Isolation and Characterization of σ70-Retaining Transcription Elongation Complexes from Escherichia coli

Abstract

sigma(70) subunit is thought to be released from the core RNA polymerase (RNAP) upon the transition from initiation to elongation or shortly afterward. Here, we identify a population of RNAP from E. coli that retains sigma(70) throughout elongation. The relative amount of this population appears to depend on cellular growth and reaches its maximum during the stationary phase. The proportion of sigma(70)-retaining elongation complexes (EC-sigma(70)) is invariant with various promoters or distances from the transcription start site. EC-sigma(70) responds to pauses, intrinsic terminators, and the elongation factor NusA similarly to EC without sigma(70). However, EC-sigma(70) has a substantially higher ability to support multiple rounds of transcription at certain promoters, suggesting its profound role in gene expression and regulation in bacteria.

The interaction between RNA polymerase and the elongation factor NusA

“…5,23,24 This would allow NusA to bind resulting in a transition complex comprising both factors, 23 consistent with data derived from in vitro experiments. 19 Binding of NusA is likely to induce structural changes in RNAP, which in turn could further reduce the affinity of σ interaction to RNAP. Our model is consistent with the findings that through NusA competition, the σ binding affinity dropped dramatically between initiating RNAP core and the elongation complex (from K d 10 -10 to 10 -6 M), 25 and that NusA is incorporated into the elongation complexes before σ is fully dissociated.…”

“…18 The resultant model accounted for the mutually exclusive binding of the two proteins and the C-terminal domains of NusA were also ideally situated around the β-flap region to interact with the emerging RNA for transcription regulation. 19 However, several other studies have indicated that transcription complexes can contain both σ and NusA in certain circumstances, 20,21 suggesting that alternative conformations of σ and NusA may be adopted permitting their simultaneous interaction with RNAP and that this working model would require some modification to account for all the experimental observations.…”

Altering the interaction between σ 70 and RNA polymerase generates complexes with distinct transcription-elongation properties

“…Transcripts were quantitated by using a PhosphorImager scan and IMAGEQUANT software (Molecular Dynamics). Retention of 70 His in stalled A29-TECs was determined by an indirect method (25).…”

“…The simplest explanation for these differences is that r-E 70 and n-E 70 differentially retain 70 in the TEC and that the presence of 70 alters the pausing and elongation properties of the enzyme. Therefore, we determined the amounts of 70 present in each TEC halted upstream of the pause site by using a procedure described by Bar-Nahum and Nudler (25). Paused ternary complexes with short radiolabeled transcripts were immobilized on Ni-NTA resin by virtue of their 70 His (25), and the fraction of the radiolabeled transcript retained on Ni-NTA was determined.…”

“…It is thought that dissociates shortly after passing the reiterated Ϫ10 region (23). E 70 from stationary cells may be refractory to dissociation as a significant fraction (Ϸ30%) of RNA polymerase purified from such cells retained during elongation (25).…”