Interaction of bacteriophage T4 AsiA protein with Escherichia coliσ70 and its variant

Pahari, Susanta; Chatterji, Dipankar

doi:10.1016/s0014-5793(97)00668-6

Cited by 27 publications

(33 citation statements)

References 13 publications

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“…Transcription by E 70 from the galP1 promoter or from a promoter sequence lacking a Ϫ35 hexamer has been shown to be essentially insensitive to inhibition by AsiA (12,13,15). In agreement with this, we did not observe the marked lags described above with lacUV5 when we used a consensus galP1 promoter that lacks a Ϫ35 consensus sequence (data not shown).…”

Section: Existence Of a Ternary Complex Formed At The Lacuv5 Promotersupporting

confidence: 86%

“…These observations suggested the existence of a transcriptionally active ternary complex at lacUV5, a typical bacterial promoter with Ϫ10 and Ϫ35 recognition sequences. Active ternary complexes containing AsiA have been previously observed at several promoters that do not require a Ϫ35 motif for open complex formation (12,13,15). A number of convergent observations support the hypothesis of a ternary complex at a ؊10/Ϫ35 promoter.…”

Section: Discussionmentioning

confidence: 62%

“…We have studied the mechanism of inhibition exerted by AsiA when it is bound to E 70 acting upon a ؊10/Ϫ35 promoter in greater detail (13,15,16). By runoff transcription analysis on the lacUV5 promoter, we show that the species E 70 ⅐AsiA is much less inhibited in KGlu than in KCl (Fig.…”

Section: Discussionmentioning

confidence: 93%

“…Transcription inhibition by AsiA of E 70 , the RNA polymerase holoenzyme, has been conveniently analyzed with well defined DNA templates (13,15,16). Here, we performed single round transcriptions with a DNA linear fragment containing the lacUV5 promoter upstream of a strong transcriptional terminator and generating a 178-nucleotide transcript.…”

Section: Single Round Transcription Analysis In Kglu Buffers-mentioning

confidence: 99%

“…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 contain an "extended Ϫ10" motif (12,13,15,16). These observations led to a simple model in which the interactions of domain 4.2 in 70 with the Ϫ35 promoter element or with AsiA are mutually exclusive.…”

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confidence: 99%

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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: Existence Of a Ternary Complex Formed At The Lacuv5 Promotersupporting

confidence: 86%

Section: Discussionmentioning

confidence: 62%