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1.
Cleavage of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA-RNA junction with prokaryotic RNases HII 总被引:1,自引:0,他引:1
We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA-RNA-DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5'-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA-RNA junction (5'-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA. 相似文献
2.
Use of specific endonuclease cleavage in RNA sequencing-an enzymic method for distinguishing between cytidine and uridine residues. 总被引:7,自引:3,他引:4
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The extracellular ribonuclease I of the common slime mold physarum polycephalum (RNase Phy1), which has recently been purified to homogeneity, has been used to distinguish between C and U residues in 3'-end-labeled oligoribonucleotides. As shown by Bargetzi and coworkers, this enzyme exhibits strong cleavage preference for U-N over C-N and N-C over N-U bonds. In the present paper, conditions are being detailed, which enable one to deduce the sequences of rather large, pyrimidine-rich, terminally labeled oligonucleotides by partial digestion with RNases U2, A, and Phy1, followed by resolution of the cleavage products by size. The techniques described in this and a previous communication provide a direct means for identifying A, G, C, and U residues in end-labeled polyribonucleotides. 相似文献
3.
In order to investigate the effects of mutation of Gln32, a component of a base recognition site (B2 site) of a base-nonspecific RNase from Rhizopus niveus, we prepared several enzymes mutant at this position, Q32F, Q32L, Q32V, Q32T, Q32D, Q32N, and Q32E, and their enymatic activities toward RNA and 16 dinucleoside phosphates were measured. Enzymatic activities of the mutant enzymes towards RNA were between 10-125% of the native enzyme. From the rates of hydrolysis of 16 dinucleoside phosphates by mutant enzymes, we estimated the base specificity of both B1 and B2 sites. The results indicated that mutation of Gln32 to Asp, Asn, and Glu caused the B2 site to prefer cytosine more and to a less extent, to prefer uracil (Q32N), and that Q32F made the enzyme more guanine-base preferential. The results suggested that we are able to construct an enzyme that preferentially cleaves internucleotidic linkages, at the 5'-side of cytosine residues (Q32D, Q32N, and Q32E) and guanine residues (Q32F and Q32T), thus, cleaves purine-C(Q32D, Q32N, Q32E) and GpG and ApG (Q32F, and Q32T) most easily. The results seemed to suggest converting a base-non-specific RNase to a base-specific one. 相似文献
4.
V Both G P Moiseyev J Sevcik 《Biochemical and biophysical research communications》1991,177(2):630-635
Kinetic parameters kcat and KM were measured for cleavage of poly I, poly A, poly U, poly C and poly I poly C by guanyl-specific RNases Sa, Pb1 and T1 and compared with that of guanyl-preferential RNase Bi. Catalytic efficiencies of the investigated enzymes to polynucleotide substrates vary considerably. The structural basis for specificity of these RNases is discussed. A hypothesis is suggested that Ser-56 plays an important role in recognition of poly A by RNase Bi. 相似文献
5.
《Bioscience, biotechnology, and biochemistry》2013,77(8):1604-1608
Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate. The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom. RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2. In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified. Since RNase Le2, which has very similar N-terminal sequence to RNase Le 37 and RNase Le 45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism. 相似文献
6.
Suzuki R Kanno S Ogawa Y Iwama M Tsuji T Ohgi K Irie M 《Bioscience, biotechnology, and biochemistry》2005,69(2):343-352
A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS-PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes. 相似文献
7.
Tadao Kamikawa Fujito Higuchi Makoto Taniguchi Yukihiro Asaka 《Bioscience, biotechnology, and biochemistry》2013,77(3):691-692
Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH4)2SO4 fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach. 相似文献
8.
Nathalie Mathy Agnès Hébert Peggy Mervelet Lionel Bénard Audrey Dorléans Inés Li de la Sierra‐Gallay Philippe Noirot Harald Putzer Ciarán Condon 《Molecular microbiology》2010,75(2):489-498
Ribonucleases J1 and J2 are recently discovered enzymes with dual 5′‐to‐3′ exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild‐type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5′‐to‐3′ exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication. 相似文献
9.
Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively. 相似文献
10.
Human non-secretory ribonucleases. I. Purification, peptide mapping and lectin blotting analysis of the kidney, liver and spleen enzymes 总被引:2,自引:1,他引:1
Lawrence Cris W.; Little Pamela A.; Little Brian W.; Miller Marsha J.; Bazel Shamideh; Alhadeff Jack A. 《Glycobiology》1993,3(3):241-248
Human non-secretory neutral ribonucleases (RNases) from kidney,liver and spleen have been purified and characterized. SDSPAGEindicates that all three RNases are highly purified and haveapparent mol. wts of 1718 kDa. Kinetic analysis indicatesthat all three RNases have a broad pH optimum centred around6.5, and all three have similar substrate specificities withsignificant preference for RNA and poly(U) when compared topoly(C), poly (A) and poly(G). All of the above data, as wellas immunoblotting data using three polyclonal antibodies (anti-humanliver RNase, anti-human pancreatic RNase, anti-human eosino-phil-derivedneurotoxin), indicate that the three proteins are highly purifiedand are non-secretory RNases (IIN). Further characterizationby cyanogen bromide peptide mapping and extensive lectin blottingindicated no significant differences between the three humanRNases. All three RNases appear to have very similar, if notidentical, protein backbones and all three are glycoproteinswhich are recognized by lectins with specificity for GlcNAc,Fuc and, to a lesser extent, with specificity for Galß(14)GlcNAc.No significant tissuespecific differences were found among thethree human non-secretory RNases. lectin blotting non-secretory RNases peptide mapping 相似文献
11.
Replacement and insertion of nucleotides at the anticodon loop of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides. 总被引:1,自引:1,他引:0
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Insertion of the four major nucleotides at the 5'-side of the anticodon triplet of E. coli tRNAMetf was performed by joining of the half molecules obtained by limited digestion with RNase A and the chemically synthesized tetranucleotide pN-C-A-U using RNA ligase. Insertion of U-U at the 5'-side or A and A-A at the 3'-side of the anticodon were also performed using U-U-C-A-U, C-A-U-A and C-A-U-A-A. The constant U next to the 5'-side of the anticodon was replaced with A and C by ligation of A-C-A-U and C-C-A-U to the 5'-half molecule which had been treated with periodate plus lysine, followed by joining to the 3'-half. These modified tRNAs were tested for their ability to accept methionine with the methionyl-tRNA synthetase of E. coli. The affinity of these analogs for the synthetase decreased more extensively when the insertion was at the 3'-side of the anticodon triplet. Insertion of mononucleotides at the 5'-side or replacement of the constant U next to the 5'-side of the anticodon did not affect aminoacylation drastically. This may mean that the 3'-side of the anticodon loop of tRNA is one of the major recognition sites for the methionyl-tRNA synthetase. 相似文献
12.
Anna Perederina Olga Esakova Chao Quan Elena Khanova Andrey S Krasilnikov 《The EMBO journal》2010,29(4):761-769
Ribonuclease (RNase) P is a site‐specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix‐loop‐helix protein‐binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 Å. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family. 相似文献
13.
Nonradioactive RNA fragments may be sequenced by incorporation of (3H)-label into 3'-terminal positions, controlled digestion with specific ribonucleases, and separation according to size of the digestion products on polyethyleneimine- (PEI-) cellulose thin layers. This combination of techniques allows one to measure accurately distances of specific cleavage sites from the labeled terminal positions. The cleavage specificities of RNases T1, U2, and A are utilized to identify the positions of G, A, and pyrimidine residues respectively. C and U may be distinguished by mobility differences on PEI-cellulose thin layers at ph 2.6. The procedure is simple, rapid, and highly sensitive; as little as 0.5 - 1 microgram of a RNA of the size of tRNA will be needed to sequence all fragments in a complete RNase digest. 相似文献
14.
N. I. Menzorova Ju. T. Sibirtsev V. A. Rasskazov 《Applied Biochemistry and Microbiology》2009,45(4):369-373
Three enzyme preparations, two acid and one alkaline RNases, were isolated from the hepatopancreas of the red king crab Paralithodes camtschatica using DEAE-cellulose chromatography and gel chromatography. The alkaline RNase was activated by Mg2+ ions and had a pH optimum of 7.2; the acid RNases, a pH optimum of 5.5. The molecular weight of the alkaline RNase was 19 kDa; two acid RNases, 33 and 70 kDa, respectively. The enzymes exhibited a sufficiently high thermostability (IT50 = 53–55°C) and were strongly inhibited by NaCl (IC50, 0.1–0.25 M). The alkaline RNase exhibited no specificity for heterocyclic bases, whereas the acid RNases hydrolyzed poly(U) and poly(A) at maximum rates. 相似文献
15.
16.
Three ribonucleases (RNases) with different molecular masses were isolated from human kidney. The enzymes were purified to an electrophoretically homogeneous state, and their respective molecular masses were found to be 18,000 (tentatively named RNase HK-1), 20,000 (RNase HK-2A), and 22,000 (RNase HK-2B) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the amino acid compositions, amino-terminal sequences, and enzymological properties of the enzymes indicate that RNase HK-1 is related to "nonsecretory" RNase, and that RNases HK-2A and HK-2B are both related to "secretory" RNase. Furthermore, RNase HK-1 showed cross-reactivity with an antibody specific to nonsecretory RNase from human urine, whereas RNases HK-2A and HK-2B showed cross-reactivity with another antibody specific to human urine secretory RNase. However, the carbohydrate compositions of RNases HK-2A and HK-2B were markedly different from that of the secretory urine RNase. This finding seems to indicate that the kidney is not the origin of the urine enzyme. 相似文献
17.
The level of biosynthesis of secreted guanyl-specific ribonucleases (RNases) of Bacillus intermedius (binases) and Bacillus circulans (RNases Bci) by recombinant B. subtilis strains increases under nitrogen starvation. The promoter of the binase gene carries the sequences homologous to the recognition sites of the regulatory protein TnrA, which regulates gene expression under growth limitation by nitrogen. Using the B. subtilis strain defective in protein TnrA, it has been shown that the regulatory protein TnrA is involved in the regulation of expression of the binase gene and the gene of RNase Bci. The TnrA regulation of expression of the RNase Bci gene is indirect, probably by means of the regulatory protein PucR. Thus, it has been established that at least two regulatory mechanisms activate the expression of the genes encoding the secreted RNases of spore-forming bacteria: a system of proteins homologous to the B. subtilis PhoP-PhoR, and regulation by a protein similar to the B. subtilis TnrA regulatory protein. 相似文献
18.
Nathan L. Ponzar Razia Tajwar Nicola Pozzi John E. Tavis 《The Journal of biological chemistry》2022,298(4)
The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds’ reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme–substrate–inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes. 相似文献
19.
Stacking of Crick Wobble pair and Watson-Crick pair: stability rules of G-U pairs at ends of helical stems in tRNAs and the relation to codon-anticodon Wobble interaction. 总被引:5,自引:2,他引:3
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The occurrence of the noncomplementary G-U base pair at the end of a helix is found to be governed by stacking interactions. As a rule, a G-U pair with G on the 5'-side of a Watson-Crick base pair exhibits strikingly greater stacking overlap with the Watson-Crick base pair than a G-U pair on the 3'-side of a Watson-Crick base pair. The former arrangement is expected to be more stable and indeed is observed 29 times out of 32 in the known transfer RNA molecules. In accordance with this rule, the major wobble base pairs G-U or I-U in codon-anticodon interactions have G or I on the 5'-side of the anticodon. Similarly, in initiator tRNAs, this rule is obeyed where now the G is the first letter of the codon (5'-side). In the situation where U is in the wobble position of the anticodon, it is usually substituted at C(5) andmay also have a 2-thio group and it can read one to four codons depending on its modifications. A G at the wobble position of the anticodon can recognize the two codons ending with U or C and modification of G (unless it is I) does not change its reading properties. 相似文献
20.
Devos JM Tomanicek SJ Jones CE Nossal NG Mueser TC 《The Journal of biological chemistry》2007,282(43):31713-31724
Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes. 相似文献