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Reverse transcriptase

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Reverse transcriptase (RT) is a specialized enzyme that performs reverse transcription. This is the process of converting RNA into DNA. In the classical central dogma of biology, information typically flows from DNA to RNA. Reverse transcriptase expands this understanding by allowing information to move from RNA back to DNA. This enzyme is vital for many life forms. It helps certain viruses replicate their genomes within a host. It also helps retrotransposons move within a genome. In eukaryotic cells, a specific version helps maintain the ends of chromosomes.

Reverse transcription
Reverse transcription

The mechanism of reverse transcription is a complex, multi-step biochemical process. It begins when a lysyl tRNA acts as a primer. This primer attaches to a specific site on the viral RNA called the primer-binding site (PBS). The enzyme then uses its RNA-dependent DNA polymerase activity to add DNA nucleotides. These are added to the 3′ end of the primer to create a DNA strand complementary to the RNA. Following this, a domain called RNase H degrades parts of the original RNA template. This degradation is necessary to allow the synthesis of the second DNA strand.

HIV-1 Reverse Transcriptase with Active Sites
HIV-1 Reverse Transcriptase with Active Sites

To create a complete double-stranded cDNA, the enzyme performs several specific actions. First, the tRNA primer "jumps" to the 3′ end of the viral genome. This allows the newly synthesized DNA to hybridize with the complementary R region on the RNA. The enzyme then performs DNA-dependent DNA polymerase activity. This activity copies the first DNA strand into a second, antisense strand. This sequence of events results in a double-stranded DNA molecule. Once formed, this DNA can be integrated into the host genome by an enzyme called integrase. This allows the virus to use the host's own machinery to make new RNA copies.

There are several distinct types of reverse transcriptases studied by scientists. The HIV-1 reverse transcriptase, used by the human immunodeficiency virus, consists of two subunits. These subunits have molecular weights of 66 kDa and 51 kDa. In contrast, the M-MLV reverse transcriptase from the Moloney murine leukemia virus is a single 75 kDa monomer. Another example is the AMV reverse transcriptase from the avian myeloblastosis virus. It is composed of two subunits weighing 63 kDa and 95 kDa. Finally, telomerase is a reverse transcriptase found in eukaryotes that maintains telomeres.

Zidovudine
Zidovudine

The history of this discovery is tied to two major scientific achievements in 1970. Howard Temin discovered reverse transcriptases at the University of Wisconsin–Madison using Rous sarcoma virions. Independently, David Baltimore isolated the enzyme at MIT from murine leukemia virus and Rous sarcoma virus. Their work changed how we understand genetic flow. Because of these discoveries, Temin and Baltimore shared the 1975 Nobel Prize in Physiology or Medicine. This prize was shared with Renato Dulbecco. Before the term "reverse transcriptase" was used, the enzyme was called RNA-dependent DNA polymerase.

Reverse transcription is known for being an extremely error-prone process. This is because the enzyme lacks proofreading ability, which most DNA polymerases possess. These errors lead to mutations, which can result in things like drug resistance in viruses. For example, the error rate for AMV is approximately 1 in 17,000 bases. For M-MLV, the error rate is about 1 in 30,000 bases. These mutations accumulate quickly due to the lack of a correction mechanism. Interestingly, this error-prone nature might even contribute to the diversity of life through template switching.

Reverse transcription
Reverse transcription

Today, reverse transcriptase has many important connections to modern science and medicine. In laboratories, it is used for molecular cloning, RNA sequencing, and polymerase chain reaction (PCR). It is also essential for genome analysis. In medicine, drugs like zidovudine (AZT) are used to inhibit this enzyme. By stopping the enzyme, the drug can help prevent the virus from replicating. Scientists are also working on new tools like the reverse transcribing xenotranscriptase (RTX). This engineered enzyme can both copy and proofread RNA, which could improve the accuracy of future sequencing tests.

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File:HIV-1 Reverse Transcriptase with Active Sites.png
HIV-1 Reverse Transcriptase with Active Sites.png
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Reverse transcription.svg
File:Zidovudine.svg
Zidovudine.svg
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