Antagonistic Regulation of Escherichia coli Ribosomal RNA rrnB P1 Promoter Activity by GreA and DksA

Katarzyna Potrykus,Daniel Vinella,Helen Murphy,Agnieszka Szalewska-Palasz,Richard D'Ari,Michael Cashel

doi:10.1074/jbc.m601531200

Abstract

The Escherichia coli proteins DksA, GreA, and GreB are all structural homologs that bind the secondary channel of RNA polymerase (RNAP) but are thought to act at different levels of transcription. DksA, with its co-factor ppGpp, inhibits rrnB P1 transcription initiation, whereas GreA and GreB activate RNAP to cleave back-tracked RNA during elongational pausing. Here, in vivo and in vitro evidence reveals antagonistic regulation of rrnB P1 transcription initiation by Gre factors (particularly GreA) and DksA; GreA activates and DksA inhibits. DksA inhibition is epistatic to GreA activation. Both modes of regulation are ppGpp-independent in vivo but DksA inhibition requires ppGpp in vitro. Kinetic experiments and studies of rrnB P1-RNA polymerase complexes suggest that GreA mediates conformational changes at an initiation step in the absence of NTP substrates, even before DksA acts. GreA effects on rrnB P1 open complex conformation reveal a new feature of GreA distinct from its general function in elongation. Our findings support the idea that a balance of the interactions between the three secondary channel-binding proteins and RNAP can provide a new mode for regulating transcription.

Highlights

The Escherichia coli proteins DksA, GreA, and GreB are all structural homologs that bind the secondary channel of RNA polymerase (RNAP) but are thought to act at different levels of transcription
Ally distinct from GreA and GreB [10], positions a pair of conserved acidic amino acid residues near the catalytic center. These acidic residues activate an intrinsic RNA phosphodiesterase activity of RNAP paused during elongation, leading to cleavage of nascent RNA, whose 3Ј end has threaded backwards relative to the catalytic center; this cleavage creates a 3Ј-hydroxyl near the catalytic center and restores the possibility of polymerization [11,12,13]
DksA and ppGpp can function both in vitro and in vivo as synergistic co-factors for negative as well as positive regulation of transcription initiation (19 –22). It requires reorientation of the pair of acidic residues, it has been proposed that DksA might anchor (p)ppGpp in one of two possible orientations near the catalytic center through Mg2ϩ ions jointly coordinated by ppGpp pyrophosphate residues, thereby stabilizing the bound co-factor and potentiating its regulatory effects [19]