Reverse Transcription Basics

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Reverse Transcription Basics

The process of creating DNA from an RNA template is known as reverse transcription. Reverse transcriptases, or RNA-dependent DNA polymerases, are the enzymes that propel this process. 

Reverse transcriptases are found in retroviruses and prokaryotic and eukaryotic organisms in their natural states.

When a viral particle enters a target cell's cytoplasm, reverse transcription starts. As a component of an as-yet-uncharacterized nucleoprotein complex, the viral RNA genome penetrates the cytoplasm. Through a complex set of steps, reverse transcription creates a linear DNA duplex in the cytoplasm. Although the DNA and its RNA template are colinear, the DNA has terminal duplications called long terminal repeats (LTRs) that are absent from viral RNA. 



Current reverse transcription models suggest that in order to produce the LTRs, two specific template switches called "jumps" or strand-transfer reactions are needed.

The two unique enzymatic activities of RT—a DNA polymerase that can use either DNA or RNA as a template and a nuclease known as ribonuclease H (RNase H), which is specialized for the RNA strand of RNA:DNA duplexes—are absolutely necessary for the synthesis of retroviral DNA. 

While other proteins might play a part, and certain viral proteins (like nucleocapsid, NC) probably boost reverse transcription efficiency, only the DNA polymerase or RNase H of RT is responsible for all the enzymatic activities needed to finish the sequence of events leading to the creation of a retroviral DNA. Retroviral DNA synthesis is thought to proceed according to the plan.

The 3′end of a partially unraveled transfer RNA is used as a primer to start the synthesis of minus-strand DNA when it anneals to the genomic RNA's primer-binding site (PBS). 

Minus-strand DNA synthesis produces a discrete-length DNA intermediate known as minus-strand strong-stop DNA (–sssDNA) when it reaches the 5′end of genomic RNA. -sssDNA is only a brief sequence of about 100–150 bases since the binding site for the tRNA primer is located close to the 5′ end of viral RNA.

After the RNA strand of the RNA:sssDNA duplex is broken down by RNase-H, the sssDNA anneals to the 3′end of a viral genomic RNA due to the first strand transfer. At the 5′ and 3′ ends of the RNA genome, identical sequences known as repeating (R) sequences mediate this transfer. Sequences complementary to R are present at the 3′end of -sssDNA because it was cloned from the R sequences at the 5′end of the viral genome. -sssDNA can anneal to the R sequences at the 3′end of the RNA genome once the RNA template has been removed. The NC seems to be helping the annealing reaction.

After the -sssDNA has been moved to the viral RNA's 3′R region, RNase H digestion of the template strand and minus-strand DNA synthesis commence. Still, this degeneration is not finished.

 A brief polypurine tract (PPT) found in the RNA genome is largely resistant to RNase H destruction. a specific RNA segment that comes from plus-strand DNA synthesis primed by PPT. After a piece of the primer tRNA is reverse-transcribed, plus-strand synthesis stops, producing a DNA known as plus-strand strong-stop DNA (+sssDNA). 

Certain viruses produce multiple plus-strand primers from the RNA genome, even though all strains of retroviruses produce a specific plus-strand primer from the PPT.

RNase H eliminates the primer tRNA, revealing complimentary sequences in +sssDNA that are located at or close to

The second strand transfer is the annealing of the complementary PBS segments in the minus-strand and +sssDNA.

After that, the plus and minus strands of DNA are synthesized, with one strand acting as a template for the other.

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