An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription

Gerber, Jeffrey S.; Hinton, Deborah M.

doi:10.1128/jb.178.21.6133-6139.1996

Cited by 40 publications

(51 citation statements)

References 41 publications

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“…This modification plays a major role in regulating promoter utilization since it irreversibly blocks the use of the rescued pathway documented here. The efficiency of the E 70 ⅐AsiA complex will now crucially depend on the binding of MotA to those promoters containing the Ϫ30 middle promoter motif (47,48) and on the establishment of the proper contacts between the 70 subunit and MotA (49).…”

Section: Discussionmentioning

confidence: 99%

The Escherichia coli RNA Polymerase·Anti-ς70 AsiA Complex Utilizes α-Carboxyl-terminal Domain Upstream Promoter Contacts to Transcribe from a −10/−35 Promoter

Orsini

Kolb

Buc

2001

Journal of Biological Chemistry

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During infection of⅐AsiA to the lacUV5 promoter.In Escherichia coli RNA polymerase, the core enzyme E (subunit composition ␣ 2 ␤␤Ј) associates with the subunit to form the holoenzyme E, the species able to initiate transcription at specific promoter sites on bacterial or phage genomes. The nature and properties of the subunit present in a holoenzyme at a given promoter, together with the ability of the ␣-carboxyl-terminal domain (␣-CTD) 1 to recognize an upstream element of the promoter, determine whether and how often transcription will start at this site (1). These principles are nicely illustrated by the regulation of transcription during the development of phage T4 in E. coli. Here, all the phage genes are transcribed by the host RNA polymerase, the structure and molecular properties of which are modified by phagecoded proteins, resulting in the sequential utilization of early, middle, and late promoters (2, 3). Immediately after infection, T4 early promoters, with their bacterium-like Ϫ10 and Ϫ35 recognition sequences, are transcribed by E 70 , the host holoenzyme harboring 70 , the major host factor. T4 middle promoters contain a Ϫ10 element closely matching the Ϫ10 consensus sequence for 70 , but the Ϫ35 element is replaced by a so-called MotA box, a Ϫ30 binding site for the phage-coded middle transcriptional activator MotA (4, 5). In addition to E 70 and MotA, middle mode transcription also requires the presence of another phage early protein, the anti-factor AsiA (6). Upon binding the MotA box, MotA protein activates recognition of middle promoters by a holoenzyme E 70 ⅐AsiA complex (7, 8). Here, AsiA appears to act as a molecular device that switches 70 from the early to the middle class of T4 promoters. Transcription at late promoters is closely coupled with T4 DNA replication and requires a novel T4-encoded subunit, gp55 (9).Because it is a coactivator of transcription from T4 middle promoters and, simultaneously, a transcription inhibitor of bacterial or T4 early promoters (3), AsiA might also be able to cause the rapid arrest of transcription from early promoters, concomitant with the start of MotA-dependent middle transcription. Assigning this function is a long-standing and unresolved question in phage T4 biology (10). It has been recently shown, however, that this transcription shutoff also occurs in the absence of AsiA (11), although the same study confirmed that transcription of E. coli genes is rapidly and strongly inhibited in vivo when AsiA is overproduced.In vitro transcription studies have helped to outline the mechanism of inhibition by AsiA. This 10-kDa protein binds to region 4.2 of 70 . In the E 70 ⅐AsiA complex, this binding blocks the normal interaction between 70 and the Ϫ35 upstream promoter element (12)(13)(14). Although AsiA strongly inhibits open promoter complex formation and transcription by E 70 from a Ϫ10/Ϫ35 promoter like lacUV5 or the T4 early promoter P15.0, the holoenzyme is resistant to AsiA inhibition at promoters that, like galP1, lack a Ϫ35 consensus motif and conta...

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Section: Discussionmentioning

confidence: 99%

The Escherichia coli RNA Polymerase·Anti-ς70 AsiA Complex Utilizes α-Carboxyl-terminal Domain Upstream Promoter Contacts to Transcribe from a −10/−35 Promoter

Orsini

Kolb

Buc

2001

Journal of Biological Chemistry

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“…Purification of 70 was done as described (18,35) using E. coli BL21(DE3)/pLysS (36) containing the plasmid pLHN12 (35), which expresses rpoD. Wt MotA was purified as described (Ref.…”

Section: Methodsmentioning

confidence: 99%

“…1A). The basic, hydrophobic cleft formed from this arrangement interacts with 70 H5 (12), and MotA with mutations within the NTD bind DNA but fail to activate transcription (12,18,19). MotA CTD consists of a saddleshaped, double-wing motif generated by three ␣-helices interspersed with six ␤-strands (17) (Fig.…”

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confidence: 99%

Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation

Hsieh

James

Knipling

et al. 2013

Journal of Biological Chemistry

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Background:No structure exists of the bacteriophage T4 activator MotA with DNA. Results: Using FeBABE, physical models, and ICM Molsoft, we determined how MotA interacts with DNA within the transcription complex. Conclusion:The unusual "double-wing" motif in MotA CTD sits within the DNA major groove. Significance: FeBABE analyses together with structures can be used to determine protein-DNA architecture within multiprotein complexes.

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“…The best known target of these interactions is the ␣ subunit of RNAP (Ishihama 1992;Russo and Silhavy 1992;Ebright and Busby 1995;Niu et al 1996). Some activators, however, interact with the subunit (Hochschild 1994;Kuldell and Hochschild 1994;Li et al 1994;Artsimovitch et al 1996;Gerber and Hinton 1996), and evidence suggests that the ␤ subunit may also serve as an activation target in at least one case (Lee and Hoover 1995). Finally the ␤Ј subunit has been identified as the target of action of an activator that functions without binding to the DNA (Miller et al 1997).…”

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confidence: 99%

Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target

Dove¹,

Hochschild²

1998

Genes & Development

148

136

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Evidence obtained in both eukaryotes and prokaryotes indicates that arbitrary contacts between DNA-bound proteins and components of the transcriptional machinery can activate transcription. Here we demonstrate that the Escherichia coli protein, which copurifies with RNA polymerase, can function as a transcriptional activator when linked covalently to a DNA-binding protein. We show further that can function as an activation target when this covalent linkage is replaced by a pair of interacting polypeptides fused to the DNA-binding protein and to , respectively. Our findings imply that the protein is associated with RNA polymerase holoenzyme in vivo, and provide support for the hypothesis that contact between a DNA-bound protein and any component of E. coli RNA polymerase can activate transcription.[Key Words: subunit; E. coli; RNA polymerase; transcriptional activator] Received December 3, 1997; revised version accepted January 15, 1998. Recent findings in both eukaryotes and prokaryotes indicate that arbitrary protein-protein contacts can trigger gene activation provided one of the protein partners is tethered to the DNA and the other is a component (or is tethered to a component) of RNA polymerase (RNAP) (Barberis et al. 1995;Chatterjee and Struhl 1995;Klages and Strubin 1995;Xiao et al. 1995;Apone et al. 1996;Farrell et al. 1996;Dove et al. 1997;Gaudreau et al. 1997;Gonzalez-Couto et al. 1997;Lee and Struhl 1997; for review, see Ptashne and Gann 1997). Experiments in yeast have shown further that direct fusion of a DNA-binding domain to a component of the RNAP II holoenzyme can activate transcription from a promoter bearing a recognition site for the DNA-binding domain (Barberis et al. 1995;Farrell et al. 1996;Gaudreau et al. 1997; for review, see Ptashne and Gann 1997), but analogous experiments have not been performed previously in bacteria.RNAP in Escherichia coli consists of an enzymatic core composed of subunits ␣, ␤, and ␤Ј in the stoichiometry ␣ 2 ␤␤Ј, and one of several alternative factors that confer on the enzyme the ability to recognize specific promoters (Burgess 1976;Hellman and Chamberlin 1988). An additional protein, omega ( ), has been called a subunit of RNAP on the basis of its copurification with RNAP core and holoenzyme in near stoichiometric amounts (Burgess 1969). The function of is unknown and, unlike the other subunits, is not required for transcription either in vitro (Heil and Zillig 1970) or in vivo (Gentry and Burgess 1989;Gentry et al. 1991). Cells deleted for the gene encoding (rpoZ) have no discernible mutant phenotypes (Gentry and Burgess 1989;Gentry et al. 1991).Many natural activators in bacteria bind the DNA near the promoters they regulate and interact directly with one or more subunits of RNAP (Busby and Ebright 1994). The best known target of these interactions is the ␣ subunit of RNAP (Ishihama 1992;Russo and Silhavy 1992;Ebright and Busby 1995;Niu et al. 1996). Some activators, however, interact with the subunit (Hochschild 1994;Kuldell and Hochschild 1994;Li et al. 1...

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An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription

Cited by 40 publications

References 41 publications

The Escherichia coli RNA Polymerase·Anti-ς70 AsiA Complex Utilizes α-Carboxyl-terminal Domain Upstream Promoter Contacts to Transcribe from a −10/−35 Promoter

The Escherichia coli RNA Polymerase·Anti-ς70 AsiA Complex Utilizes α-Carboxyl-terminal Domain Upstream Promoter Contacts to Transcribe from a −10/−35 Promoter

Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation

Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target

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