RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations.

Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate. The extracellular ribonuclease from Bacillus intermedius (binase, RNase Bi) shares a common mechanism for RNA hydrolysis with mammalian RNases. Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage.

Ribonuclease A mutant His119 Asn: the role of histidine in catalysis.

Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies. The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates. Three amino acid residues located at the active site of RNase A (His12, His119, and Lys41) are known to be involved in catalysis.

Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase).

Members of the microbial guanyl-specific ribonuclease family show a high level of structural homology. The structural basis for guanyl base binding by microbial ribonucleases has been established for all members of the family and the existence of a guanine recognition loop was shown. However, bacillar RNases such as binase and barnase show far less specificity towards the guanyl base in hydrolysing oligonucleotides composed of more than 4 or 5 nucleotides.

An efficient system for active bovine pancreatic ribonuclease expression in Escherichia coli.

Bovine pancreatic ribonuclease (RNase A) is a member of a homologous group of extensively studied proteins. It is a small, basic protein, containing 124 amino acid residues and four stabilizing disulfide bridges. Ribonuclease A catalyzes the hydrolysis of the phosphodiester bonds in ribonucleic acids.

Formation and properties of S-protein complex with S-peptide-containing fusion protein.

A fusion protein (FP) comprised of the RNase A S-peptide and human epidermal growth factor was shown to form a stable noncovalent catalytically active complex with the RNase A S-protein at a stoichiometric ratio 1:1 with Kdiss = 5.0 x 10(-7) M. The S-protein complex with FP exhibits the pyrimidine specificity toward substrates in both reactions catalyzed by RNase S, transesterification and hydrolysis.

Biological activities of human interferon and 2'-5' oligoadenylates in plants.

Exogenous human interferon 2 (IFN) and 2'-5' oligoadenylates (2-5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1-1 u/ml and 2-5A at 1-10 nM.

Intramolecular interactions in pancreatic ribonucleases.

A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters in pancreatic ribonuclease A (RNase A) has been carried out. Distance calculations for all noncovalently bonded atoms revealed that the average number of nonpolar contacts between a side chain of an amino acid and its neighbors is substantially larger if it involves hydrophobic residues rather than nonhydrophobic ones. However, the difference decreased when the number of contacts per nonpolar group and/or atom were calculated.

Analysis of non-polar regions in proteins.

A new method for finding hydrophobic nuclei and microclusters in protein structure is proposed. The method uses simple and clear-cut criteria based on an analysis of distances between the hydrocarbon groups of all residues. A detailed analysis of the composition and properties of hydrophobic nucleic and microclusters for proteins of different types has been carried out.

A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases.

The rates of enzymatic hydrolysis of 2',5'-oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3'-terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2'-5')adenylyl(2'-5')adenosine.

A new scheme for the synthesis of 5'-nucleotide phosphonate analogs.

A convenient and general method is proposed for the synthesis of 5'-nucleotide phosphonate analogs starting from 5-deoxy-1,2-O-isopropylidene-alpha-D-xylo-hexofuranose.

A new synthesis of 2'-C-methylnucleosides starting from D-ribose.

A general method have been developed for the synthesis of 2'-C-methylnucleosides from 2,3-O-isopropylidene-2-hydroxymethyl-5-O-trityl-D-ribofuranose (2).

The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction.

Regulation of poly(ADP-ribose) transferase activity by 2',5'-oligoadenylates.

pppA2'pA2'pA appears to be a potent natural noncompetitive inhibitor of poly (ADP-ribose) transferase activity in the histone dependent reaction of ADP-ribosylation with Ki=5 microM. Moreover, it is a noncompetitive inhibitor of the Mg2+ dependent reaction of autoADPRT-ribosylation with Ki=20 microM. The activity of ADPRT falls down abruptly both in the cytoplasm and nuclei of mouse L-cells treated with interferon.