions as an RNA-dependent ATPase that causes transcript release at specific rho-dependent termination sites on the DNA template. Rho exists as a hexagon of identical subunits, physically organized as a trimer of dimers with D3 symmetry. The structural asymmetry of the dimer is reflected in the binding properties of rho; each dimer has a strong and a weak binding site for both the ATP substrate and the RNA cofactor. Here we use homopolynucleotides in competition and complementation experiments to characterize the ATPase activation properties of the cofactor binding sites of the functional rho dimer. We show that (i) no ATPase activity is observed unless both the high- and the low-affinity cofactor binding sites of the functional rho dimer are occupied; (ii) saturating levels of poly(rC), poly(rC) in combination with poly(rU), or poly(rU) alone can fully activate the ATPase of rho; and (iii) poly(dC) can serve as a fully competitive inhibitor of half of the ATPase activity of rho when one of the cofactor sites is filled with poly(rC). These observations lead to a set of phenomenological rules that describe the cofactor dependence of the ATPase activation of the functional dimer of rho and help to define a mechanistic basis for interpreting rho function in termination.
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PMID:ATPase activity of transcription-termination factor rho: functional dimer model. 143 33

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.
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PMID:A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. 146 25

Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.
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PMID:Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover. 171 28

RNA-dependent ATPase activity of Rho + and two mutant proteins Rho15 and Rho301 was studied. It was shown that monomeric Rho forms oligomers in the presence of ATP. This ATP-induced structural change of Rho allows protection of the protein from heat inactivation. Poly(C), which highly activates Rho ATPase, was found to potentiate heat inactivation of Rho301, but no Rho + and Rho15, only under optimal conditions of ATP hydrolysis. It was also shown that Rho301 is defective in interaction with RNA. The molecular model postulating that Rho-catalysed ATP hydrolysis with free RNA involves the cyclic process of protein dissociation and reassembly is postulated.
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PMID:Free RNA-dependent ATPase activity of transcription termination factor Rho: a model of cyclic dissociation and reassembly of Rho protein. 242 24

The domain structure of rho protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods. The single cysteine at position 202, previously thought to be essential for rho function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities. A 151-residue amino-terminal fragment (N1), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDod-SO4/polyacrylamide gels and renatured. The N1 fragment binds poly(C) and mRNA corresponding to the rho-dependent terminator sequence trp t', but not RNA unrecognized by rho; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein. Uncleaved rho renatures to regain its RNA-dependent ATPase activity, but neither N1 nor N2 exhibits any detectable ATP hydrolysis. Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP. Sequence homology to the alpha subunit of the E. coli F1 membrane ATPase, and to consensus elements of other nucleotide-binding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164. The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination.
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PMID:Structure of rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains. 245 28

Eukaryotic initiation factors (eIF)-4A, -4B, and -4F isolated from wheat germ were tested for their ability to catalyze ATP hydrolysis in the absence and presence of RNA. We find that eIF-4B or eIF-4F alone exhibit ATPase activity in the presence of poly(U), satellite tobacco necrosis virus (STNV) RNA, or globin mRNA but not in the absence of RNA. eIF-4A alone exhibits ATPase activity in the absence of RNA, but this activity is not stimulated by the addition of RNA. eIF-4A does, however, enhance RNA-dependent ATP hydrolysis in the presence of either eIF-4B or eIF-4F. The RNA-dependent ATPase activities of eIF-4B and eIF-4F are additive, not synergistic. At saturating concentrations of eIF-4F, little or no stimulation of ATP hydrolysis is obtained upon the addition of eIF-4B and at saturating concentrations of eIF-4B little or no stimulation is obtained upon the addition of eIF-4F. This observation is in agreement with our previous finding (Lax, S., Fritz, W., Browning, K., and Ravel, J. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 330-333) that initiation of polypeptide synthesis is obtained in vitro with either eIF-4F or eIF-4B.
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PMID:ATPase activities of wheat germ initiation factors 4A, 4B, and 4F. 294 76


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