Mutations at position -10 in the lambda PR promoter primarily affect conversion of the initial closed complex (RPc)...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• The lambda P(R) promoter is crucial for initiating transcription.
• Understanding promoter mutations aids in deciphering gene regulation mechanisms.

Purpose of the Study:

• To investigate the impact of specific mutations in the --10 region of the lambda P(R) promoter on transcriptionally competent open complex formation.
• To elucidate the role of base-pair recognition in promoter function.

Main Methods:

• DNAse I footprinting
• Potassium permanganate (KMnO4)-sensitivity assays
• Abortive initiation kinetic analysis

Main Results:

• Mutations at --10A and --10C reduced the rate of closed-to-open complex conversion (k(f)) but not closed complex formation equilibrium (K(B)).
• The --10G mutation significantly decreased promoter occupancy and, when accounted for, showed a 20-fold reduction in k(f).
• All mutations primarily affected the isomerization step preceding DNA strand separation, indicating double-stranded DNA recognition in the --10 region.

Conclusions:

• RNA polymerase holoenzyme recognizes specific base pairs in the --10 region of P(R) on double-stranded DNA.
• The --10G mutation may also influence the equilibrium between intermediate and open complexes.
• Mutant promoters formed less stable open complexes compared to wild-type P(R).