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:3.1.27.3 (RNase T1)
1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Single amino acid residue substitutions rarely destroy the structural integrity of proteins. Substitution of glycine residues, however, is among the few sorts of alterations that can have such an effect. Here, we seek to understand what accounts for the extreme functional impairment of the bacterial ribonuclease barnase upon substitution of Gly52 or Gly53. We find that inactivation is caused by overall disruption of the folded state that manifests itself in three ways: (1) dramatically reduced stability (by 5.2 to 8.4 kcal mol-1 for mutants showing inactivation in vivo); (2) progressive loss of folded-state activity with increasing temperature, indicating a less well formed fold; and (3) substantial proteolytic degradation of mutant enzymes in vivo. Examination of two deletion mutants, missing either Gly53 or Asp54, shows that the irregular beta-bulge formed by these two residues is of vital importance to the structural integrity of barnase. The parallel behaviour of mutants carrying replacements of either of the two glycine residues therefore appears to arise from a common mechanism: disruption of local structure at the beta-bulge. The importance of this structural element to the function of barnase raises the question of whether it may be present in other RNases. The Streptomyces enzymes RNase Sa and RNase St differ considerably from barnase in both sequence and structure, yet both show significant sequence similarity to barnase over a region beginning at Gly53. Structural comparison indicates that the Streptomyces enzymes do have the barnase-like irregular beta-bulge, making this an important characteristic feature of a group of bacterial ribonucleases. The sensitivity of this feature demonstrates that detailed aspects of local structure may have a major role in determining the overall structural and functional properties of an enzyme, even where no explanation for this role is readily apparent. If this is a general characteristic of the structure-function relationship, it may pose a formidable obstacle to the de novo design of new enzymes.
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PMID:An irregular beta-bulge common to a group of bacterial RNases is an important determinant of stability and function in barnase. 1006 10

Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate.
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PMID:[Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells]. 1019 Jan 4

Promoters of the genes for guanyl-specific ribonucleases, secreted by B. intermedius (binase) and B. pumilus (Rnase Bp) in phosphate deficient conditions, contain regions similar to appropriate consensus sequences in promoters of the PHO regulated genes of B. subtilis. A number of genes expressed in response to phosphate starvation in B. subtilis are regulated by the two component signal transduction system PhoP-PhoR. Expression of recombinant genes for binase and RNase Bp in B. subtilis strains with mutations in the regulatory protein genes of the PHO regulon was studied. Their expression is strongly regulated by the regulatory proteins of the B. subtilis PHO regulon.
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PMID:Expression of the genes for guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus is regulated by the two component signal transduction system PhoP-PhoR in B. subtilis. 1022 Aug 98

Promoters of the genes of guanyl-specific ribonucleases of Bacillus intermedius (binase) and Bacillus pumilus (RNase Bp) were found to contain sequences homologous to those recognizable by the regulatory protein PhoP in the promoters of the PHO regulon of B. subtilis, as well as regions partially homologous to the binding sites of another regulatory protein, PhoB, in the promoters of the PHO regulon of Escherichia coli. The role of the two-component regulatory systems PhoP-PhoR and PhoB-PhoR in the regulation of expression of the genes of binase and RNase Bp in recombinant strains of B. subtilis and E. coli was studied by using mutant strains. It was established that the expression of these genes in recombinant B. subtilis cells is stringently controlled by the PhoP-PhoR two-component regulatory system, whereas the expression of these genes in E. coli cells is not controlled by the regulatory proteins PhoB or PhoR. Presumably, regulatory systems of the response to phosphate starvation, analogous to the PHO regulon of B. subtilis, also function in other representatives of the genus Bacillus.
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PMID:[Expression of genes for guanyl-specific ribonucleases in Bacillus intermedius and Bacillus pumilus is regulated by the PhoP-PhoR two-component signal transduction system of PHO regulon of Bacillus subtilis]. 1049 72

A targeted RNase would be ideal for gene therapy of several acquired and inherited disorders. Such an RNase may be engineered to contain a ribonucleolytic domain and a specific target RNA binding domain. To demonstrate the feasibility of this approach, an RNase targeted against human immunodeficiency virus (HIV) RNA--Tev-RNase T1--was designed and tested for its use in HIV-1 gene therapy. A human CD4+ T lymphoid (MT4) cell line and human peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors lacking or expressing the tevT1 gene. Expression of enzymatically functional Tev-RNase T1 protein and its lack of toxicity was demonstrated in stable MT4 transductants. Compared with control cells lacking this protein, both transduced MT4 cells and PBLs expressing Tev-RNase T1 delayed HIV-1 replication. Tev-RNase T1 was shown to act after integration, since HIV-1 proviral DNA could be detected, but the amount of HIV-1 RNA produced in MT4 cells and PBLs was significantly decreased. This study demonstrates the feasibility of a targeted RNase strategy for therapeutic use.
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PMID:Targeted RNases: a feasibility study for use in HIV gene therapy. 1050 17

RNase Sa3 produced by Streptomyces aureofaciens strain CCM 3239 belongs to the T1 family of microbial ribonucleases. It is closely related both to RNase Sa, studied in detail earlier, and to RNase Sa2 produced by the same microorganism. The most important property of RNase Sa3 is the relatively high cytotoxic activity, which was not observed for RNase Sa and Sa2. Recombinant RNase Sa3 was overexpressed in Escherichia coli and purified to high homogeneity. The hanging-drop vapour-diffusion method was used for crystallization. The two crystal forms are trigonal P3(1)21 and tetragonal P4(1)2(1)2, with unit-cell parameters a = b = 64.7, c = 69.6 A, gamma = 120 degrees and a = b = 34.0, c = 147.2 A, respectively. They diffract to 2.0 and to 1.7 A resolution, respectively, using synchrotron radiation. The asymmetric units of crystal forms I and II contain one molecule of the enzyme, which corresponds to V(M) = 3.8 A(3) Da(-1) with a solvent content of 68% and V(M) = 1.9 A(3) Da(-1) with a solvent content of 37%, respectively.
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PMID:Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3. 1132 Mar 22

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.
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PMID:Solution structure and dynamics of ribonuclease Sa. 1145 93

Mutation detection based on ribonuclease cleavage of basepair mismatches in single-stranded RNA probes hybridized to DNA targets was first described over 15 years ago. The original methods relied on RNase A for mismatch cleavage; however, this enzyme fails to cleave many mismatches and has other drawbacks. More recently, a new method for RNase-cleavage-based mutation scanning has been developed, which takes advantage of the ability of RNase 1 and RNase T1 to cleave mismatches in duplex RNA targets, when these enzymes are used in conjunction with nucleic acid intercalating dyes. The method, called NIRCA, is relatively low-cost in terms of materials and equipment required. It is being used to detect mutations and SNPs in a wide variety of genes involved in human genetic disease and cancer, as well as in disease-related viral and bacterial genes. This review describes historical and recently developed RNase cleavage-based methods for mutation/SNP scanning.
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PMID:RNase cleavage-based methods for mutation/SNP detection, past and present. 1152 30

An RNA sequence showing high stability with respect to digestion by ribonuclease T1 (RNase T1) was isolated by in vitro selection from an RNA library. Although ribonuclease T1 cleaves single-stranded RNA specifically after guanosine residues, secondary structure calculations predict several guanosines in single-stranded areas. Two of these guanosines are part of a GGCA-tetraloop, a recurring structure element in the secondary structure predictions. Molecular dynamics simulations of the conformation space of the nucleotides involved in this tetraloop show on the one hand that the nucleic acid backbone of the guanosines cannot realise the conformation required for cleavage by RNase T1. On the other hand, it could be shown that an RNA molecule not forced into a tetraloop occupies this conformation several times in the course of the simulation. The simulations confirm the GGCA-tetraloop as an RNase-stable secondary structure element. Our results show that, besides the known prerequisite of a single-stranded RNA, RNase T1 has additional demands on the substrate conformation.
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PMID:RNase-stable RNA: conformational parameters of the nucleic acid backbone for binding to RNase T1. 1153 Sep 31

The aim of this study was to gain a better understanding of the contribution of hydrogen bonds by tyrosine -OH groups to protein stability. The amino acid sequences of RNases Sa and Sa3 are 69 % identical and each contains eight Tyr residues with seven at equivalent structural positions. We have measured the stability of the 16 tyrosine to phenylalanine mutants. For two equivalent mutants, the stability increases by 0.3 kcal/mol (RNase Sa Y30F) and 0.5 kcal/mol (RNase Sa3 Y33F) (1 kcal=4.184 kJ). For all of the other mutants, the stability decreases with the greatest decrease being 3.6 kcal/mol for RNase Sa Y52F. Seven of the 16 tyrosine residues form intramolecular hydrogen bonds and the average decrease in stability for these is 2.0(+/-1.0) kcal/mol. For the nine tyrosine residues that do not form intramolecular hydrogen bonds, the average decrease in stability is 0.4(+/-0.6) kcal/mol. Thus, most tyrosine -OH groups contribute favorably to protein stability even if they do not form intramolecular hydrogen bonds. Generally, the stability changes for equivalent positions in the two proteins are remarkably similar. Crystal structures were determined for two of the tyrosine to phenylalanine mutants of RNase Sa: Y80F (1.2 A), and Y86F (1.7 A). The structures are very similar to that of wild-type RNase Sa, and the hydrogen bonding partners of the tyrosine residues always form intermolecular hydrogen bonds to water in the mutants. These results provide further evidence that the hydrogen bonding and van der Waals interactions of polar groups in the tightly packed interior of folded proteins are more favorable than similar interactions with water in the unfolded protein, and that polar group burial makes a substantial contribution to protein stability.
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PMID:Tyrosine hydrogen bonds make a large contribution to protein stability. 1155 95


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