Question

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

Short answer

Reverse transcriptase produces double-stranded DNA by first synthesizing a DNA strand from an RNA template and then using the newly formed DNA as a template for a second DNA strand.

Step-by-step solution

RNA Template Binding

Reverse transcriptase binds to the RNA template strand, preparing to synthesize a complementary DNA strand.

DNA Synthesis

Reverse transcriptase synthesizes the first DNA strand by adding complementary DNA nucleotides to the RNA template strand in the 5' to 3' direction.

Template Degradation

The RNA template is degraded, usually by the RNase H activity of reverse transcriptase, leaving the newly synthesized single-stranded DNA.

Second Strand Synthesizer

Reverse transcriptase synthesizes a second complementary DNA strand using the initial DNA strand as a template, resulting in double-stranded DNA.

Key concepts

RNA template

In the process of DNA synthesis by reverse transcriptase, the RNA template plays a crucial role. An RNA template is a single-stranded RNA molecule that serves as a guide for the synthesis of complementary DNA. When reverse transcriptase binds to the RNA template, it starts the process by aligning with the RNA's nucleotide sequence.
The enzyme identifies the starting point on the RNA strand, making sure it correctly binds to initiate the synthesis. This binding step is essential since the RNA template acts as the blueprint for creating the new DNA strand.
Without a proper RNA template, the reverse transcription process wouldn't occur accurately, leading to errors in the DNA synthesis.

DNA synthesis

DNA synthesis is the core function of reverse transcriptase. In this step, the enzyme starts adding complementary DNA nucleotides to the RNA template, working in the 5' to 3' direction. Each RNA nucleotide guides the addition of a specific DNA nucleotide: for example, adenine (A) in RNA pairs with thymine (T) in DNA.
The synthesis continues until a complete DNA strand is formed, complementary to the RNA template.
It's important to note that this process is highly accurate, but still prone to occasional errors, which can lead to mutations. The enzyme's efficiency and fidelity are crucial for correct DNA synthesis.

RNase H activity

Once the initial DNA strand is synthesized, the RNA template must be removed. RNase H activity is a critical feature of reverse transcriptase that degrades the RNA strand. RNase H is a ribonuclease enzyme that specifically targets the RNA in an RNA-DNA hybrid.
By degrading the RNA template, RNase H makes way for the completion of double-stranded DNA. This activity ensures that the new single-stranded DNA is freed from its RNA guide, making it available for the synthesis of the complementary DNA strand.
RNase H activity must be precise to avoid damage to the newly synthesized DNA.

complementary DNA strand

The final step involves the synthesis of the complementary DNA strand. Here, reverse transcriptase uses the newly synthesized single-stranded DNA as a template.
The enzyme sequentially adds DNA nucleotides, creating a second DNA strand that is complementary to the first.
This results in a double-stranded DNA molecule, which faithfully represents the original RNA template.
This double-stranded DNA can then be integrated into the host genome or used for further applications, such as cloning or sequencing. The formation of this complementary DNA strand is essential for the complete and functional replication of genetic information.