Introduction to the characteristics of reverse transcriptase

Reverse transcriptase is also known as RNA-dependent DNA polymerase. In 1970, Temin et al. discovered a special DNA polymerase in oncogenic RNA viruses. The enzyme uses RNA as a template, dNTP as substrate, tRNA as a primer, and on the 3'-OH end of tRNA, according to the base pairing. In principle, a single strand of DNA that is complementary to the RNA template is synthesized in the 5'-3' direction, and this single strand of DNA is called complementary DNA (cDNA).

Reverse transcriptase is an enzyme that guides deoxynucleotide triphosphates to synthesize complementary DNA (cDNA) as a template. The reverse transcriptase of mammalian type C virus and the reverse transcriptase of murine type B virus is both polypeptide chains. The reverse transcriptase of avian RNA viruses consists of two upper subunits. Reverse transcriptases with different structures have also been isolated from eukaryotes.

Multiple reverse transcriptases have numerous enzymatic activities

①RNA-guided DNA polymerase activity; using RNA as a template to catalyze the polymerization of dNTPs into DNA. This enzyme requires RNA as a primer, mostly tRNA of tryptophan, and synthesizes DNA in the 5'→3' direction at the 3'-end of the primer tRNA. Reverse transcriptase does not have 3′→5′ exonuclease activity, so it has no correction function, so the error rate of DNA synthesized by reverse transcriptase is relatively high.

② RNase H activity; the hybrid molecule formed by the cDNA synthesized by reverse transcriptase and the template RNA will be hydrolyzed by RNase H from the 5' end of RNA.

③ DNA-guided DNA polymerase activity; the first DNA single strand synthesized by reverse transcription is used as the template, and the dNTP is used as the substrate to synthesize the second DNA molecule.

In addition, some reverse transcriptases also have DNA endonuclease activity, which may be related to the integration of viral genes into the chromosomal DNA of host cells. The discovery of reverse transcriptase has greatly promoted genetic engineering technology, and it has become an important tool enzyme. Extract mRNA from tissue cells and use it as a template to synthesize complementary DNA (cDNA) under the action of reverse transcriptase, from which a cDNA library (cDNA library) can be constructed, from which specific target genes can be screened. The most commonly used method in genetic engineering technology is to obtain the target gene.

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