Oriented DNA binding by one-armed lambda repressor heterodimers and contacts between repressor and RNA polymerase at...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Bacteriophage lambda repressor protein is crucial for regulating gene expression by interacting with E. coli RNA polymerase.
• The specific subunit within the repressor dimer responsible for transcriptional activation has remained experimentally undetermined.
• Previous studies identified mutations affecting repressor activation but did not pinpoint the interacting subunit.

Purpose of the Study:

• To experimentally determine which subunit of the bacteriophage lambda repressor dimer is responsible for activating transcription at the P(RM) promoter.
• To elucidate the precise interaction between the repressor dimer and the sigma subunit of E. coli RNA polymerase during transcriptional activation.

Main Methods:

• Development and utilization of an oriented heterodimer approach employing one-armed repressor-leucine zipper fusion proteins.
• In vitro assessment of operator DNA binding using methylation protection assays with both one-armed and two-armed repressor heterodimers.
• In vivo analysis of P(RM) promoter activation using one-armed heterodimers with a specific mutation (pc2) in a single subunit.

Main Results:

• The subunit incorporating the leucine zipper arm in one-armed repressor heterodimers demonstrated preferential binding to the consensus half-site of lambda operators.
• In vivo experiments revealed that the promoter-proximal subunit of the repressor dimer is the one that interacts with RNA polymerase at the P(RM) promoter.
• The N-terminal domain of the promoter-proximal subunit was identified as the specific contact point for RNA polymerase.

Conclusions:

• The promoter-proximal subunit of the bacteriophage lambda repressor dimer, specifically its N-terminal domain, directly contacts E. coli RNA polymerase to activate transcription at P(RM).
• This study resolves the long-standing question regarding subunit-specific function in lambda repressor-mediated transcriptional activation.
• The oriented heterodimer approach provides a powerful tool for dissecting protein-protein interactions in transcriptional regulation.