Mutational and Functional Analysis of a Segment of the Sigma Family Bacteriophage T4 Late Promoter Recognition Protein gp55

Wong, Kevin R.; Kassavetis, George A.; Leonetti, Jean-Paul; Geiduschek, E. Peter

doi:10.1074/jbc.m211447200

Cited by 16 publications

(13 citation statements)

References 48 publications

(61 reference statements)

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“…S5C), it copurified with RNAP to a much lesser extent than WT YvrHa. This supports the hypothesis that this region 2.2 motif in YvrHa is involved in core binding, consistent with results for conventional -factors (13)(14)(15).…”

Section: A P -T I P H E L I Xsupporting

confidence: 79%

“…3, Bottom), implying functional importance. Previously, mutations in -factor that negatively influence holoenzyme formation (presumably by disrupting interaction with the ␤Ј-subunit coiled coil) were mapped to this subsequence (13)(14)(15). To determine if this region is also critical for YvrHa function, we engineered mutations in this motif.…”

Section: Yvri Regionmentioning

confidence: 99%

“…Amino acids in regions 2.1 and 2.2 form a pair of ␣-helices that mediate core binding by virtue of interaction with a coiled-coil element in the amino terminus of the ␤Ј-subunit of core RNAP (8). This interaction is critical for holoenzyme formation, because a number of mutations in these interacting elements negatively influence -binding to core (9)(10)(11)(12)(13)(14)(15). This interaction is also important for -region 2.3-2.4 recognition of the single-and double-stranded -10 element within promoter DNA (16,17).…”

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confidence: 99%

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A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

MacLellan

Guariglia-Oropeza

Gaballa

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The -like factor YvrI and coregulator YvrHa activate transcription from a small set of conserved promoters in Bacillus subtilis. We report here that these two proteins independently contribute -region 2 and -region 4 functions to a holoenzyme-promoter DNA complex. YvrI binds RNA polymerase (RNAP) through a region 4 interaction with the ␤-subunit flap domain and mediates specific promoter recognition but cannot initiate DNA melting at the -10 promoter element. Conversely, YvrHa possesses sequence similarity to a conserved core-binding motif in -region 2 and binds to the N-terminal coiled-coil element in the RNAP ␤-subunit previously implicated in interaction with region 2 of -factors. YvrHa plays an essential role in stabilizing the open complex and interacts specifically with the N-terminus of YvrI. Based on these results, we propose that YvrHa is situated in the transcription complex proximal to the -10 element of the promoter, whereas YvrI is responsible for -35 region recognition. This system presents an unusual example of a two-subunit bacterial -factor.

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Section: A P -T I P H E L I Xsupporting

confidence: 79%

Section: Yvri Regionmentioning

confidence: 99%

mentioning

confidence: 99%

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A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

MacLellan

Guariglia-Oropeza

Gaballa

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…However, to date, only a few examples of regulatory proteins that target the ␤-flap have been described, including the bacteriophage T4-encoded coactivator of late-gene transcription gp33 (46) and the lambdoid phage-encoded Q antiterminator proteins (31). Gp33 functions in the context of a holoenzyme containing the phage-encoded factor, gp55, a truncated member of the 70 family that bears weak homology to region 2 (47). When bound to the ␤-flap, gp33 serves as a structural analogue to region 4, linking RNAP to the DNA indirectly, via an interaction with the sliding clamp protein gp45 (the activator of late-gene transcription) (46).…”

Section: Discussionmentioning

confidence: 99%

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

Yuan

Nickels

Hochschild

2009

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Second, the RNAP β' coiled coil and β flap of the T4 late holoenzyme are occupied by separate proteins: gp55 and gp33 bind to the RNAP core at sites that are the principal attachment points of σ 70 domain 2 (Wong et al, 2003) and domain 4 (Nechaev et al, 2004), respectively. Gp55 is the promoter recognition subunit of T4 late transcription and gp55-RNAP holoenzyme suffices for accurately initiating basal late transcription in vitro (Kassavetis and Geiduschek, 1984); a limited similarity of gp55 residues ∼42-122 with σ 70 domain 2 can be discerned (Gribskov and Burgess, 1986).…”

Section: Introductionmentioning

confidence: 99%

Dissection of the Bacteriophage T4 Late Promoter Complex

Nechaev

Geiduschek

2008

Journal of Molecular Biology

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Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator gp45 is the viral replisome's sliding clamp; two sliding clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) σ subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The C-terminal sliding clamp-binding epitopes of gp33 and gp55 are perfectly interchangeable, but the functions of these two RNAP-sliding clamp connections differ, with only the gp33-gp45 linkage essential for activation. Formation of transcription-ready promoter complexes by the sliding clamp-activated wild-type T4 RNAP resists competition by high concentrations of the polyanion heparin. This avid formation of promoter complexes requires both linkages of the T4 late RNAP to the sliding clamp. Preopening the promoter compensates for loss of the gp55-gp45 but not the gp33-gp45 linkage. We interpret these findings in relation to the common model of transcriptional initiation in bacteria and highlight the roles of individual interactions of the promoter complex.

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Mutational and Functional Analysis of a Segment of the Sigma Family Bacteriophage T4 Late Promoter Recognition Protein gp55

Cited by 16 publications

References 48 publications

A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

A two-subunit bacterial σ-factor activates transcription in Bacillus subtilis

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

Dissection of the Bacteriophage T4 Late Promoter Complex

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