RNA Polymerase Overview and Structure

Transcription: Overview

• Transcription and DNA replication are similar processes, both involve enzymes synthesizing new strands of nucleic acid complementary to DNA strands
• Key difference: DNA polymerase adds deoxyribonucleotides (dNTPs) while RNA polymerase adds ribonucleotides (rNTPs) to the template DNA

RNA Polymerase Structure

• Bacterial RNA polymerase contains 5 subunits: β’, β, α (two), and ω
• β’ is the largest subunit and contains the active site for RNA synthesis
• β is the second largest subunit involved in RNA synthesis
• α subunits (α1 and α2) are the third lightest subunit
  • Each α subunit has two domains: the N-terminal domain and the C-terminal domain
  • N-terminal domain is involved in RNA polymerase assembly
  • C-terminal domain is involved in interaction with promoters and nonspecific DNA sequence binding
• ω is the smallest subunit and facilitates the assembly and stabilization of RNA polymerase

Sigma Factor

• RNA polymerase requires a sigma factor for transcription initiation
• Sigma factor is a protein that facilitates specific binding of RNA polymerase to promoters
• The complete RNA polymerase holoenzyme has 6 subunits

Transcription Initiation

• Transcription initiation occurs in 3 steps: formation of closed complex, open complex, and abortive initiation
• Closed complex: RNA polymerase binds to the promoter region (DNA is double-stranded)
• Open complex: DNA strands around the transcription start site unwind
• Abortive initiation: Once the open complex forms, RNA polymerase adds rNTPs to the template DNA
  • Unlike DNA polymerase, RNA polymerase does not require a primer for transcription initiation
  • Short RNA stretches are formed and released, a process called abortive initiation.
  • The sigma factor blocks the RNA exit channel of the RNA polymerase enzyme, leading to abortive initiation

Transcription Elongation

• Once the sigma factor is released, RNA polymerase proceeds with elongation

Transcription Termination

• Termination is the process of stopping transcription and releasing the RNA product

• Prokaryotes have two modes of termination: Rho-independent (intrinsic) and Rho-dependent

• Rho-independent termination:

  • Does not require external proteins or factors
  • Also called intrinsic termination
  • Contains two key sequence elements in the DNA:
    • Short sequence of inverted repeats ~20 nucleotides long
    • Stretch of 8-10 A-T base pairs
  • After transcription, the inverted repeat sequence forms a stem-loop structure (hairpin)
  • The hairpin structure halts the RNA polymerase
  • The A-T rich sequence forms weak A-U base pairs which are easily broken, releasing the RNA transcript and terminating transcription.

• Rho-dependent termination

  • Requires the Rho protein
  • Rho is an ATP-dependent hexameric helicase
  • Rho binds to single-stranded RNA rich in cytosine (Rho utilization site)
  • When RNA polymerase reaches ~100 nucleotides from the terminator sequence, it pauses
  • The sequence that holds the RNA polymerase is called the Rho-sensitive pause site
  • Rho protein, bound to the RNA, translocates to the RNA-DNA hybrid using energy from ATP
  • Rho unwinds the hybrid, separating the RNA transcript from the DNA template and terminating transcription.

This quiz covers the fundamental aspects of RNA polymerase, comparing it to DNA replication. It details the structure of bacterial RNA polymerase, including its subunits and their functions, as well as the role of the sigma factor in transcription. Test your knowledge on these essential biological processes.