Question

Outline a series of steps by which reverse transcriptase produces DNA on an RNA template.

Short answer

Reverse transcriptase binds to RNA and uses a primer to synthesize cDNA, degrades the RNA, then synthesizes the second DNA strand, resulting in double-stranded DNA.

Step-by-step solution

- Binding of Reverse Transcriptase

Reverse transcriptase first binds to the RNA template. This enzyme has a strong affinity for RNA and specifically attaches to the 3' end of the RNA.

- Primer Attachment

A primer, usually a short RNA strand called a tRNA, binds to the RNA template. This primer provides a free 3'-OH group that is necessary for the polymerization process.

- Synthesis of Complementary DNA (cDNA)

Reverse transcriptase begins to synthesize a complementary DNA strand starting from the primer. It adds DNA nucleotides complementary to the RNA template one by one, extending from the 3' end of the primer.

- RNA Degradation

As the cDNA strand is synthesized, the original RNA template is degraded by the RNase H activity of reverse transcriptase. This degradation occurs in a step-wise manner concurrent with the synthesis of cDNA.

- Synthesis of Second DNA Strand

Once the RNA template is degraded, the single-stranded cDNA acts as a template for synthesizing the second DNA strand. The reverse transcriptase synthesizes this strand, creating a double-stranded DNA.

- Final Product

The end result is a double-stranded DNA molecule complementary to the original RNA template. This DNA can then be integrated into the host genome by other enzymes.

Key concepts

RNA template

An RNA template is a strand of RNA that serves as a template for synthesizing a complementary DNA strand. In the context of reverse transcriptase, the enzyme uses the RNA template to produce cDNA. This is vital since reverse transcriptase has an affinity for RNA and specifically attaches to the 3' end of the RNA template. Understanding the RNA template is crucial because it provides the genetic information that reverse transcriptase will convert into DNA. This process is essential in retroviruses like HIV, where the RNA genome needs to be converted into DNA to integrate into the host genome.

cDNA synthesis

cDNA synthesis refers to the creation of a complementary DNA strand from an RNA template, a process catalyzed by reverse transcriptase. This begins once a primer is attached to the RNA template. The reverse transcriptase then adds DNA nucleotides complementary to the RNA sequence, growing the cDNA strand from the primer's 3' end. The process ensures that the genetic information in the RNA is accurately copied into a stable DNA form. This is a crucial step because it allows the genetic material initially present in RNA format to be used by standard DNA-based cellular machinery.

RNase H activity

RNase H activity is an essential function of reverse transcriptase. As cDNA synthesis progresses, the original RNA strand needs to be removed to make way for the full DNA strand. RNase H is the component responsible for degrading the RNA strand in a step-wise manner during cDNA synthesis. This makes it possible for the single-stranded cDNA to serve as a template for synthesizing the second, complementary DNA strand. Without RNase H activity, the RNA could interfere with the formation of stable double-stranded DNA, a crucial step in the lifecycle of retroviruses.

double-stranded DNA

Double-stranded DNA (dsDNA) is the final product of the reverse transcription process. After RNase H degrades the RNA template, the single-stranded cDNA serves as a template for synthesizing a second DNA strand. The reverse transcriptase enzyme extends this second strand to create a complete, double-stranded DNA molecule. This dsDNA is complementary to the original RNA template and can now be integrated into the host genome, allowing the viral genetic material to be expressed and replicated alongside host DNA. This step is pivotal for the virus's ability to hijack host cell machinery for its own replication.

tRNA primer

A tRNA primer is a small RNA molecule that initiates cDNA synthesis by providing a free 3'-OH group for the reverse transcriptase. This primer anneals to a specific site on the RNA template, creating the starting point for the enzyme to begin adding complementary DNA nucleotides. The presence of this primer is crucial because reverse transcriptase cannot start the DNA synthesis process on its own. The tRNA primer ensures that the enzyme has a foothold to start converting RNA into cDNA effectively.