Sigma subunit of Escherichia coli RNA polymerase loses contacts with the 3′ end of the nascent RNA after synthesis of a tetranucleotide

Abstract

We have used photocrosslinking to analyze the contacts between the 3′ end of the RNA and Escherichia coli RNA polymerase during the early steps of RNA synthesis using the nucleotide analog 8-azido-ATP (8-N 3-ATP). The crosslinking group on 8-N 3-ATP contacts the β, β' and σ subunits when the analog is bound to the holoenzyme. We show here that 8-N 3-ATP is a substrate for E. coli RNA polymerase and acts as an RNA chain terminator when incorporated into the 3′ end of nascent RNA. 8-N 3-AMP was incorporated uniquely at the 3′ end of tri-, tetra- and pentanucleotides synthesized from a poly[d(A-T)] template and at the 3′ end of pentanucleotides from two promoters (λ PSpr′ and E. coli rrnB P1). The oligonucleotides were covalently attached to the RNA polymerase by irradiation of transcription complexes with ultraviolet light. All RNAs labeled the β and β' subunits, but σ was contacted only by the trinucleotide and tetranucleotide on poly[d(A-T)]. Sigma is still present in transcription complexes containing the pentanucleotide on poly[d(A-T)], despite the lack of labeling. Neither pentanucleotide from the authentic promoters contacted σ. We conclude that as holoenzyme moves downstream, either two separate conformational changes occur, after synthesis of the trinucleotide and tetranucleotide, which result in movement of σ away from the nucleotide binding site or, alternatively, σ remains fixed relative to the DNA while the domain on core polymerase forming the nucleotide binding site moves downstream.