The "Swiss knife" of the viral army: the secret of reverse transcriptase unraveled. Reverse transcriptase Revertase reverse transcriptase

It is called so because most of the transcription processes in living organisms occur in a different direction, namely, an RNA transcript is synthesized from a DNA molecule.

Story

Reverse transcriptase was discovered by Howard Temin at the University of Wisconsin-Madison, and independently by David Baltimore in 1970. Both researchers received the Nobel Prize in Physiology or Medicine in 1975 with Renato Dulbecco.

Accuracy of transcription

Reverse transcription from RNA to DNA is accompanied by a high level of translation errors, which distinguishes reverse transcriptase from other DNA polymerases. These errors can lead to mutations responsible for the drug resistance of viruses.

Significance for viruses

Reverse transcription is necessary, in particular, for the life cycle of retroviruses, for example, human immunodeficiency viruses and human T-cell lymphoma types 1 and 2. After the viral RNA enters the cell, the reverse transcriptase contained in the viral particles synthesizes a complementary DNA to it, and then completes the second strand on this DNA strand, as on a template.

Significance for eukaryotes

Application

Antiretroviral therapy

Role in genetic engineering

In genetic engineering, reverse transcriptase is used to produce cDNA, a copy of a eukaryotic gene that does not contain introns. For this, mature mRNA (encoding the corresponding gene product: protein, RNA) is isolated from the body and reverse transcription is carried out with it as a template. The resulting cDNA can be transformed into bacterial cells to obtain a transgenic product.

see also

Write a review on "Reverse Transcriptase"

Notes (edit)

Excerpt Characterizing Reverse Transcriptase

- O! O! O!
- Well, goodbye, Bolkonsky! Goodbye, prince; Come to dinner earlier, ”came the voices. - We take care of you.
“Try to praise the order in the delivery of provisions and routes as much as possible when you talk to the emperor,” said Bilibin, escorting Bolkonsky to the front.
“And I would like to praise, but I cannot, for as long as I know,” answered Bolkonsky, smiling.
- Well, in general, talk as much as possible. His passion is audiences; but he himself does not like to speak and does not know how, as you will see.

At the exit, Emperor Franz only gazed intently into the face of Prince Andrew, who was standing in the appointed place between the Austrian officers, and nodded to him with his long head. But after leaving yesterday's wing, the adjutant courteously conveyed to Bolkonsky the emperor's desire to give him an audience.
Emperor Franz received him, standing in the middle of the room. Before starting the conversation, Prince Andrew was struck by the fact that the emperor seemed to be confused, not knowing what to say, and blushed.
- Tell me, when did the battle begin? He asked hastily.
Prince Andrew answered. This question was followed by other, equally simple questions: “Is Kutuzov healthy? how long ago did he leave Krems? " etc. The Emperor spoke with such an expression as if his whole purpose consisted only in asking a certain number of questions. The answers to these questions, as it was all too obvious, could not interest him.
- At what time did the battle begin? The emperor asked.
`` I can't tell your Majesty at what time the battle began from the front, but in Durenstein, where I was, the army launched an attack at 6 o'clock in the evening, '' said Bolkonsky, becoming animated and in this case assuming that he would be able to present the ready-made in his head a true description of everything that he knew and saw.
But the emperor smiled and interrupted him:
- How many miles?
- Where and to where, Your Majesty?
- From Durenstein to Krems?
“Three and a half miles, your majesty.
- Did the French leave the left bank?
- As the scouts reported, the last ones crossed the rafts at night.
- Is there enough forage in Krems?
- Forage was not delivered in that quantity ...
The emperor interrupted him.
- At what time was General Schmitt killed? ...
- At seven o'clock, I think.
- At 7:00. Very sad! Very sad!
The emperor said he was grateful and bowed. Prince Andrew went out and was immediately surrounded by courtiers from all sides. Affectionate eyes looked at him from all sides and gentle words were heard. Yesterday's outbuilding adjutant reproached him for not staying in the palace, and offered him his house. The Minister of War came up, congratulating him on the Order of Maria Theresa of the 3rd degree, which the emperor had bestowed on him. The Empress's chamberlain invited him to her Majesty. The Archduchess also wanted to see him. He did not know who to answer, and for several seconds collected his thoughts. The Russian envoy took him by the shoulder, took him to the window and began to talk to him.
Contrary to Bilibin's words, the news he brought was received with joy. A thanksgiving service was appointed. Kutuzov was awarded the Grand Cross by Maria Theresa, and the entire army received awards. Bolkonsky received invitations from all sides and had to make visits to the main dignitaries of Austria all morning. Having finished his visits at five o'clock in the evening, mentally composing a letter to his father about the battle and about his trip to Brunn, Prince Andrey returned home to Bilibin. At the porch of the house occupied by Bilibin, there was a chaise half packed with things, and Franz, Bilibin's servant, with difficulty dragging a suitcase, went out the door.

Revertase is an enzyme that synthesizes cDNA on an RNA template.

In some viruses, the genome is not DNA, as usual, but RNA. Such viruses were called retroviruses (retro - reverse). In 1970, D. Baltimore and H.M. Temin discovered a mechanism for transferring information from viral RNA to DNA, i.e. on the contrary to what takes place in the cells of higher organisms. This process is called reverse transcription, and the enzyme that performs it was called reverse transcriptase, or revertase (revertase).

Reverse transcriptase, or reverse transcriptase, [lat. transcriptio- rewriting) - the enzyme RNA-dependent DNA polymerase, with the help of which reverse transcription is carried out - DNA synthesis on the RNA matrix; encoded by the genomes of some RNA-containing viruses and mobile genetic elements of the genome of higher organisms, an important "tool" for genetic engineering. Reverse transcriptase has at least three enzymatic activities:

1) DNA polymerase, using both RNA and DNA as a template;

2) the activity of RNase H, which hydrolyzes RNA as part of an RNA-DNA hybrid, but not single- or double-stranded RNA, and

3) DNA endonuclease activity.

Discovered independently of each other by D. Baltimore and H. Temin in 1970 in RNA-containing tumor-bearing viruses (Nobel Prize for 1975 together with R. Dulbecco).

So, reverse transcriptases are able to synthesize DNA on an RNA template by polymerizing four deoxyribonucleoside triphosphates. And just like DNA polymerases, they function only when primed.

Reverse transcriptases are used in the synthesis of double-stranded DNA, complementary to RNA (especially mRNA), for its subsequent cloning in plasmid vectors when obtaining cDNA libraries (clones). Reverse transcriptases, like DNA polymerases, can be used to introduce radioactive or fluorescent labels into DNA probes in appropriately labeled deoxyribonucleoside triphosphates.

The ability to synthesize DNA on an RNA template under certain conditions has been demonstrated for the thermostable DNA polymerase Thermus thermophilus. This allows it to be used for the direct detection of specific RNAs in biological samples by PCR. Modern modifications of this approach make it possible in one reaction mixture (and test tube) to synthesize in a reverse transcription reaction a small number of copies of the amplified DNA fragment on an RNA template, which are immediately used by the same enzyme as a template in a conventional PCR (one tube PCR).

When studying retroviruses, the genome of which is represented by single-stranded RNA molecules, it was found that in the process of intracellular development they go through the stage of integration of their genome in the form of double-stranded DNA into the chromosomes of the host cell. In 1964, H. Temin put forward a hypothesis about the existence of a virus-specific enzyme capable of synthesizing complementary DNA on an RNA template. In 1970 X. Temin and S. Mizutani, as well as independently of them D. Baltimore, discovered such an enzyme in a preparation of extracellular virions of the Rous sarcoma virus. This RNA-dependent DNA polymerase is called reverse transcriptase (reverse transcriptase).

The most detailed study is the revertase of avian retroviruses. Each virion contains about 50 molecules of this enzyme. Reverse transcriptase consists of two subunits - ά (65 kDa) and β (95 kDa), which are present in equimolar amounts. The ά -subunit is the N-terminal part (two thirds) of the β-subunit.

Reverse transcriptase has at least three enzymatic activities:

· DNA polymerase, using both RNA and DNA as a template;

· The activity of RNase H, which hydrolyzes RNA in the RNA-DNA hybrid, but not single or double-stranded RNA;

· DNA endonuclease.

The first two activities are required for the synthesis of viral DNA, and the endonuclease appears to be important for the integration of viral DNA into the genome of the host cell. The β-subunit of revertase has all three activities, while the-subunit has only polymerase and RNase H.

Purified reverse transcriptase synthesizes DNA on both RNA and DNA templates. To start synthesis, revertase, like other polymerases, needs a short double-stranded region - a primer. The primer can be a single-stranded segment of both RNA and DNA, which during the reaction turn out to be covalently linked to the newly synthesized DNA strand.

Reverse transcriptase is predominantly used to transcribe messenger RNA into complementary DNA (cDNA). The reverse transcription reaction is carried out in the presence of potent inhibitors of RNase activity. In this case, it is possible to obtain full-length DNA copies of the target RNA molecules. Oligo (dT) is used as a primer for reverse transcription of poly (A) -containing mRNA (Fig.), And for RNA molecules without 3 "poly (A) ends, chemically synthesized oligonucleotides complementary to the 3" end of the studied RNA. In addition, the latter type of RNA molecules can be converted to poly (A) -containing by means of E. coli poly (A) -polymerase.

After the synthesis of the complementary DNA strand on the mRNA and the destruction of the RNA (usually alkali treatment is used), the second DNA strand is synthesized. This uses the ability of reverse transcriptase to form self-complementary hairpins at the 3'-ends of single-stranded cDNAs that can act as a primer. The template is the first strand of cDNA. This reaction can be catalyzed by both reverse transcriptase and DNA polymerase I. The combination of these two enzymes allows increase the yield of full-fledged double-stranded cDNA molecules.

At the end of the synthesis, the first and second strands of cDNA remain covalently linked by the loop of the hairpin, which served as a primer in the synthesis of the second strand. This loop is cleaved with endonuclease S1, which specifically destroys single-stranded regions of nucleic acids. The resulting ends are not always blunt, and to increase the efficiency of subsequent cloning, they are repaired to blunt using the Klenow fragment of E. coli DNA polymerase I. The resulting double-stranded cDNA can then be inserted into cloning vectors, expanded within hybrid DNA molecules, and used for further research.

Reverse transcriptase (revertase or RNA-dependent DNA polymerase) is an enzyme that catalyzes the synthesis of DNA on an RNA template in a process called “ reverse transcription "... The name of the process reflects the opposite of the process transcriptions carried out in a different direction: an RNA transcript is synthesized from the DNA template molecule.

These enzymes were isolated from RNA viruses ( retroviruses). Reverse transcriptase is used by tumorigenic viruses to transcribe mRNA into the complementary DNA strand. When studying retroviruses, the genome of which is represented by single-stranded RNA molecules, it was found that in the process of intracellular development, the retrovirus goes through the stage of integration of its genome in the form of double-stranded DNA into the chromosomes of the host cell. In 1964, Temin put forward a hypothesis about the existence of a virus-specific enzyme capable of synthesizing complementary DNA on an RNA template. Efforts aimed at isolating such an enzyme were crowned with success, and in 1970 Temin and Mizutani, and independently of them Baltimore, discovered the desired enzyme in a preparation of extracellular virions of the Rous sarcoma virus. This RNA-dependent DNA polymerase is called reverse transcriptase, or reverse transcriptase.

The most detailed study is the revertase of avian retroviruses. Each virion contains about 50 molecules of this enzyme. Reverse transcriptase consists of two subunits, a (65 kDa) and b (95 kDa), present in equimolar amounts. Reverse transcriptase has at least three enzymatic activities:

1) DNA polymerase, using both RNA and DNA as a template;

2) the activity of RNase H, which hydrolyzes RNA in the RNA-DNA hybrid;

3) DNA endonuclease activity.

The first two activities are required for the synthesis of viral DNA, and the endonuclease appears to be important for the integration of viral DNA into the genome of the host cell. Purified reverse transcriptase synthesizes DNA on both RNA and DNA templates (Fig. 11).

Rice. 11. Scheme of synthesis of double-stranded DNA copies of RNA molecules

To start synthesis, revertase, like other polymerases, needs a short double-stranded region (primer). The primer can be a single-stranded segment of both RNA and DNA, which during the reaction turn out to be covalently linked to the newly synthesized DNA strand. In genetic engineering, both oligo- (dT) primers complementary to the 3'-polyA ends of mRNA and a set of hexanucleotides “random” in composition and sequence (random primers) are used. poly (A) ends, chemically synthesized oligonucleotides complementary to the 3 "end are used

Reverse transcriptase is predominantly used to transcribe messenger RNA into complementary DNA (cDNA). The reverse transcription reaction is carried out under specially selected conditions using strong inhibitors of RNase activity. In this case, it is possible to obtain full-length DNA copies of the target RNA molecules. After the synthesis of the complementary DNA strand on the mRNA and the destruction of the RNA (usually alkali treatment is used), the second DNA strand is synthesized. In this case, the ability of reverse transcriptase to form self-complementary hairpins at the 3'-ends of single-stranded cDNAs, which can act as a primer, is used.

The template is the first strand of cDNA. This reaction can be catalyzed by both revertase and E. coli DNA polymerase I. The combination of these two enzymes has been shown to increase the yield of full-fledged double-stranded cDNA molecules. At the end of the synthesis, the first and second strands of cDNA remain covalently linked by the loop of the hairpin, which served as a primer in the synthesis of the second strand. This loop is cleaved with endonuclease S1, which specifically destroys single-stranded regions of nucleic acids. The resulting ends are not always blunt, and to increase the efficiency of subsequent cloning, they are repaired to blunt using the Klenow fragment of E. coli DNA polymerase I. The resulting double-stranded cDNA can then be inserted into cloning vectors, expanded within hybrid DNA molecules, and used for further research.