Application | Comment | Organism |
---|---|---|
drug development | an active recombinant His6-tagged HBV RNaseH is suitable for low-throughput drug screening and can be used to discover enzyme inhibitors, many of which work against viral replication in cells | Hepatitis B virus |
Cloned (Comment) | Organism |
---|---|
HBV RNaseH sequences from genotype C isolate V01460 encoding residues corresponding to amino acids 688 to 844 of the HBV polymerase (reference strain ADW2, genotype A) are cloned by gene synthesis into pMAL-c5X-His with sequences encoding a His6-tag appended to the 3' end of the RNaseH gene to create pMal-HRHgtC. Sequences encoding Ala-Gly-Ala are inserted between the maltose binding protein (MBP) and HBV RNaseH sequences, and sequences encoding Gly-Ala-Gly are inserted between sequences for the RNaseH and the His6-tag. Recombinant expression of wild-type and mutant His6-tagged enzymes in Escherichia coli strain BL21(DE3) | Hepatitis B virus |
Protein Variants | Comment | Organism |
---|---|---|
D702A/E731A | sequences encoding HBV RNaseH residues 809-844 are deleted from pMal-HRHgtC to create pMal-HRHgtCDELTA5. Active site residues D702 and E731 are mutated to alanines to create pMAL-HRHgtC(D702A/E731A) which encodes an inactive RNaseH | Hepatitis B virus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required, four RNaseH active site conserved carboxylates (the DEDD motif) coordinate two divalent cations, usually Mg2+ | Hepatitis B virus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Hepatitis B virus | - |
HBV, genotype C isolate V01460 | - |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) cell 54000 x g supernatant by nickel affinity chromatography and dialysis. Purification in the presence of ATP and Mg2+ greatly increases the specific activity of the RNaseH in an DNA oligonucleotide-directed RNA cleavage assay | Hepatitis B virus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | DNA oligonucleotide (ODN)-directed RNA cleavage assays are conducted. Substrate specificity of the HBV RNaseH, overview. HBV RNaseH activity requires an DNA:RNA heteroduplex of 14 bp or more for efficient cleavage. The full-length HBV RNaseH efficiently cleaves the RNA when the ODN ranges from 20-14 nt, but little to no cleavage is observed with the 13mer. HBV RNaseH cannot cut RNA:RNA duplexes. The HBV RNaseH enzyme also shows 3'-5' exoribonuclease activity, Ec 3.1.13.2 | Hepatitis B virus | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
More | cf. EC 3.1.13.2 | Hepatitis B virus |
ribonuclease H | - |
Hepatitis B virus |
RNaseH | - |
Hepatitis B virus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
42 | - |
assay at | Hepatitis B virus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Hepatitis B virus |
General Information | Comment | Organism |
---|---|---|
evolution | RNaseH enzymes belong to the nucleotidyl transferase superfamily whose members share a similar protein fold and catalytic mechanism | Hepatitis B virus |
additional information | the RNaseH active site contains four conserved carboxylates (the DEDD motif) that coordinate two divalent cations, usually Mg2+. The RNA cleavage mechanism requires both cations to promote a hydroxyl-mediated nucleophilic scission reaction | Hepatitis B virus |
physiological function | the endonucleolytic RNaseH activity (EC 3.1.26.4) requires an DNA:RNA duplex 14 nt or more and cannot tolerate a stem-loop in either the RNA or DNA strands. It tolerates a nick in the DNA strand but not a gap. The RNaseH has no obvious sequence specificity or positional dependence within the RNA, and it cuts the RNA at multiple positions even within the minimal 14 nt duplex. The RNaseH also possesses a processive 3'-5' exoribonuclease activity (EC 3.1.13.2) that is slower than the endonucleolytic reaction. The HBV reverse transcription mechanism features an initial endoribonucleolytic cut, 3'-5' degradation of RNA, and a sequence-independent terminal RNA cleavage | Hepatitis B virus |