Bacterial Genes and Transcription

Bacterial Genes

• A gene is a segment of DNA that codes information to produce protein or functional RNA
• The expression of a gene is regulated by cellular mechanisms
• A gene has two major components:
  • Information to make functional protein or RNA
  • Regulatory elements for expression

Bacterial Transcription, Schematic

• RNA polymerase recognizes and binds to promoter elements (-35 and -10) causing DNA to open up
• Transcription starts at the +1 site
• RNA polymerase moves along the DNA template strand in the 5’ to 3’ direction
• The non-template strand has the same sequence as the newly synthesized RNA (except for T replaced with U)

Template vs Non-template DNA strands

• Template strand (non-coding): used as a template for transcription, does not code for the protein sequence
• Non-template strand (coding): codes for the protein sequence, is not used as a template for transcription

Sigma Factors

• Sigma factors assist RNA polymerase core to bind to specific promoters
• Different types of sigma factors exist in a single cell
• Sigma 70 (σ70) recognizes the typical bacterial promoter sequence (-35 and -10) and helps RNA polymerase core bind
• RNA polymerase core bound to a sigma factor is called RNA polymerase Holoenzyme

Bacterial Transcription Initiation and Elongation

• RNA polymerase creates a transcription bubble (approximately 10-20 bp) as it moves along the DNA template strand
• The bubble exposes the template strand between positions -10 to +10

Three Stages of Transcription

• Initiation: binding of RNA polymerase to a promoter sequence
• Elongation: sequential addition of nucleotides to the 3’ end of the growing RNA using DNA as a template
• Termination: Dissociation of RNA polymerase and release of a primary transcript from template

Transcription Initiation in Bacteria

• RNA polymerase Holoenzyme carrying σ70 recognizes -35 and -10 elements
• α subunits of RNA polymerase core recognize and bind to the UP element
• When DNA binds to RNA polymerase, it gets bent. Initially, the DNA is not separated (Closed Complex)
• RNA polymerase opens up the DNA (Open Complex) and generates the transcription bubble

Transcription Initiation: Abortive Initiation

• RNA polymerase initiates synthesis on the opened template strand starting at the +1 site
• Sigma factor blocks RNA polymerase core from moving too far from the promoter
• Sigma factor disrupts polymerization and brings the core back to the +1 site
• After a few attempts, RNA polymerase core breaks off from the sigma factor and enters the elongation phase

Transcription Elongation in Bacteria

• RNA polymerase moves down the DNA and synthesizes the new RNA strand
• The sigma factor is absent during elongation

Transcription Termination - Rho-independent

• Occurs in regions of DNA where there is a highly GC-rich sequence followed by a U-rich region.
• The GC-rich sequences are complementary to each other, resulting in a hairpin structure.
• The U-rich region forms a weaker hydrogen bond with the template DNA.
• The hairpin structure pulls the RNA away from the template DNA, destabilizing the bond and causing transcription to terminate.

Transcription Termination - Rho-dependent

• Rho is a homohexameric protein that binds to the Rho Utilization Site (RUT) on the growing RNA.
• Rho uses ATP hydrolysis to move along the RNA in the 5' to 3' direction.
• When Rho approaches RNA polymerase, it interacts with the stem-loop structure of the RUT site on RNA, slowing down RNA polymerase.
• This interaction causes RNA polymerase to dissociate from the DNA and RNA.

RNA Polymerase and Transcription

• Initiation: RNA polymerase binds to a promoter sequence.
• Elongation: RNA polymerase adds nucleotides to the growing RNA chain using DNA as a template.
• Termination: RNA polymerase dissociates from the DNA, releasing the RNA transcript.