Cloned (Comment) | Organism |
---|---|
recombinant expression of both enzyme subunits, N-terminally His-tagged p66 and p51 subunit mutants C282S of HIV-1, in Escherichia coli strain BL21 Rosetta | Human immunodeficiency virus 1 |
Protein Variants | Comment | Organism |
---|---|---|
C282S | site-directed mutagenesis, the mutation avoids non-specific cross-linking in both subunits, p66 and p51 subunits of HIV-1 | Human immunodeficiency virus 1 |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
heparin | inhibits substrate binding by the enzyme, in the samples that are pretreated with DTT, the addition of heparin strongly inhibits the reaction and completely eliminates off-register cleavage | Human immunodeficiency virus 1 | |
NaCl | inhibits substrate binding by the enzyme, in the samples that are pretreated with DTT, the addition of salt strongly inhibits the reaction and completely eliminates off-register cleavage | Human immunodeficiency virus 1 |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Human immunodeficiency virus 1 |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Human immunodeficiency virus 1 | - |
HIV-1 | - |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His-tagged p66 and p51 subunit mutants C282S from Escherichia coli strain BL21 Rosetta by nickel affinity chromatography, dialysis, heparin affinity chromatography, and gel filtration | Human immunodeficiency virus 1 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | when the RNA/DNA hybrid is immobilized at the polymerase active site, RNase H cleavage occurs, experimentally verifying that the substrate can simultaneously interact with both active sites, analysis of the mechanism of the coordination of the two activities. Cross-linking of HIV-1 RT with RNA/DNA hybrids, inhibition of complex formation by salt and heparin. Purified HIV-1 RT D186C, M184C and Q258C variants are tethered to appropriately modified CL3 substrates, and RNase H activity within the cross-linked complexes is determined, overview | Human immunodeficiency virus 1 | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
RNase H | - |
Human immunodeficiency virus 1 |
RNase H activity of HIV-1 reverse transcriptase | - |
Human immunodeficiency virus 1 |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
RNase H assay at | Human immunodeficiency virus 1 |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
enzyme domain-substrate cross-linking assay at | Human immunodeficiency virus 1 |
8 | - |
RNase H assay at | Human immunodeficiency virus 1 |
General Information | Comment | Organism |
---|---|---|
additional information | molecular dynamics simulations and determination of the conformation of the complex in which the unwound RNA/DNA substrate simultaneously interacts with the polymerase and RNase H active sites. Existence of a transient conformation of the HIV-1 by reverse transcriptase (RT) substrate complex, which is important for modulating and coordinating the enzymatic activities of HIV-1 RT | Human immunodeficiency virus 1 |
physiological function | replication of human immunodeficiency virus 1 (HIV-1) involves conversion of its single-stranded RNA genome to double-stranded DNA, which is integrated into the genome of the host. This conversion is catalyzed by reverse transcriptase (RT), which possesses DNA polymerase and RNase H domains. When the RNA/DNA hybrid is immobilized at the polymerase active site, RNase H cleavage occurs, experimentally verifying that the substrate can simultaneously interact with both active sites | Human immunodeficiency virus 1 |