Modern biology amazes with the uniqueness and scale of its discoveries. Today, this science studies most of the processes that are hidden from our eyes. This is remarkable for molecular biology - one of the promising areas that helps to unravel the most complex mysteries of living matter.
What is reverse transcription
Reverse transcription (abbreviated as RT) is a specific process characteristic of most RNA viruses. Its main feature is the synthesis of a double-stranded DNA molecule based on messenger RNA.
OT is not characteristic of bacteria or eukaryotic organisms. The main enzyme, reversetase, plays a key role in the synthesis of double-stranded DNA.
Discovery history
The idea that a ribonucleic acid molecule could become a template for DNA synthesis was considered absurd until the 1970s. Then B altimore and Temin, working separately from each other, almost simultaneously discovered a new enzyme. They called it RNA-dependent-DNA polymerase, or reverse transcriptase.
The discovery of this enzyme unconditionally confirmed the existence of organismscapable of reverse transcription. Both scientists received the Nobel Prize in 1975. After some time, Engelhardt proposed an alternative name for reverse transcriptase - revertase.
Why OT contradicts the central dogma of molecular biology
The Central Dogma is the concept of sequential protein synthesis in any living cell. Such a scheme is built from three components: DNA, RNA and protein.
According to the central dogma, RNA can be synthesized exclusively on the DNA template, and only then RNA is involved in building the primary structure of the protein.
This dogma was officially accepted in the scientific community before the discovery of reverse transcription. Not surprisingly, the idea of reverse synthesis of DNA from RNA has long been rejected by scientists. Only in 1970, along with the discovery of reversetase, was an end to this issue, which was reflected in the concept of protein synthesis.
Revertase of avian retroviruses
The process of reverse transcription is not complete without the participation of RNA-dependent-DNA polymerase. Revertase of the avian retrovirus has been studied to the maximum extent to date.
Only about 40 molecules of this protein can be found in one virion of this family of viruses. The protein consists of two subunits that are in equal numbers and perform three important functions of reversease:
1) Synthesis of a DNA molecule both on a single-stranded/double-stranded RNA template and on the basis of deoxyribonucleic acids.
2) RNase H activation, the main role of which is tocleavage of the RNA molecule in the RNA-DNA complex.
3) Destruction of sections of DNA molecules for insertion into the eukaryotic genome.
Mechanism OT
The reverse transcription steps may vary depending on the family of viruses, i.e. on the type of their nucleic acids.
Let's first consider those viruses that use reversetase. Here the OT process is divided into 3 steps:
1) Synthesis of the “-” RNA strand on the template “+” of the RNA strand.
2) Destruction of the "+" strand of RNA in the RNA-DNA complex using the enzyme RNase H.
3) Synthesis of a double-stranded DNA molecule on the template "-" of the RNA chain.
This method of virion reproduction is typical for some oncogenic viruses and the human immunodeficiency virus (HIV).
It is worth noting that for the synthesis of any nucleic acid on an RNA template, a seed or primer is needed. A primer is a short sequence of nucleotides that is complementary to the 3' end of an RNA molecule (template) and plays an important role in initiating synthesis.
When ready-made double-stranded DNA molecules of viral origin are integrated into the eukaryotic genome, the usual mechanism of virion protein synthesis starts. As a result, the cell “captured” by the virus becomes a virion production factory, where the necessary protein and RNA molecules are formed in large quantities.
Another way of reverse transcription is based on the action of RNA synthetase. This protein is active in paramyxoviruses, rhabdoviruses, picornoviruses. In this case, there is no third stage of OT - the formationdouble-stranded DNA, and instead, a “+” RNA chain is synthesized on the template of the viral “-” RNA chain and vice versa.
The repetition of such cycles leads both to the replication of the virus genome and to the formation of mRNA capable of protein synthesis under the conditions of an infected eukaryotic cell.
Biological significance of reverse transcription
The OT process is of paramount importance in the life cycle of many viruses (primarily retroviruses such as HIV). The RNA of a virion that attacked a eukaryotic cell becomes a template for the synthesis of the first DNA strand, on which it is not difficult to complete the second strand.
The obtained double-stranded DNA of the virus is integrated into the eukaryotic genome, which leads to the activation of the processes of virion protein synthesis and the appearance of a large number of its copies inside the infected cell. This is the main mission of Revertase and OT in general for the virus.
Reverse transcription can also occur in eukaryotes in the context of retrotransposons, mobile genetic elements capable of being independently transported from one part of the genome to another. Such elements, according to scientists, caused the evolution of living organisms.
Retrotransposon is a stretch of eukaryotic DNA that codes for several proteins. One of them, reversetase, is directly involved in the delocalization of such a retrotransporozone.
Use of OT in science
From the moment it was possible to isolate reversetase in its pure form, the process of reverse transcription was adopted by biologists. The study of the OT mechanism still helps to read the sequences of the most important human proteins.
The fact is that the genome of eukaryotes, including us, contains non-informative regions called introns. When a nucleotide sequence is read from such DNA and a single-stranded RNA is formed, the latter loses introns and codes exclusively for protein. If DNA is synthesized using reversetase on an RNA template, then it is easy to sequence it and find out the order of nucleotides.
Nucleic acid that has been formed by reverse transcriptase is called cDNA. It is often used in the polymerase chain reaction (PCR) to artificially increase the copy number of the resulting cDNA copy. This method is used not only in science, but also in medicine: laboratory assistants determine the similarity of such DNA with the genomes of various bacteria or viruses from a common library. The synthesis of vectors and their introduction into bacteria is one of the promising areas of biology. If RT is used to form the DNA of humans and other organisms without introns, such molecules can easily be introduced into the bacterial genome. So the latter become factories for the production of substances necessary for a person (for example, enzymes).
