Transcription Process and Termination in Prokaryotes

Overview

• Transcription is similar to DNA replication; both involve enzymes synthesizing new nucleic acid strands complementary to DNA.
• Key Difference: DNA replication uses DNA polymerase, while transcription uses RNA polymerase.

RNA Polymerase Structure in Bacteria

• RNA polymerase in bacteria has five subunits: beta prime, beta, alpha 1, alpha 2, and omega.
  • Beta Prime: Largest, has active site for RNA synthesis.
  • Beta: Second largest, involved in RNA synthesis.
  • Alpha 1 & Alpha 2: Each with two domains (N-terminal and C-terminal).
    • N-terminal domain: Involved in assembly of RNA polymerase.
    • C-terminal domain: Interacts with the promoter and binds non-specifically to DNA sequences.
  • Omega: Smallest, facilitates assembly and stabilizes RNA polymerase.

Sigma Factor

• RNA polymerase needs Sigma factor for transcription initiation.
• Sigma factor allows RNA polymerase to bind specifically to the promoter.
• Complete RNA polymerase holoenzyme = RNA polymerase + Sigma factor.

Transcription Initiation Steps

1. Formation of Closed Complex
   • RNA polymerase binds to the promoter while DNA is double-stranded.
2. Formation of Open Complex
   • DNA strands near the start site unwind.
   • RNA polymerase starts adding ribonucleotides, no primer needed.
3. Abortive Initiation
   • Short RNA stretches are formed and released.
   • Occurs due to Sigma factor blocking RNA exit channel.

Elongation

• Sigma factor released.
• RNA polymerase proceeds with elongation.

Termination Mechanisms

Rho-Independent Termination

• Intrinsic terminators with two sequence elements:
  1. Short inverted repeats (~20 nucleotides).
  2. Stretch of ~8 A-T base pairs.
• RNA forms a stem-loop (hairpin) structure halting polymerase.
• Weak A-U base-pairing leads to RNA release, ending transcription.

Rho-Dependent Termination

• Requires Rho factor (ATP-dependent helicase).
• Rho binds to single-stranded RNA rich in cytosines (Rho-utilization site or rut site).
• RNA polymerase pauses at Rho-sensitive pause site.
• Rho uses ATP to translocate to RNA-DNA hybrid, unwinding and ending transcription.