A mutation in region 1.1 of σ70 affects promoter DNA binding by Escherichia coli RNA polymerase holoenzyme

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Abstract

The sigma subunit of eubacterial RNA polymerase is essential for initiation of transcription at promoter sites. It directs recognition of DNA sequences by holoenzyme (α2ββ′σ) and facilitates subsequent steps in the initiation pathway. The primary σ factor from Escherichia coli, σ70, has four regions that are conserved among members of the σ70 family. Previous work has shown that region 1.1 modulates DNA binding by regions 2 and 4 when σ is separated from the core subunits, and is required for efficient progression through the later steps of initiation in the context of holoenzyme. In this report, we show that an amino acid substitution at position 53 in region 1.1, which converts isoleucine to alanine (I53A), creates a σ factor that associates with the core subunits to form holoenzyme, but the holoenzyme is severely deficient for promoter binding. The I53A phenotype can be suppressed by truncation of five amino acids from the C-terminus of σ70. We propose that the behavior of σ70-I53A is a consequence of impaired ability to undergo a critical conformational change upon binding to the core subunits, which is needed to expose the DNA-binding domains and confer promoter recognition capability upon holoenzyme.

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