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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A detailed structural and functional model of E. coli RNase T was generated based on sequence analysis, homology modeling, and experimental observation. In the accompanying article, three short sequence segments (nucleic acid binding sequences (NBS)) important for RNase T substrate binding were identified. In the model, these segments cluster to form a positively charged surface patch. However, this patch is on the face of the RNase T monomer opposite the DEDD catalytic center. We propose that by dimerization, the NBS patch from one subunit is brought to the vicinity of the DEDD center of the second monomer to form a fully functional RNase T active site. In support of this model, mutagenetic studies show that one NBS1 residue, Arg(13), sits at the catalytic center despite being on the opposite side of the monomer. Second, the complementarity of the RNase T subunits through the formation of homodimers was demonstrated by reconstitution of partial RNase T activity from monomers derived from two inactive mutant proteins, one defective in catalysis and one in substrate binding. These data explain why RNase T must dimerize to function. The model provides a detailed framework on which to explain the mechanism of action of RNase T.
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PMID:Mechanism of action of RNase T. II. A structural and functional model of the enzyme. 1236 33

Escherichia coli RNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis. Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues. Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases. We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues. Site-directed mutagenesis of these regions indicated that they are involved in substrate binding. Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity. These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T.
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PMID:Mechanism of action of RNase T. I. Identification of residues required for catalysis, substrate binding, and dimerization. 1236 34

In Saccharomyces cerevisiae, a large complex, known as the Ccr4-Not complex, containing two nucleases, is responsible for mRNA deadenylation. One of these nucleases is called Pop2 and has been identified by similarity with PARN, a human poly(A) nuclease. Here, we present the crystal structure of the nuclease domain of Pop2 at 2.3 A resolution. The domain has the fold of the DnaQ family and represents the first structure of an RNase from the DEDD superfamily. Despite the presence of two non-canonical residues in the active site, the domain displays RNase activity on a broad range of RNA substrates. Site-directed mutagenesis of active-site residues demonstrates the intrinsic ability of the Pop2 RNase D domain to digest RNA. This first structure of a nuclease involved in the 3'-5' deadenylation of mRNA in yeast provides information for the understanding of the mechanism by which the Ccr4-Not complex achieves its functions.
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PMID:X-ray structure and activity of the yeast Pop2 protein: a nuclease subunit of the mRNA deadenylase complex. 1461 57

The mouse CAF1 (mCAF1) is an ortholog of the yeast (y) CAF1 protein, which is a component of the CCR4-NOT complex, the major cytoplasmic deadenylase of Saccharomyces cerevisiae. Although CAF1 protein belongs to the DEDDh family of RNases, CCR4 appears to be the principle deadenylase of the CCR4-NOT complex. Here, we present evidence that mCAF1 is a processive, 3'-5'-RNase with a preference for poly(A) substrates. Like CCR4, increased length of RNA substrates converted mCAF1 into a processive enzyme. In contrast to two other DEDD family members, PAN2 and PARN, mCAF1 was not activated either by PAB1 or capped RNA substrates. The rate of deadenylation in vitro by yCCR4 and mCAF1 were both strongly influenced by secondary structures present in sequences adjacent to the poly(A) tail, suggesting that the ability of both enzymes to deadenylate might be affected by the context of the mRNA 3'-untranslated region sequences. The ability of mCAF1 to complement a ycaf1 deletion in yeast, however, did not require the RNase function of mCAF1. Importantly, yCAF1 mutations, which have been shown to block its RNase activity in vitro, did not inactivate yCAF1 in vivo, and mRNAs were deadenylated in vivo at nearly the same rate as found for wild type yCAF1. These results indicate that at least in yeast the CAF1 RNase activity is not required for its in vivo function.
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PMID:Mouse CAF1 can function as a processive deadenylase/3'-5'-exonuclease in vitro but in yeast the deadenylase function of CAF1 is not required for mRNA poly(A) removal. 1504 70

Poly(A)-specific ribonuclease (PARN) is a highly poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN belongs to the DEDD family of nucleases, and four conserved residues are essential for PARN activity, i.e. Asp-28, Glu-30, Asp-292, and Asp-382. Here we have investigated how catalytically important divalent metal ions are coordinated in the active site of PARN. Each of the conserved amino acid residues was substituted with cysteines, and it was found that all four mutants were inactive in the presence of Mg2+. However, in the presence of Mn2+, Zn2+, Co2+, or Cd2+, PARN activity was rescued from the PARN(D28C), PARN(D292C), and PARN(D382C) variants, suggesting that these three amino acids interact with catalytically essential metal ions. It was found that the shortest sufficient substrate for PARN activity was adenosine trinucleotide (A3) in the presence of Mg2+ or Cd2+. Interestingly, adenosine dinucleotide (A) was efficiently hydrolyzed in the presence of Mn2+, Zn2+, or Co2+, suggesting that the substrate length requirement for PARN can be modulated by the identity of the divalent metal ion. Finally, introduction of phosphorothioate modifications into the A substrate demonstrated that the scissile bond non-bridging phosphate oxygen in the pro-R position plays an important role during cleavage, most likely by coordinating a catalytically important divalent metal ion. Based on our data we discuss binding and coordination of divalent metal ions in the active site of PARN.
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PMID:Coordination of divalent metal ions in the active site of poly(A)-specific ribonuclease. 1535 88

Poly(A)-specific ribonuclease (PARN), a member of the DEDD family, is a key enzyme involved in the deadenylation of mRNA in higher eukaryotic cells. In this research, it was found that Mg(2+) could protect PARN against thermal inactivation by increasing the midpoint of inactivation and decreasing the inactivation rate. This protective effect was unique to Mg(2+) in a concentration-dependent manner. However, the thermal unfolding and aggregation was promoted by the addition of Mg(2+) at high temperatures. These results revealed that Mg(2+) might have dual effects on PARN stability: protecting the active site but endangering the overall structural stability.
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PMID:Effect of magnesium ions on the thermal stability of human poly(A)-specific ribonuclease. 1730 97

Chlamydophila pneumoniae AR39 contains two different ORFs (CP0654 and CP0782) encoding ribonuclease H (RNase H) homologues, Cpn-RNase HII and Cpn-RNase HIII. Sequence alignments show that the two homologues both contain the conserved motifs of type 2 RNase H, and Cpn-RNase HII has the conserved active-site motif (DEDD) of RNase HII. Cpn-RNase HIII also contains a unique active-site motif (DEDE), common to other RNase HIIIs. Complementation assays indicated that Cpn-RNase HII can complement both Escherichia coli RNase HII and RNase HI, but Cpn-RNase HIII can only complement the latter. In vitro enzyme activity experiments showed that neither Cpn-RNase HII nor Cpn-RNase HIII is thermostable and their optimum pH values were 9.0 and 10.0, respectively. Cpn-RNase HII cleaves a 12 bp RNA-DNA substrate at multiple sites, but Cpn-RNase HIII at only one site. When a 35 bp DNA-RNA-DNA/DNA chimeric substrate was used, cleavage was only observed with Cpn-RNase HII. These results indicate that the RNase H combination of C. pneumoniae AR39 is not simple substitution of E. coli RNase H, perhaps representing a more primordial type. This is believed to be the first in vivo functional study of Chlamydophila RNase Hs and the results should contribute to the analysis of RNase Hs of other parasite species.
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PMID:Biochemical characterization and functional complementation of ribonuclease HII and ribonuclease HIII from Chlamydophila pneumoniae AR39. 1732 99

The 3' processing of most bacterial precursor tRNAs involves exonucleolytic trimming to yield a mature CCA end. This step is carried out by RNase T, a member of the large DEDD family of exonucleases. We report the crystal structures of RNase T from Escherichia coli and Pseudomonas aeruginosa, which show that this enzyme adopts an opposing dimeric arrangement, with the catalytic DEDD residues from one monomer closely juxtaposed with a large basic patch on the other monomer. This arrangement suggests that RNase T has to be dimeric for substrate specificity, and agrees very well with prior site-directed mutagenesis studies. The dimeric architecture of RNase T is very similar to the arrangement seen in oligoribonuclease, another bacterial DEDD family exoribonuclease. The catalytic residues in these two enzymes are organized very similarly to the catalytic domain of the third DEDD family exoribonuclease in E. coli, RNase D, which is monomeric.
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PMID:Crystal structure of RNase T, an exoribonuclease involved in tRNA maturation and end turnover. 1743 14

The nsp14 protein, an exoribonuclease of the DEDD superfamily encoded by severe acute respiratory syndrome coronavirus (SARS-CoV), was expressed in fusion with different affinity tags. The recombinant nspl4 proteins with either GST fusion or 6-histidine tag were shown to possess ribonuclease activity but nspl4 with a short MGHHHHHHGS tag sequence at the N-terminus increased the solubility of nspl4 protein and facilitated the protein purification. Mutations of the conserved residues of nspl4 resulted in significant attenuation but not abolishment of the ribonuclease activity. Combination of fluorescence and circular dichroism spectroscopy analyses showed that the conformational stability of nsp14 protein varied with many external factors such as pH, temperature and presence of denaturing chemicals. These results provide new information on the structural features and would be helpful for further characterization of this functionally important protein.
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PMID:[Synthesis in Escherichia coli cells and characterization of the active exoribonuclease of severe acute respiratory syndrome coronavirus]. 1954 31

RNA maturation relies on various exonucleases to remove nucleotides successively from the 5' or 3' end of nucleic acids. However, little is known regarding the molecular basis for substrate and cleavage preference of exonucleases. Our biochemical and structural analyses on RNase T-DNA complexes show that the RNase T dimer has an ideal architecture for binding a duplex with a short 3' overhang to produce a digestion product of a duplex with a 2-nucleotide (nt) or 1-nt 3' overhang, depending on the composition of the last base pair in the duplex. A 'C-filter' in RNase T screens out the nucleic acids with 3'-terminal cytosines for hydrolysis by inducing a disruptive conformational change at the active site. Our results reveal the general principles and the working mechanism for the final trimming step made by RNase T in the maturation of stable RNA and pave the way for the understanding of other DEDD family exonucleases.
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PMID:Structural basis for RNA trimming by RNase T in stable RNA 3'-end maturation. 2131 4


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