Reverse transcriptase | Its basic mechanism with 10 important FAQs

https://commons.wikimedia.org/wiki/File:HIV-1_Reverse_Transcriptase_with_Active_Sites.png

Reverse transcriptase | Its basic mechanism with 10 important FAQs

Contents

Transcription

Transcription is the beginning towards “Gene Expression.”

In this process, the information contained in the “four-letter genetic code of DNA” is transferred into the three-letter genetic code of RNA.”

, we can explain transcription as it makes a copy of RNA from DNA, or the genetic information is transferred from DNA to RNA.

The transcription process involves an enzyme known as RNA polymerase; this enzyme uses DNA as a template and facilitates the linkage of nucleotides to form RNA (corresponding and complementary to the template DNA sequence).

The transcription process is completed in three stages.

Stage 1: Initiation

Stage 2: Elongation

Stage 3: Termination

Figure: Three basic steps of transcription (process of transfer of genetic information from DNA to RNA); Initiation, Elongation and termination https://commons.wikimedia.org/wiki/File:Eukaryotic_Transcription.png

In eukaryotes, a few stages are known as post-translational modifications also occur in which the RNA undergoes modifications such as:

Splicing

5’-capping

Poly A tail incorporation

Important note: Transcription occurs for every single gene present in our genome separately.

DNA has enormous genes, each gene codes for a gene. Transcription is copying the information of the protein-coding gene into an RNA transcript.

Transcriptase

Transcriptases are the enzymes that catalyze RNA synthesis from the template DNA; this process is known as transcription. Reverse transcriptases (RTs) are the enzymes that catalyze the formation of cDNA (complementary DNA) from the RNA template; this process is known as reverse transcription.

Reverse Transcription

In the process of reverse transcription, DNA is synthesized from an RNA template (opposite of the transcription process, in which RNA is synthesized from the DNA template). The DNA produced in reverse transcription is known as complementary DNA (cDNA). Reverse transcription is carried out by an enzyme known as reverse transcriptase. 

Requirements of reverse transcriptase:

           RNA template

           Short primer (for binding with the 3′ end of RNA)

The short primer binds to the complementary region at the 3′ end and starts the synthesis of complementary DNA. This cDNA is utilized in the PCR (Polymerase chain reaction) as the template DNA. cDNA is then converted into double-stranded DNA by DNA polymerase and DNA ligase action. This combination of PCR and RT-PCR (a combination of polymerase chain reaction and reverse transcription) enables detecting minute amounts of RNAs present in the sample. Reverse transcription is often used in cloning to increase the copy number. 

Reverse transcription
Figure: Reverse transcription scheme, the RT binds to the ssRNA and directs the synthesis of cDNA which later converts into dsDNA. https://commons.wikimedia.org/wiki/File:Reverse_transcription_polymerase_chain_reaction.jpg

Several engineered RTs are often used to increase product formation efficiency. The engineered RT ensures the complete copying of the 5′ end of the mRNA. Thermostable RTs are also engineered for their use at higher temperatures.

What reverse transcriptase synthesizes?

The reverse transcriptase facilitates the synthesis of DNA from the viral RNA genome. This newly formed DNA is known as complementary DNA (cDNA). RT is mostly present in viruses containing RNA genome. The virus-containing RNA genome is known as a retrovirus.

What is the function of reverse transcriptase?

Reverse transcriptase is an RNA dependent DNA polymerase (it requires RNA as a template to synthesize cDNA). The RT identifies and binds the RNA to synthesize single stood RNA (ssRNA). After synthesizing the cDNA, RT cleaves the RNA and start synthesizing double-stranded DNA (dsDNA).   

­reverse transcriptase has two active sites (active sites) to perform two separate functions:

Polymerase active site: it comprises two finger-like domains; one domain can identify the RNA and forms hydrogen bonds with phosphate groups by using side chains.

Hydrogen bond formation brings about the conformational changes and facilitates the closure of the recognition hole; this results in the start of the transcription process (by the addition of nucleotides) with the help of the second domain magnesium ions.

The flexible zone governs the conformational change process; this flexible zone is present between the polymerase active site domains. It is often considered a target site to inhibit RT activity during biochemical and pharmacological studies. The amino acid sequence present in the flexible zone is not conserved (change a lot during the course of evolution), which enables the virus to develop resistance.

Ribonuclease H domain: it is responsible for cleaving the RNA and ssDNA release. Magnesium ion helps ribonuclease H domain in recognition of phosphate groups. The second chain of ribonuclease H domain does not have enzymatic activity. It only interacts and stabilizes the active site.

Research is still going on the RT’s exact catalytic mechanism; the above-mentioned scheme is the basic idea of the catalysis done by RT.

Unlike DNA polymerase, RT lacks proofreading activity because of the absence of Klenow fragment. The lack of proofreading activity produces more errors in the transcript, which leads to enormous possibilities of mutations to the virus.

Retrovirus reverse transcriptase

The viral particles of all retroviruses contain a multifunctional enzyme known as RT. This RT is required to synthesize the DNA copy of a retrovirus’s RNA genome as soon as it enters into a host cell. 

Determination of the 3D structure of reverse transcriptase revealed the interactions between the two active sites, including binding and catalysis. The enzyme’s structural knowledge provides us with an opportunity to study novel anti-viral drugs for treatment against retroviruses.

HIV reverse transcriptase

The human immune deficiency virus (HIV) is responsible for spreading acquired immune deficiency syndrome (AIDS) . HIV encodes for mainly four types of protein subunits exhibiting DNA polymerase, RNase H, integrase and protease like activity. DNA polymerase and RNase H activity are possessed by a single sub-unit of HIV reverse transcriptase (Heterodimeric enzyme).

To convert the virus’s genomic RNA into the dsDNA in a host cell, HIV reverse transcriptase exhibits both RNase H and DNA polymerase activity. Research is going on to know the mechanism of how HIV reverse transcriptase deploys its sub-unit (Ribonuclease H domain) to hydrolyze the genome (RNA) of retrovirus. The sites and reverse transcriptase conformation for DNA synthesis and RNA hydrolysis are different. This distinction in the conformation and revelation of the structural cavity makes it a potential target candidate for reverse transcriptase inhibition.

Figure: Structure of HIV reverse transcriptase during the synthesis of cDNA, Usually the HIV RT has two active sites. one catalytic site has polymerase function while other has nuclease activity. https://commons.wikimedia.org/wiki/File:Reverse_transcriptase_3KLF_labels.png

Important note: The RNase H activity of the HIV reverse transcriptase totally depends on the active site’s amino acid sequence. The actual mechanism is still yet to be discovered.

Conclusions

This article discussed the events of transcription, reverse transcription, and the importance of reverse transcriptase in retroviruses. For more details on related topics, click here.

FAQs

Q1 What is the role of reverse transcriptase

Answer: The enzyme RT is also known as RNA dependent DNA polymerase; it catalyzes the transcription of retroviral genomic RNA into cDNA.

Q2 Where does reverse transcriptase come from

Answer: The process of reverse transcription occurs in retroviruses, prokaryotes and even eukaryotes to some extent. It is involved in the synthesis of cDNA from RNA. Retroviruses have RTs, which are dependent on RNA to synthesize cDNA. Nuclear division does not involve the use of RT. An enzyme known as Telomerase is present in eukaryotes. Telomerase is an specialized RT which synthesize telomeric DNA (present at the tips of chromosomes) from RNA.

Q3 What organisms have reverse transcriptase

Answer: The enzyme RT is present in many organisms such as plants, animals, bacteria and viruses. The enzyme RT role is to convert an RNA sequence to a cDNA sequence. It is general in all organisms. The specific functions of RT include:

Retrovirus propagation

Diversity of eukaryotic mobile transposons or retrotransposons

Replication or synthesis of telomeres

Synthesis of multi-copy single-stranded DNA (msDNA), extrachromosomal DNA/RNA and chimeric elements.

Q4 Do humans make reverse transcriptase.

Answer: Yes, humans express reverse transcriptases; telomerase is a reverse transcriptase type. It is found in many eukaryotes. Humans carry an RNA template of telomerase to form telomeric DNA.

Q5 How do you prevent reverse transcriptase

Answer: Reverse transcriptase activity can be prevented by using potent enzyme inhibitors. These inhibitors (specifically for RTs) are generally anti-retroviral agents. Inhibitors restrict the RT by forming cDNA from viral RNA, and hence it stops the propagation of the virus.

Q6 What is the difference between real-time PCR and reverse transcriptase

Answer: The reverse transcriptase PCR (RT-PCR) is much more sensitive than regular PCR. RT-PCR is often used to detect a particular gene’s expression, DNA sequencing, DNA cloning, monitoring hereditary diseases, and analysis of functional genes. In contrast, general PCR is much extensively used for DNA amplification purposes.

Q7 Why is RNA converted to cDNA

Answer: reverse transcriptase usually transcribe template RNA into cDNA. 

cDNA is generally used in molecular biology for cloning eukaryotic genes into the prokaryotic cell factories. Whenever we want to express a protein (of external source) into a cell that normally does not synthesize such protein, we require a cDNA transformation into the prokaryotic cell factory.

Q8 What is the difference between QPCR and RT-PCR

Answer: both the techniques are inter-related and utilized to produce/amplify DNA copies. RT-PCR amplifies the RNA code’s reverse transcription, while Q-PCR measures the amplification process. Q-PCR is for quantification, while RT-PCR is for amplifications. Q-PCR is quantitative, while RT-PCR is non-quantitative in nature.

Q9 Can reverse transcriptase use DNA as a template.

Answer: Reverse transcriptase uses single-stranded RNA as a template to synthesize double-stranded DNA copy. RTs do not use DNA as a template.

Q10 What is the distinctive feature of reverse transcriptase

Answer: The distinctive feature of reverse transcriptase is to transfer the genetic information from RNA to DNA. 

About Dr. Abdullah Arsalan

I am Abdullah Arsalan , Completed my PhD in Biotechnology. I have 7 years of research experience. I have published 6 papers so far in the journals of international repute with an average impact factor of 4.5 and few more are in consideration. I have presented research papers in various national and international conferences. My subject area of interest is biotechnology and biochemistry with special emphasis on Protein chemistry, enzymology, immunology, biophysical techniques and molecular biology.

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