What is Reverse transcriptases?

Reverse transcriptases, or shorted in RTs,   are RNA dependent DNA pols initially isolated from retroviruses. In addition, RTs are coded by dsRNA viruses that utilize reverse transcription such as hepatitis B virus; and various retroelements in eukaryotes and prokaryotes. The enzyme telomerase maintaining the ends of the eukaryotic chromosomes is technically also a reverse transcriptase, although its mechanism is very distinct from conventional RTs. Historically, the discovery of RT revolutionized molecular biology leading to the revision of the central dogma and enabling scientists to develop new research tools that heavily influenced cloning, analysis of gene expression and RNA biology. HIV RT is one of the most extensively studied polymerases in the context of understanding the biology of this devastating virus and designing RT inhibitors as drugs to manage HIV infections.

RTs exhibit three key enzyme activities (Fig. 1):

(1) RNA-dependent DNA pol that uses ssRNA template and a primer (tRNALys for HIV RT) to synthesize ssDNA/cDNA, which remains hybridized to its RNA template;

(2) RNAse H endonuclease, which selectively degrades the RNA strand of DNA/RNA hybrids and

(3) DNA dependent DNA polymerase activity, converting the single-stranded cDNA into dsDNA. Conventional RT enzymes have two active sites, one executing the polymerase activities and another executing the endonuclease activity. RT are monomeric or dimeric proteins and some lack intrinsic RNAse H activity.

The RTs of Moloney murine leukemia virus (M-MLV) and Avian myeloblastosis virus (AMV) are most frequently used as molecular tools in RT-PCR, RT-qPCR, cDNA cloning, RNA sequencing and any other experimental technique/approach that requires conversion of RNA to DNA. Site-directed mutagenesis and protein evolution have been utilized to optimize those enzymes improving thermostability and modulating RNAseH activity. Using thermostable version of RT is beneficial for lowering the nonspecific nucleic acid amplification and minimizing impact of complex secondary structures. Robust RNAseH activity is an advantage in RT-PCR, whereas lower RNAseH activity is beneficial in cDNA cloning protocols, especially when very long mRNA transcripts are reverse transcribed. In some cases RT is used only to produce the RNA/DNA hybrid and a conventional DNA pol carries out the cDNA to dsDNA polymerization step.

The mechanism of reverse transcriptases

Figure 1. Reverse transcriptase activities and mechanism of action.

Related Products

Related topic

An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2