RNA Reverse Transcriptase

What is RNA Reverse Transcriptase?

Reverse transcriptase, also known as RNA-dependent DNA polymerase, first discovered in some RNA viruses, is an enzyme with reverse transcription activity that is capable of synthesizing DNA using single-stranded RNA as a template. Reverse transcriptase was first discovered in RNA viruses in 1970 by Howard Temin and David Baltimore. They were awarded the 1975 Nobel Prize in Physiology or Medicine.

Reverse transcriptase activities and mechanism of action. Figure 1. Reverse transcriptase activities and mechanism of action. (M, Boriana, 2018)

Reverse Transcription Processes of HIV Virus

1. Formation of DNA-RNA heteroduplexes

  • Template: genome of retroviral single-stranded RNA
  • Primer: tRNA (mainly tryptophan tRNA)
  • Enzyme: reverse transcriptase (RNA-directed DNA polymerase activity)
  • Process: synthesize a single strand of DNA under the action of template, primer and enzyme, and form a DNA-RNA heteroduplex with the template for complementary DNA (cDNA).

2. Heteroduplex RNA hydrolysis

  • Enzyme: RNase H activity of reverse transcriptase
  • Process: Hydrolysis of RNA in the heteroduplex to yield a single strand of DNA (cDNA) that is complementary to the RNA.

3. Double-stranded DNA formation

  • Template: newly synthesized complementary single-stranded cDNA
  • Substrate: dNTP
  • Enzyme: reverse transcriptase (DNA-directed DNA polymerase activity)
  • Process: cDNA single strand as template, dNTP as substrate, then synthesize double stranded DNA.

Application of Reverse Transcriptase

Reverse transcriptases not only play a functional role in biological systems, but are also important in the study of RNA populations. One of the first molecular biology experiments using reverse transcriptase is the preparation of cDNAs, which are used to build libraries containing DNA copies of mRNAs from cells and tissues. These cDNA libraries can help to understand the genes that are actively expressed at a given point in time and their functions. Although the construction of cDNA libraries is an important step in achieving the identification of expressed genes, there are still many challenges in conducting low abundance RNA studies. These problems have since been solved with the advent of polymerase chain reaction (PCR), a technique for amplifying small amounts of genetic material. Reverse transcription-conjugated PCR, also known as reverse transcription PCR (RT-PCR), can detect even RNAs with extremely low levels of gene expression, thus opening the way for the detection of free RNAs, RNA viruses, and oncogene fusions in molecular diagnostic applications. In addition, cDNAs can be used as templates for a range of applications, such as microarray and RNA sequencing applications, to characterize unknown RNAs in a high-throughput manner.

Properties of Reverse Transcriptase

Reverse transcriptases are essential reagents in the process of synthesizing complementary DNA (cDNA) strands from RNA templates. Therefore, an in-depth understanding of the nature of these enzymes and their effect on reverse transcription is essential for DNA/RNA-related biological studies.

  • DNA polymerase activity

Reverse transcriptase is composed of different structural enzymes with different biochemical activities. Although the functions of reverse transcriptases from different organisms vary to a greater or lesser extent, including, for example, DNA-dependent DNA polymerase activity, the main functions of reverse transcriptases depend on RNA-dependent DNA polymerase and RNase H activity.

  • RNase H activity

An intrinsic property of reverse transcriptase is RNase H activity, which allows simultaneous cleavage of RNA templates in heterodimeric strands of RNA such as cDNA during synthesis. Since the RNA template may be degraded before full-length reverse transcription is completed, RNase H activity is a factor to be circumvented during the synthesis of long fragmented cDNAs. For better cDNA synthesis, the RNase H activity of reverse transcriptase is reduced or even eliminated completely by introducing mutations in the RNase H structural domain of reverse transcriptase. Such mutations can often increase the yield of long-stranded cDNAs and facilitate their synthesis.

  • Thermal stability

The ability of reverse transcriptase to tolerate high temperatures is an important influence on cDNA synthesis. Elevated reaction temperatures help to denature RNAs with robust secondary structures and/or high GC content, allowing reverse transcriptase to read the sequence. Thus, reverse transcription at higher reaction temperatures enables full-length cDNA synthesis with higher yields, which in turn allows for better reverse transcription of RNA to cDNA.

  • Ability to sustain synthesis

The synthetic capacity of a reverse transcriptase is the number of nucleotides bound into a single binding site of the enzyme. Therefore, a reverse transcriptase with a high synthesis capacity can synthesize longer cDNA strands in a shorter reaction time. Some genetically engineered modified MMLV reverse transcriptases can bind up to 1,500 nucleotides in a single binding site, a binding capacity roughly 65 times greater than that of wild-type MMLV reverse transcriptase. The enzyme synthesis capacity is also related to its affinity for the template. Thus, reverse transcriptases with high synthesis capacity are resistant to common inhibitors that may be of RNA origin.

  • High fidelity

The fidelity of a reverse transcriptase refers to the sequence accuracy during the reverse transcription of RNA to DNA. Fidelity is inversely proportional to the reverse transcription error rate. MMLV-based reverse transcriptases have been reported to have an error rate of one incorrect nucleotide out of 15,000 to 27,000 nucleotides synthesized, whereas AMV reverse transcriptase exhibits a higher error rate.

  • Terminal nucleotidyl transferase (TdT) activity

Reverse transcriptase exhibits terminal nucleotide transferase (TdT) activity, which results in the non-template specific addition of nucleotides to the 3' end of the synthesized DNA. TdT activity is only exhibited when the reverse transcriptase comes into contact with the 5' end of the RNA template, at which point it adds 1-3 additional nucleotides to the end of the cDNA and exhibits specificity for double-stranded nucleic acid substrates. TdT activity can also be demonstrated by reverse transcriptase in the presence of the 5' end of the RNA template, where it adds 1-3 additional nucleotides to the end of the cDNA, and is specific for double-stranded nucleic acid substrates.

Reference

  1. M, Boriana. Viral Tools for In Vitro Manipulations of Nucleic Acids. Harnessing the Power of Viruses. 2018, 27–67.
* Only for research. Not suitable for any diagnostic or therapeutic use.
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