Role of Escherichia coli RNA polymerase alpha subunit in modulation of pausing, termination and anti-termination by the transcription elongation factor NusA.

Abstract

The alpha subunit (alpha) of RNA polymerase (RNAP) is critical for assembly of polymerase and positive control of transcription initiation in Escherichia coli. Here, we report that alpha also plays a role in transcription elongation, and this involves a direct interaction between alpha and NusA factor. During in vitro transcription without NusA, alpha interacts with the nascent RNA, as revealed by photocrosslinking. When NusA is present, RNA crosslinks to NusA rather than to alpha. We show that this NusA-RNA interaction is diminished during transcription with an RNAP mutant that lacks the C-terminus of alpha beyond amino acid 235, including the so-called alpha CTD. The absence of alpha CTD also affects NusA's ability to enhance transcription pausing, termination at intrinsic terminators and anti-termination by the phage lambda Q anti-terminator, but not anti-termination by the lambda N anti-terminator. NusA functions are not recovered even when transcription with mutant RNAP is done with excess NusA, a condition which does restore NusA-RNA crosslinking. By affinity chromatography, we show that NusA interacts directly with alpha, and also with beta and beta', but not with mutant alpha. Hence, alpha-NusA interaction is vital for the control of transcript elongation and termination.