Regulation of bacterial RNA polymerase σ factor activity: a structural perspective

To initiate transcription from specific promoters, the bacterial RNA polymerase (RNAP) core enzyme must associate with the initiation factor , which contains determinants that allow sequence-specific interactions with promoter DNA. Most bacteria contain several factors, each of which directs recognition of a distinct set of promoters. A large and diverse family of proteins known as ''antifactors'' regulates promoter utilization by targeting specific factors. The founding member of this family is the AsiA protein of bacteriophage T4. AsiA specifically targets the primary factor in Escherichia coli, 70 , and inhibits transcription from the major class of 70 -dependent promoters. AsiA-dependent transcription inhibition has been attributed to a well-documented interaction between AsiA and conserved region 4 of 70 . Here, we establish that efficient AsiA-dependent transcription inhibition also requires direct protein-protein contact between AsiA and the RNAP core. In particular, we demonstrate that AsiA contacts the flap domain of the RNAP ␤-subunit (the ␤-flap). Our findings support the emerging view that the ␤-flap is a target site for regulatory proteins that affect RNAP function during all stages of the transcription cycle.anti-factor ͉ transcription initiation ͉ transcription regulation T he bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytically-active multisubunit core enzyme (␣ 2 ␤␤Ј ) in complex with a factor, which confers on the core enzyme the ability to initiate promoter-specific transcription (1). Bacteria typically contain a number of factors, each of which specifies recognition of a distinct class of promoters (2). The primary factor in Escherichia coli is 70 , and the 70 -containing holoenzyme is responsible for most transcription that occurs during the exponential phase of growth. In the context of the RNAP holoenzyme, 70 makes direct contact with 2 conserved promoter elements that are separated by Ϸ17 bp, the Ϫ10 and Ϫ35 elements (consensus sequences TATAAT and TTGACA, respectively). RNAP holoenzyme can also initiate transcription from promoters that lack a recognizable Ϫ35 element, but carry an extended Ϫ10 element (consensus TGnTATAAT) (3). At extended Ϫ10 promoters, additional contacts between 70 and the TG dinucleotide of the extended Ϫ10 element compensate for the lack of a Ϫ35 element (4). Primary factors share 4 regions of conserved sequence (regions 1-4), which have been further subdivided (1, 5). Structural work indicates that comprises 4 flexibly-linked domains: 1.1 (containing region 1.1), 2 (containing regions 1.2-2.4), 3 (containing regions 3.0 and 3.1), and 4 (containing regions 4.1 and 4.2) (1, 5-8). Regions 2, 3, and 4 contain DNA-binding domains responsible for recognition of the promoter Ϫ10 element, extended Ϫ10 element, and Ϫ35 element, respectively (1, 4, 5).Holoenzyme formation critically depends on a high-affinity interaction between 70 region 2 and a coiled-coil motif in the ␤Ј-subunit (the ␤Ј coiled coil, also referred to as the clamp helices) (9, 10). The in...

Section: Discussioncontrasting

confidence: 56%

mentioning

confidence: 99%

The bacteriophage T4 AsiA protein contacts the β-flap domain of RNA polymerase

Yuan

Nickels

Hochschild

2009

“…In complex with PhyR SL , NepR forms two helices that contact the solventaccessible surfaces of PhyR SL σ 2 and σ 4 , as well as the short helix α4 of the σ 2 -σ 4 linker. By comparison, in the X-ray crystal structures of both the Escherichia coli σ E -RseA complex (28) and the Rhodobacter sphaeroides σ E -ChrR complex (30), the anti-sigma domain (ASD) of RseA and ChrR is composed of four helices, the first three of which are sandwiched between σ 2 and σ 4 , occluding region σ 4.1 , where the RNA polymerase β flaptip-helix would bind, whereas helix 4 of the ASD occludes the RNA polymerase β coiled-coil binding surface of σ 2 (7,(31)(32)(33)(34) (corresponding to σ 2.1 and σ 2.2 ; Fig. 6A).…”

Section: Discussionmentioning

confidence: 99%

“…ECF sigma factors are often regulated through sequestration by cognate anti-sigma factors, which interfere with their ability to bind the RNA polymerase core enzyme or promoter regions by occluding their respective molecular determinants or by altering their conformations (5)(6)(7). The activity of anti-sigma factors must itself be regulated, and this is achieved by a variety of mechanisms, such as direct stimulus sensing followed by conformational changes, regulated intramembrane proteolysis by sensor proteases, or partner switches with anti-sigma factor antagonists (6,(8)(9)(10)(11).…”

mentioning

confidence: 99%

Structural basis for sigma factor mimicry in the general stress response of Alphaproteobacteria

Campagne

Damberger

Kaczmarczyk³

et al. 2012

Reprogramming gene expression is an essential component of adaptation to changing environmental conditions. In bacteria, a widespread mechanism involves alternative sigma factors that redirect transcription toward specific regulons. The activity of sigma factors is often regulated through sequestration by cognate anti-sigma factors; however, for most systems, it is not known how the activity of the anti-sigma factor is controlled to release the sigma factor. Recently, the general stress response sigma factor in Alphaproteobacteria, σ EcfG , was identified. σ EcfG is inactivated by the anti-sigma factor NepR, which is itself regulated by the response regulator PhyR. This key regulator sequesters NepR upon phosphorylation of its PhyR receiver domain via its σ EcfG sigma factor-like output domain (PhyR SL ). To understand the molecular basis of the PhyR-mediated partner-switching mechanism, we solved the structure of the PhyR SL –NepR complex using NMR. The complex reveals an unprecedented anti-sigma factor binding mode: upon PhyR SL binding, NepR forms two helices that extend over the surface of the PhyR SL subdomains. Homology modeling and comparative analysis of NepR, PhyR SL , and σ EcfG mutants indicate that NepR contacts both proteins with the same determinants, showing sigma factor mimicry at the atomic level. A lower density of hydrophobic interactions, together with the absence of specific polar contacts in the σ EcfG –NepR complex model, is consistent with the higher affinity of NepR for PhyR compared with σ EcfG . Finally, by reconstituting the partner switch in vitro, we demonstrate that the difference in affinity of NepR for its partners is sufficient for the switch to occur.

“…Concomitant with retraction of α11-β5, we observe that several water molecules infiltrate the binding interface between the PhyR receiver domain and PhyR-SL (Movie S1). An analysis of different σ/anti-σ complex structures in the Protein Data Bank (PDB) reveals that interaction between a σ factor and its cognate anti-σ factor can occur in a number of different ways (23)(24)(25). A flexible linker between regions σ 2 and σ 4 has been described as important for anti-σ interaction but also as a general structural feature required for proper association of σ with RNAP and with -10 and -35 sequences in the promoter (16,(26)(27)(28)(29).…”

Section: Discussionmentioning

confidence: 99%

Structural basis of a protein partner switch that regulates the general stress response of α-proteobacteria

Herrou¹,

Rotskoff²,

Luo³

et al. 2012

α-Proteobacteria uniquely integrate features of two-component signal transduction (TCS) and alternative sigma factor (σ) regulation to control transcription in response to general stress. The core of this regulatory system is the PhyR protein, which contains a σ-like (SL) domain and a TCS receiver domain. Aspartyl phosphorylation of the PhyR receiver in response to stress signals promotes binding of the anti-σ factor, NepR, to PhyR-SL. This mechanism, whereby NepR switches binding between its cognate σ factor and phospho-PhyR (PhyR∼P), controls transcription of the general stress regulon. We have defined the structural basis of the PhyR∼P/NepR interaction in Caulobacter crescentus and characterized the effect of aspartyl phosphorylation on PhyR structure by molecular dynamics simulations. Our data support a model in which phosphorylation of the PhyR receiver domain promotes its dissociation from the PhyR-SL domain, which exposes the NepR binding site. A highly dynamic loop–helix region (α3-α4) of the PhyR-SL domain plays an important role in PhyR∼P binding to NepR in vitro, and in stress-dependent activation of transcription in vivo. This study provides a foundation for understanding the protein-protein interactions and protein structural dynamics that underpin general stress adaptation in a large and metabolically diverse clade of the bacterial kingdom.