RNase H
Encyclopedia
The enzyme RNase H is a non-specific endonuclease
and catalyzes the cleavage of RNA
via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from archaea
to bacteria
and eukaryota.
RNase H’s ribonuclease activity cleaves the 3’-O-P bond of RNA
in a DNA
/RNA
duplex to produce 3’-hydroxyl and 5‘-phosphate terminated products. In DNA replication
, RNase H is responsible for removing the RNA primer, allowing completion of the newly synthesized DNA.
is missing one of the helices known as the C-helix, a positively charged α-helix whose protrusive shape increases substrate binding capacity. The active site
of the enzyme is centered around a conserved DEDD motif (composed of residues: D443, E478, D498, and D549) which performs the hydrolysis of the RNA substrate. A magnesium
ion is commonly used as a cofactor
during the hydrolysis
step. It is also a potential but unconfirmed mechanism in which multiple ions are necessary for to perform the hydrolysis. The enzyme also contains a nucleic acid binding cleft about 60 Å in length that can encompass a region of 18 bound RNA/DNA base pairs.
laboratory
, as RNase H specifically degrades the RNA in RNA:DNA hybrids and will not degrade DNA or unhybridized RNA, it is commonly used to destroy the RNA template after first-strand complementary DNA
(cDNA) synthesis by reverse transcription, as well as procedures such as nuclease protection assay
s. RNase H can also be used to degrade specific RNA strands when the cDNA oligo is hybridized, such as the removal of the poly(A) tail from mRNA hybridized to oligo(dT), or the destruction of a chosen non-coding RNA
inside or outside the living cell. To terminate the reaction, a chelator, such as EDTA
, is often added to sequester the required metal ions in the reaction mixture.
Retroviral RNase H, a part of the viral reverse transcriptase
enzyme, is an important pharmaceutical target, as it is absolutely necessary for the proliferation of retroviruses, such as HIV
and murine leukemia virus
. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS
. While not an effective treatment option, incorporation of 6-deoxythioguanosine has been shown to inhibit RNase H cleavage of the DNA/RNA complex.
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
and catalyzes the cleavage of RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
to bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
and eukaryota.
RNase H’s ribonuclease activity cleaves the 3’-O-P bond of RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
in a DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
/RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
duplex to produce 3’-hydroxyl and 5‘-phosphate terminated products. In DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
, RNase H is responsible for removing the RNA primer, allowing completion of the newly synthesized DNA.
Structure
The 3-D structure of RNase H commonly consists of a 5-stranded β-sheet surrounded by a distribution of α-helices. In some RNase H, such as the one found in HIV-1, the enzymeEnzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
is missing one of the helices known as the C-helix, a positively charged α-helix whose protrusive shape increases substrate binding capacity. The active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
of the enzyme is centered around a conserved DEDD motif (composed of residues: D443, E478, D498, and D549) which performs the hydrolysis of the RNA substrate. A magnesium
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...
ion is commonly used as a cofactor
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....
during the hydrolysis
Hydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
step. It is also a potential but unconfirmed mechanism in which multiple ions are necessary for to perform the hydrolysis. The enzyme also contains a nucleic acid binding cleft about 60 Å in length that can encompass a region of 18 bound RNA/DNA base pairs.
Function
In a molecular biologyMolecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
laboratory
Laboratory
A laboratory is a facility that provides controlled conditions in which scientific research, experiments, and measurement may be performed. The title of laboratory is also used for certain other facilities where the processes or equipment used are similar to those in scientific laboratories...
, as RNase H specifically degrades the RNA in RNA:DNA hybrids and will not degrade DNA or unhybridized RNA, it is commonly used to destroy the RNA template after first-strand complementary DNA
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
(cDNA) synthesis by reverse transcription, as well as procedures such as nuclease protection assay
Nuclease protection assay
Nuclease protection assay is a laboratory technique used in biochemistry and genetics to identify individual RNA molecules in a heterogeneous RNA sample extracted from cells. The technique can identify one or more RNA molecules of known sequence even at low total concentration...
s. RNase H can also be used to degrade specific RNA strands when the cDNA oligo is hybridized, such as the removal of the poly(A) tail from mRNA hybridized to oligo(dT), or the destruction of a chosen non-coding RNA
Non-coding RNA
A non-coding RNA is a functional RNA molecule that is not translated into a protein. Less-frequently used synonyms are non-protein-coding RNA , non-messenger RNA and functional RNA . The term small RNA is often used for short bacterial ncRNAs...
inside or outside the living cell. To terminate the reaction, a chelator, such as EDTA
EDTA
Ethylenediaminetetraacetic acid, widely abbreviated as EDTA , is a polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ligand...
, is often added to sequester the required metal ions in the reaction mixture.
Mechanism
Genes
The following human genes encode proteins with RNase H activity:- ERVK6ERVK6HERV-K_19q12 provirus ancestral Pol protein is a protein that in humans is encoded by the ERVK6 gene.-Further reading:...
- RNASEH1RNASEH1Ribonuclease H1 also known as RNase H1 is an enzyme that in humans is encoded by the RNASEH1 gene. The RNase H1 is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism....
- RNASEH2ARNASEH2ARRibonuclease H2 subunit A, also known as RNase H2 subunit A, is an enzyme that in humans is encoded by the RNASEH2A gene.- Function :...
- RNASEH2B
- RNASEH2C
Role in Disease
It is associated with Aicardi–Goutieres syndrome.Retroviral RNase H, a part of the viral reverse transcriptase
Reverse transcriptase
In the fields of molecular biology and biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse...
enzyme, is an important pharmaceutical target, as it is absolutely necessary for the proliferation of retroviruses, such as HIV
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
and murine leukemia virus
Murine leukemia virus
The murine leukemia viruses are retroviruses named for their ability to cause cancer in murine hosts. Some MLVs may infect other vertebrates. MLVs include both exogenous and endogenous viruses...
. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS
AIDS
Acquired immune deficiency syndrome or acquired immunodeficiency syndrome is a disease of the human immune system caused by the human immunodeficiency virus...
. While not an effective treatment option, incorporation of 6-deoxythioguanosine has been shown to inhibit RNase H cleavage of the DNA/RNA complex.