Reverse transcriptase is an enzyme that generates a strand of DNA by reading a template of ribonucleic acid (RNA) in a process called reverse transcription. The enzyme rewrites a temporary genetic message, encoded in RNA, into a more permanent and stable DNA format.
The “Reverse” in Reverse Transcription
To understand reverse transcriptase, it helps to know the usual pathway of genetic information described by the central dogma of molecular biology: information moves from DNA to RNA and then to protein. DNA holds the permanent genetic blueprint for an organism, containing the instructions for its development and function.
When a cell needs a protein, it performs transcription to create a temporary RNA copy of the relevant gene from the DNA template. This RNA molecule travels to the cytoplasm to direct protein assembly, a system that keeps the original DNA blueprint protected. Reverse transcriptase upends this standard flow by using an RNA molecule as a template to create DNA, performing the opposite of transcription.
The Step-by-Step Mechanism
Reverse transcription begins with the binding of a primer to the RNA template. In many retroviruses, this primer is a specific transfer RNA (tRNA) from the host cell that anneals to a complementary site on the viral RNA. This binding provides the starting point for the enzyme, which cannot initiate DNA synthesis on its own and requires an existing 3′-hydroxyl (-OH) group to add new nucleotides.
Once the primer is in place, the reverse transcriptase enzyme reads the RNA template and adds corresponding DNA nucleotides to the 3′ end of the primer. This process elongates the primer, resulting in a hybrid molecule consisting of one strand of RNA and one newly synthesized, complementary DNA strand.
The enzyme’s RNase H domain then becomes active. It degrades the original RNA template from the RNA-DNA hybrid by cleaving the phosphodiester bonds. This process removes the RNA portion, leaving behind a single strand of viral DNA.
Finally, the single-stranded DNA serves as a template for a second, complementary DNA strand. The reverse transcriptase uses its DNA-dependent DNA polymerase activity to build this second strand. This creates a stable, double-stranded DNA (dsDNA) molecule that contains the genetic information from the viral RNA and is ready for the next stage of the viral life cycle.
Viral Replication
Reverse transcriptase is fundamental to the life cycle of retroviruses, such as the Human Immunodeficiency Virus (HIV). After a retrovirus enters a host cell, it releases its RNA genome and the enzyme into the cytoplasm. The enzyme then performs reverse transcription, converting the viral RNA into a double-stranded DNA copy.
This newly created viral DNA is transported into the host cell’s nucleus. There, an enzyme called integrase facilitates the permanent insertion of the viral DNA into the host’s genome. The viral DNA, now known as a provirus, is hidden within the host’s chromosomes.
Whenever the infected host cell divides, it also replicates the integrated viral DNA. The cell’s machinery is used to transcribe the provirus into new viral RNA molecules. These RNA molecules are then used to synthesize new viral proteins and be packaged into new virus particles that can infect other cells.
Endogenous and Cellular Functions
While often associated with viruses, reverse transcriptase activity also occurs in normal eukaryotic cells. A prominent example is the enzyme telomerase, which acts as a specialized reverse transcriptase to maintain telomeres, the protective structures at the ends of chromosomes. These regions naturally shorten with each round of cell division.
Telomerase carries its own RNA template to add repetitive DNA sequences to the chromosome ends, counteracting the shortening process and supporting cellular longevity. Dysregulation of its activity is linked to cellular aging; insufficient activity can cause premature aging, while over-activity can contribute to the uncontrolled cell division characteristic of cancer.
Technological and Medical Applications
Scientists use reverse transcriptase for various technological and medical purposes. A widely used laboratory technique, Reverse Transcription Polymerase Chain Reaction (RT-PCR), relies on this enzyme to detect and measure RNA levels. It converts RNA in a sample into DNA, which is then amplified using PCR methods. This technique is used for diagnosing RNA viruses, like influenza and SARS-CoV-2, and for studying gene expression.
The enzyme is also a primary target for treating HIV. Antiretroviral drugs known as reverse transcriptase inhibitors (RTIs) are a component of HIV therapy that block the enzyme’s action. By inhibiting reverse transcriptase, these drugs prevent the virus from converting its RNA genome into DNA. This halts the replication cycle and prevents the virus from establishing a permanent infection in host cells.