Unit 2: Transcription

1. RNA Polymerase and Transcription Unit, Transcription Factors

Transcription is the biological process where a specific segment of DNA is used as a template to synthesize RNA. This is the first step of gene expression.

RNA Polymerase (The Core Enzyme)

In prokaryotes, a single type of RNA polymerase catalyzes the synthesis of all RNA types (mRNA, tRNA, rRNA). It is a holoenzyme consisting of a core enzyme (2 alpha, 1 beta, 1 beta prime, and 1 omega subunit) and a Sigma (σ) factor.

• Core Enzyme: Responsible for elongation.
• Sigma Factor: Essential for initiation; it recognizes the promoter region on DNA.

The Transcription Unit

A transcription unit is a stretch of DNA transcribed into an RNA molecule. It typically consists of three regions:

• Promoter: Binding site for RNA polymerase, located upstream (5' end) of the structural gene.
• Structural Gene: The actual template for RNA synthesis.
• Terminator: Defines the end of the transcription process.

Transcription Factors

In eukaryotes, transcription requires Transcription Factors (TFs) to help RNA polymerase bind to the promoter. These include General Transcription Factors (like TFIID, TFIIB) and specific regulatory factors.

2. Mechanism of Transcription in Prokaryotes

In prokaryotic organisms like E. coli, the process occurs in three main stages: Initiation, Elongation, and Termination.

Step 1: Initiation

The Sigma (σ) factor guides the RNA polymerase holoenzyme to the promoter region (containing specific sequences like the Pribnow box at -10 and the -35 sequence). Once bound, the DNA double helix unwinds to form an "open complex."

Step 2: Elongation

The sigma factor is released, and the core enzyme moves along the template strand (3' to 5' direction). It adds ribonucleotides (NTPs) complementary to the DNA template to synthesize the RNA strand in the 5' to 3' direction.

Step 3: Termination

Termination occurs via two mechanisms:

• Rho-independent (Intrinsic): A GC-rich hairpin loop forms in the RNA, followed by a string of Uracils, causing the polymerase to stall and detach.
• Rho-dependent: A protein called the Rho factor binds to the growing RNA and moves toward the polymerase to pull the transcript away from the DNA.

3. Basic Difference between Prokaryotic and Eukaryotic Transcription

While the fundamental mechanism is similar, eukaryotes exhibit significantly higher complexity.

4. Post Transcriptional Modifications

In eukaryotes, the primary transcript (pre-mRNA) must undergo several modifications to become functional, mature mRNA.

A. 5' Capping

A 7-methylguanosine cap is added to the 5' end of the transcript. This protects the mRNA from degradation by exonucleases and serves as a recognition signal for ribosomes during translation initiation.

B. 3' Poly-A Tailing

The 3' end is cleaved, and a string of 200–300 Adenine residues (Poly-A tail) is added by the enzyme Poly-A polymerase. This tail aids in mRNA stability and its export from the nucleus to the cytoplasm.

C. Splicing

Definition: The process of removing non-coding regions called Introns and joining together the coding regions called Exons.

This is carried out by a large complex called the Spliceosome, composed of small nuclear ribonucleoproteins (snRNPs).

5. Exam Focus Enhancements