DNA Replication and RNA Transcription Processes

DNA Replication: Semiconservative Process

DNA replication is a semiconservative process. It begins at replication origins, forming "bubbles."

Key Enzymes and Proteins in DNA Replication

• Helicases: Unwind the DNA double helix, moving in opposite directions from the origin.
• Topoisomerases: Act ahead of the replication fork to relieve supercoiling that arises from unwinding.
• SSB Proteins (Single-Strand Binding Proteins): Bind to and stabilize the separated single DNA strands, preventing them from re-annealing.

DNA synthesis always proceeds in the 5' to 3' direction, leading to two distinct modes of synthesis:

Continuous Synthesis (Leading Strand)

On the leading strand, synthesis is continuous. An RNA primase (a type of RNA polymerase) synthesizes a short RNA primer (approximately 10 nucleotides). Subsequently, DNA polymerase adds nucleotides continuously to the 3' end of this primer, extending the new DNA strand.

Discontinuous Synthesis (Lagging Strand)

On the lagging strand, synthesis is discontinuous, occurring in short segments. RNA primase synthesizes multiple RNA primers along the template strand. DNA polymerase III then synthesizes DNA segments, extending from these primers, forming what are known as Okazaki fragments. These fragments are typically around 1000-2000 nucleotides long in prokaryotes and 100-200 nucleotides in eukaryotes.

After the Okazaki fragments are formed:

• DNA Polymerase I: Removes the RNA primers and fills the resulting gaps with DNA nucleotides.
• DNA Ligase: Joins the adjacent Okazaki fragments, creating a continuous DNA strand.

In eukaryotes, there are multiple origins of replication, leading to several replication bubbles. Additionally, Okazaki fragments are generally shorter in eukaryotes compared to prokaryotes.

Transcription: RNA Synthesis from DNA

Transcription is the process of synthesizing RNA from a DNA template. It is characterized by being complementary to the DNA template strand, proceeding in the 5' to 3' direction, and being asymmetric (only one DNA strand serves as a template for a given gene).

The DNA strand that serves as the template for RNA synthesis is called the template strand or antisense strand. The other DNA strand, which has a sequence similar to the RNA transcript (with T instead of U), is called the coding strand or sense strand.

Gene Structure

A typical gene consists of three main regions:

• Promoter Region: A DNA segment that allows RNA polymerase and other transcription factors to recognize and bind to the gene, initiating transcription.
• Structural Region: Contains the actual genetic information (exons and introns) that will be transcribed into RNA and, for protein-coding genes, translated into protein.
• Terminator Region: A DNA sequence that signals the end of the gene, causing RNA polymerase to stop transcription and release the newly synthesized RNA molecule.

Phases of Transcription in Eukaryotes

1. Initiation

   Transcription begins when RNA polymerase, along with various transcription factors, binds to the promoter region. Eukaryotic promoters often contain specific sequences like the TATA box and CAAT box, which are crucial for the precise initiation of transcription.

2. Elongation

   RNA polymerase moves along the DNA template, synthesizing the RNA molecule in the 5' to 3' direction. As the RNA chain grows, a 5' cap (a modified guanosine triphosphate, 7-methylguanosine) is added to the nascent RNA. This cap protects the RNA from degradation by exonucleases and is important for translation.

3. Termination

   Transcription terminates upon reaching specific DNA sequences. In eukaryotes, a common signal for termination and polyadenylation is the AATAAA sequence. After transcription, an enzyme called poly-A polymerase adds a tail of approximately 100-250 adenine nucleotides (the poly-A tail) to the 3' end of the RNA. This tail is important for RNA stability, transport, and translation. The resulting molecule is known as pre-mRNA or nuclear RNA.

4. Maturation (RNA Processing)

   The pre-mRNA undergoes further processing before becoming mature messenger RNA (mRNA). Small nuclear ribonucleoproteins (snRNPs), along with other proteins, recognize and remove introns (non-coding sequences) through a process called splicing. RNA ligase then joins the exons (coding sequences) together, forming the mature mRNA molecule, which is ready for export from the nucleus and translation.

Transcription in Prokaryotes

Prokaryotic transcription differs from eukaryotic transcription in several ways:

• Prokaryotic RNA typically lacks a 5' cap and a poly-A tail.
• Prokaryotic genes generally do not contain introns, so their RNA does not require splicing or extensive maturation.
• Transcription and translation can occur simultaneously in prokaryotes because there is no nuclear envelope separating the processes.
• Prokaryotic genes are often polycistronic, meaning a single mRNA molecule can contain information for synthesizing multiple different proteins.
• There is typically only one type of RNA polymerase in prokaryotes, which synthesizes all types of RNA (mRNA, tRNA, rRNA).