Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA 1 1Edited by E. Ebright

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the ␤ subunit and conserved region 4 of the subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the region 4͞␤-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the ␤-flap for binding to region 4, thereby inhibiting transcription initiation by disrupting the region 4͞␤-flap interaction.T he bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytically proficient core enzyme (subunit structure ␣ 2 ␤␤Ј ) and a subunit that confers on the holoenzyme the ability to recognize specific promoter sequences (1). The primary factor in Escherichia coli is 70 , and the 70 -containing holoenzyme (E 70 ) typically recognizes promoters defined by two conserved sequence elements (the Ϫ10 and Ϫ35 hexamers) positioned roughly 10 and 35 bp upstream of the transcription start point (1). Most factors share four conserved regions (2), and of these, regions 2 and 4 interact with the Ϫ10 and Ϫ35 elements, respectively (1). Nevertheless, intact 70 recognizes specific promoter sequences only in the context of the holoenzyme because of critical conformational changes in 70 that take place when it associates with the core enzyme (3). These include an increase in the interdomain distance between regions 2 and 4 that is caused by an interaction between region 4 and the ␤-flap domain (4). The region 4͞␤-flap interaction is essential for the recognition of Ϫ10͞Ϫ35 promoters because it positions regions 2 and 4 of 70 for simultaneous interaction with the promoter Ϫ10 and Ϫ35 elements (4). This role of the region 4͞␤-flap interaction suggested that there may exist regulatory factors that inhibit transcription from Ϫ10͞Ϫ35 promoters by disrupting the region 4͞␤-flap interaction (4, 5).Here we examine the mechanism of action of the bacteriophage T4-encoded anti-factor AsiA. AsiA is known to bind tightly to region 4 of 70 and inhibit transcription from Ϫ10͞Ϫ35 promoters when complexed with the holoenzyme (6-10). The finding that AsiA binds to region 4 of 70 and inhibits transcription specifically from Ϫ10͞Ϫ35 promoters raises the possibility that AsiA works by disrupting the 70 region 4͞␤-flap interaction, a model that is consistent with recent NMR analysis (11). We provide genetic evidence that AsiA and the ␤-flap interact with overlapping determinants on 70 region 4. We then present complementary biochemical and biophysical evidence that AsiA works by a competitive binding mechanism, inhibiting transcription from Ϫ10͞Ϫ35 promoters by disrupting the region 4͞␤-flap interaction in the context of the holoenzyme. Materials and MethodsMutant Screen. Mutagenic PCR was u...

supporting

confidence: 78%

Section: Resultsmentioning

confidence: 99%

A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme

Nickels

Garrity²,

Severinova³

et al. 2004

“…Until recently, AsiA was proposed to interact exclusively with a single highly conserved region of 70 , denoted region 4.2 (approximately residues 570-599, an HTH DNA binding motif). Both the ability of AsiA to inhibit transcription at phage early promoters and its ability to facilitate transcription at phage middle promoters were considered to result from this single interaction (56,57). We have shown recently (25) that AsiA also exhibits a high affinity for conserved region 4.1 of 70 (approximately residues 540-565).…”

Section: Resultsmentioning

confidence: 99%

Solution structure and stability of the anti-sigma factor AsiA: Implications for novel functions

Urbauer

Simeonov

Urbauer

et al. 2002

Anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors. The bacteriophage T4 anti-sigma factor AsiA is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of Escherichia coli, 70 . AsiA is an all-helical, symmetric dimer in solution. The solution structure of the AsiA dimer reveals a novel helical fold for the protomer. Furthermore, the AsiA protomer, surprisingly, contains a helix-turn-helix DNA binding motif, predicting a potential new role for AsiA. The AsiA dimer interface includes a substantial hydrophobic component, and results of hydrogen͞deuterium exchange studies suggest that the dimer interface is the most stable region of the AsiA dimer. In addition, the residues that form the dimer interface are those that are involved in binding to 70 . The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to 70 , where an AsiA protomer is displaced from the dimer via the interaction of 70 with the same residues in AsiA that constitute the dimer interface. R egulation of prokaryotic transcription involves the interaction of sigma ( ) factors, which bind to the core RNA polymerase to impart promoter recognition specificity, with cognate anti-sigma (anti-) factors that inhibit function. Most of the numerous examples of ͞anti-pairs involve alternative factors, those activated in response to specific stimuli, as opposed to primary factors, which are essential for survival and responsible for the bulk of transcription, including the housekeeping genes (reviewed in refs. 1-4). An important exception is the interaction of 70 of Escherichia coli, namesake for a family of sequence conserved primary factors, with the anti-factor AsiA.The bacteriophage T4-encoded AsiA protein, product of the asiA gene (5), and the first anti-factor to be discovered, binds tightly to the 70 subunit of the E. coli RNA polymerase holoenzyme (6-10), altering the specificity of the complex toward both phage and host promoters. Following infection by bacteriophage T4, the E. coli RNA polymerase is recruited to sequentially transcribe genes from the T4 early, middle, and late promoters. T4 early promoters contain bacterial-like consensus DNA sequences that allow for their immediate recognition by the unmodified RNA polymerase, and the T4 early genes include the asiA gene. Shortly thereafter, transcription at early promoters is inhibited, along with transcription at bacterial promoters, by phage-induced modifications of the RNA polymerase, one of which is the tight association of AsiA with 70 . This interaction inhibits 70 -dependent transcription at early promoters by blocking recognition by 70 of the conserved sequence element centered at position Ϫ35 (11). Furthermore, AsiA not only has the ability to function as an anti-factor, it also has the ability to promote transcription. In conce...

“…As a control for the sensitivity for the chemical shift perturbation method, we measured the effect on the NMR spectra of adding ligands known to bind to region 4.2. Addition of T4 AsiA, a Ϸ90-residue antifactor known to bind to E. coli 70 region 4.2 (19,20), resulted in significant changes in the NMR spectra of ⌬1.1-A* (Fig. 5 A and B and Table 1).…”

Section: Methodsmentioning

confidence: 99%

Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

Camarero

Shekhtman

Campbell

et al. 2002

115

Bacterial factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequencespecific interactions with the ؊10 and ؊35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the ؊35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a 70 -like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and͞or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.T he 400-kDa bacterial core RNA polymerase (RNAP) is fully active in RNA polymerization but is incapable of promoter recognition and specific transcription initiation. Promoter recognition, promoter melting to form the open complex, and possibly other functions during transcription initiation, depend on the binding of a factor to the core RNAP (subunit composition ␣ 2 ␤␤Ј ) to form the RNAP holoenzyme (reviewed in ref. 1). The primary factor in Escherichia coli, responsible for the bulk of transcription during exponential growth, is 70 . Sequence comparisons reveal that 70 belongs to a large homologous family of proteins with four regions of highly conserved amino acid sequence (2-5) (Fig. 1).The factors direct the process of transcription initiation by first locating the promoter through sequence-specific recognition of two hexamers of consensus DNA; the Pribnow box or Ϫ10 element, centered at about Ϫ10 with respect to the transcription start site (ϩ1), and the Ϫ35 element (6, 7). Genetic and biophysical evidence strongly suggested that amino acid residues in region 4.2 recognize the Ϫ35 element (8, 9), and this was confirmed by structural studies (10). Nonetheless, most functions of , including sequence-specific DNA binding, manifest themselves only in the context of the RNAP holoenzyme. In the absence of core RNAP, 70 -like factors are unable to recognize promoter DNA in either double-or single-stranded form (11). Specific interactions between N-terminally truncated derivatives of 70 and promoter DNA were detected by using competitive filter-binding assays (11,12), leading to the hypothesis that the latent DNA-binding activity of is inhibited by the N-terminal region 1.1, and that this inhibition is relieved by conformational changes upon binding of to core RNAP. Studies of isolated 70 fragments revealed that region 1.1 inhibited region 4.2 binding to the Ϫ35 element in trans, but not region 2 binding to the Ϫ10 element. These results led to a more detailed model in which region 1.1 directly masks the DNA-binding determinants of region 4.2 (11, 12). I...