Reverse transcriptase is an enzyme that generates a strand of DNA by reading an RNA template. This process, known as reverse transcription, can be likened to a scribe translating a message from a temporary medium (RNA) to a more permanent one (DNA). The enzyme reads the sequence of RNA bases and synthesizes a corresponding DNA sequence, allowing genetic information to flow in a direction opposite to what is typically observed in cells.
The Mechanism of Reverse Transcription
The flow of genetic information in most organisms follows the central dogma of molecular biology: DNA is transcribed into RNA, which is then translated into protein. Reverse transcription presents a notable deviation from this pathway. For the enzyme to begin its work, it requires a starting point called a primer, a short nucleic acid sequence that binds to a complementary section of the RNA.
Once the primer is in place, the reverse transcriptase enzyme binds and begins synthesizing a new DNA strand complementary to the RNA template. This is its RNA-dependent DNA polymerase activity. The enzyme moves along the RNA, reading its ribonucleotides (A, U, G, C) and incorporating corresponding deoxyribonucleotides (T, A, C, G) to build the new complementary DNA (cDNA).
As the cDNA strand is synthesized, a hybrid molecule of one RNA and one DNA strand is formed. Many reverse transcriptase enzymes also possess RNase H activity, which degrades the RNA portion of this hybrid. Removing the original template is a preparatory step for synthesizing a second DNA strand, using the new cDNA as its template to create a stable, double-stranded DNA molecule.
Biological Roles in Nature
Reverse transcriptase activity is a hallmark of retroviruses, such as the human immunodeficiency virus (HIV). When a retrovirus infects a host cell, it injects its RNA genome and the reverse transcriptase enzyme. Inside the cell, the enzyme converts the viral RNA into a double-stranded DNA copy, a necessary step for the virus to replicate.
This new viral DNA is transported into the host cell’s nucleus and inserted into the host’s genome by an enzyme called integrase. Once integrated, the viral DNA, now called a provirus, is treated as one of the cell’s own genes. The host cell’s machinery then transcribes the proviral DNA back into viral RNA, which is used to produce new virus particles.
Beyond viruses, a form of reverse transcriptase is also found in eukaryotic organisms, including humans. This enzyme, called telomerase, maintains the integrity of chromosomes, which have protective caps at their ends called telomeres. Telomeres shorten with each cell division, and telomerase uses its internal RNA template to extend them, preventing the loss of genetic information. This action is particularly active in stem cells and other frequently dividing cells.
Applications in Biotechnology
Scientists have harnessed reverse transcriptase for laboratory techniques that have changed the study of RNA. One of the most widespread applications is Reverse Transcription Polymerase Chain Reaction (RT-PCR). The process begins by converting an RNA sample into more stable complementary DNA (cDNA) using reverse transcriptase.
This cDNA then serves as the template for the polymerase chain reaction (PCR), a technique that exponentially amplifies a specific DNA segment. Amplifying the cDNA allows researchers to detect very small amounts of a specific RNA, such as the genome of a virus like SARS-CoV-2, making RT-PCR a powerful diagnostic tool. When combined with quantitative PCR (qPCR), the technique can also measure the amount of a specific RNA, providing insights into gene expression levels.
Another application is the creation of cDNA libraries, which are collections of all messenger RNA (mRNA) molecules from a cell or tissue, converted into DNA form. Since mRNA represents actively expressed genes, a cDNA library provides a snapshot of cellular activity. Researchers use these libraries to identify genes, study their function, and understand how expression patterns differ between healthy and diseased states.
Therapeutic Inhibition
The reliance of retroviruses like HIV on reverse transcriptase for replication makes the enzyme an attractive target for antiviral drugs. Because reverse transcription is not a primary pathway in human cells, drugs that block this enzyme can inhibit viral replication with relatively fewer effects on the host. This strategy led to the development of a major class of antiretroviral medications known as reverse transcriptase inhibitors (RTIs).
These inhibitors disrupt the enzyme’s function and are categorized into two main types. Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) are faulty versions of the natural building blocks (nucleotides) the enzyme uses. When the reverse transcriptase incorporates an NRTI into the growing viral DNA, it acts as a chain terminator, halting the replication process.
A second category is the non-nucleoside reverse transcriptase inhibitors (NNRTIs). Instead of mimicking the building blocks, NNRTIs bind to a different site on the reverse transcriptase enzyme. This binding changes the enzyme’s shape, blocking its catalytic activity. To combat the virus’s ability to develop resistance, these different types of inhibitors are almost always used in combination therapies.