Any feedback?
Literature summary for 3.1.26.3 extracted from
- Mechanism of ribonuclease III catalytic regulation by serine phosphorylation (2016), Sci. Rep., 6, 25448 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)recA,rnc105 | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| S195A/S198A | site-directed mutagenesis, the mutant shows a slightly reduced phosphorylation level compared to wild-type | Escherichia coli |
| S33E/S34E | site-directed mutagenesis of phosphorylation sites, molecular dynamic simulations of the S33E/S34E double mutant, which formally provides the same double-negative charge as a single S33 or S34 phosphomonoester, indicate that an additional acidic residue at position 34 does not provide a stabilized interaction with R95. In contrast to the bidentate pS33-R95 side chain interaction, the observed salt bridge consists of a monodentate engagement of R95 with the E33 side chain, and no involvement of the E34 side chain. The S33E/S34E mutant shows abolished phosphorylation and cleaves R1.1 RNA with an efficiency comparable to, but not greater than unphosphorylated RNase III. The S33A/S34A double mutant is essentially fully resistant to phosphorylation | Escherichia coli |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.000163 | - |
R1.1 RNA | recombinant phosphorylated enzyme, pH 8.0, 37°C | Escherichia coli | |
| 0.000239 | - |
R1.1 RNA | recombinant non-phosphorylated enzyme, pH 8.0, 37°C | Escherichia coli |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | essential for catalysis, two Mg2+ ions in the catalytic site, and a third Mg2+ ion, which has been proposed to be involved in product release, binding structure, overview | Escherichia coli |  |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | P0A7Y0 | - |
- |
Posttranslational Modification
| Posttranslational Modification | Comment | Organism |
|---|---|---|
| phosphoprotein | Ec-RNase III is phosphorylated on serine in bacteriophage T7-infected cells, Ec-RNase III is phosphorylated on Ser33 and Ser34 in the N-terminal catalytic domain in vitro by purified serine/threonine-specific protein phosphotransferase activity (T7PK) targeting Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5fold increase in kcat and a 1.4fold decrease in Km following phosphorylation, providing a 7.4fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester. T7PK phosphorylates serine in the alpha2-alpha3 loop of the RNase III catalytic domain | Escherichia coli |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)recA,rnc105 by nickel affinity chromatography, the tag is cleaved off by thrombin | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | ribonuclease III site-specifically cleaves double-stranded(ds) structures in diverse cellular, plasmid and phage RNAs. The catalytic sites employ Mg2+ ions to hydrolyze phosphodiesters, providing products with two-nucleotide, 3'-overhangs and 5'-phosphomonoester, 3'-hydroxyl termini | Escherichia coli | ? | - |
? | |
| R1.1 RNA + H2O | internally 32P-labeled R1.1 | Escherichia coli | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | the RNase III polypeptide (~220 amino acids) consists of an N-terminal catalytic domain [RIIID, about 150 amino acids (aa)] and a C-terminal dsRNA-binding domain (dsRBD, about 65 aa) joined by a short (10 aa) flexible linker. The active form of the enzyme is a homodimer, with a functionally independent catalytic site in each subunit and two dsRBDs that assist in substrate binding | Escherichia coli |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| Ec-RNase III | - |
Escherichia coli |
| ribonuclease III | - |
Escherichia coli |
| RNase III | - |
Escherichia coli |
| rnc | - |
Escherichia coli |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Escherichia coli |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0057 | - |
R1.1 RNA | recombinant non-phosphorylated enzyme, pH 8.0, 37°C | Escherichia coli | |
| 0.029 | - |
R1.1 RNA | recombinant phosphorylated enzyme, pH 8.0, 37°C | Escherichia coli |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Escherichia coli |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | Ec-RNase III homology-modeled structure in complex with cleaved dsRNA, overview | Escherichia coli |
| physiological function | ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. Primary substrate for RNase III is the 5500 nt transcript of the rRNA operons, containing the 16S, 23S and 5S rRNAs, with the enzyme acting co-transcriptionally to provide the immediate precursors to the mature rRNAs8. RNase III also can determine mRNA half-life by catalyzing the rate-limiting cleavage step in the decay pathway. Double-helical structures that are formed by binding of small noncoding RNAs (sRNAs) provide RNase III targets, and regulate mRNA translation and/or stability. The diversity of RNase III targets and the multiple actions of the enzyme in conjunction with sRNAs and other factors underscore the global regulatory function of RNase III | Escherichia coli |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | the catalytic enhancement reflects a specific requirement for a phosphomonoester group in the alpha2-alpha3 phospholoop, rather than negative charge per se | Escherichia coli | |
| 23.85 | - |
R1.1 RNA | recombinant non-phosphorylated enzyme, pH 8.0, 37°C | Escherichia coli | |
| 177.9 | - |
R1.1 RNA | recombinant phosphorylated enzyme, pH 8.0, 37°C | Escherichia coli |
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
