Mapping the molecular interface between the σ70 subunit of E. coli RNA polymerase and T4 AsiA

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: 77%

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

“…The first anti-factor identified was the AsiA protein of bacteriophage T4, which targets 70 (7,16,17); however, unlike most other well-characterized anti-factors, AsiA binds its cognate factor in the context of the RNAP holoenzyme (18). As a component of the 70 -containing holoenzyme, AsiA inhibits transcription from the Ϫ10/Ϫ35 class of promoters, but does not inhibit transcription from extended Ϫ10 promoters (18).Prior work has established that AsiA interacts directly with 70 region 4 and that this interaction is required for AsiA-dependent transcription inhibition (7,(19)(20)(21). Two mechanistic consequences of the AsiA/ 70 region 4 interaction have been described.…”

mentioning

confidence: 99%

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

Yuan

Nickels

Hochschild

2009

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...

“…Finally, the highly conserved region of 70 that interacts with AsiA, denoted region 4, is central to the function and regulation of many aspects of transcription, including specific DNA recognition. Therefore, a detailed structural analysis of AsiA is obligatory for a comprehensive understanding of the initial stages of transcription regulation during T4 infection and prokaryotic transcription in general.Some structural details of anti-factors and their complexes with cognate factors have recently been revealed (16)(17)(18)(19)(20). However, the only available high resolution structure for any anti-factor is that of the heat shock-induced chaperone DnaK, which has been shown to bind to the alternate factor 32 to regulate the heat shock response (21-24).…”

mentioning

confidence: 99%

“…Some structural details of anti-factors and their complexes with cognate factors have recently been revealed (16)(17)(18)(19)(20). However, the only available high resolution structure for any anti-factor is that of the heat shock-induced chaperone DnaK, which has been shown to bind to the alternate factor 32 to regulate the heat shock response (21-24).…”

mentioning

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...