Twelve New MotA-dependent Middle Promoters of Bacteriophage T4: Consensus Sequence Revised

Truncaitė, Lidija; Piešiniene, Lina; Kolesinskiene, Giedre; Zajančkauskaitė, Aurelija; Driukas, Audrius; Klausa, Vytautas; Nivinskas, Rimas

doi:10.1016/s0022-2836(03)00125-6

Cited by 20 publications

(41 citation statements)

References 47 publications

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“…3, third lane of each middle promoter panel). This low level of middle promoter RNA has been observed by other investigators in T4 motA(Am) infections of wt E. coli (11,24,49,51). Previous work has demonstrated that deletion of motA is lethal (5), suggesting that the motA(Am) mutation is leaky.…”

Section: Resultssupporting

confidence: 68%

“…Amber (Am) and temperature sensitive (Ts) mutants exhibit a delay in DNA synthesis and form small plaques (25,33) while motA and asiA deletions are lethal (5,37). Analyses of RNA isolated from motA(Am) mutant infections reveal that these mutants are leaky; although transcription from many middle promoters is nearly eliminated, for particular promoters, a low level of RNA can still be detected (11,24,49,51). The mutant E. coli strain TabG was originally identified by its inability to support T4 motA mutant growth (39).…”

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confidence: 99%

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A Mutation within the β Subunit ofEscherichia coliRNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

2010

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During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coactivator AsiA, which are known to interact with 70 , the specificity subunit of RNA polymerase. T4 motA amber [motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4 motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG contains two mutations within its ␤-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of the ␤ subunit may influence interactions among MotA, AsiA, and RNA polymerase.Bacterial RNA polymerase (RNAP) is a highly conserved enzyme that shares sequence and structural homology with multisubunit polymerases from single-celled archaea to multicellular eukaryotes (19,55). In bacteria, an RNAP core consisting of five subunits (␣ 1 , ␣ 2 , ␤, ␤Ј, and ) combines with a specificity factor, , to form holoenzyme (13). A primary factor, such as 70 of Escherichia coli, is used for promoter recognition during exponential growth; alternate factors are needed under specific growth conditions or times of stress (10, 34). In addition, proteins that bind to RNAP and/or the DNA can influence the activity of polymerase (2,4,16,18,42).During infection of E. coli, bacteriophage T4 relies on the host transcriptional machinery for transcription from phage early, middle, and late promoters (reviewed in references 16, 26, and 54). Immediately after infection, synthesis of early RNA is driven by T4 early promoters, which contain strong matches to Ϫ10 and Ϫ35 DNA elements recognized by 70 . Expression of T4 middle genes is delayed, commencing after 1 to 2 min. Middle RNA is generated both from early promoters, whose transcripts extend into middle genes, and from specific middle promoters. While middle promoters contain the 70 -dependent Ϫ10 element, they have a Ϫ30 element (MotA box) rather than the Ϫ35 element recogniz...

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Section: Resultssupporting

confidence: 68%

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confidence: 99%

A Mutation within the β Subunit ofEscherichia coliRNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

2010

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“…Thus, a strong match to the core GCTT motif is not an absolute requirement for MotA function. In contrast, an excellent match to the canonical s 70 210 sequence is an invariant feature of middle promoters (Marshall et al, 1999;Stitt & Hinton, 1994;Truncaite et al, 2003). These results have suggested that a good interaction between the s 70 region 2.4 and the 210 DNA element is crucial for middle promoter usage.…”

Section: T4 Middle Promoters and A Model For Middle Promoter Activationmentioning

confidence: 99%

“…An analysis of more than 30 middle promoters has indicated that for most of these promoters, there is an excellent match to this consensus sequence, particularly in the core GCTT motif (Marshall et al, 1999;Stitt & Hinton, 1994;Truncaite et al, 2002Truncaite et al, , 2003. However, mutational analyses of a typical middle promoter sequence indicate that single base-pair changes within the MotA box are usually acceptable, and functional middle promoters with deviant sequences have been identified (Marshall et al, 1999).…”

Section: T4 Middle Promoters and A Model For Middle Promoter Activationmentioning

confidence: 99%

“…These promoters contain the s 70 210 DNA element and are dependent on RNA polymerase containing s 70 for transcription, but they lack the s 70 235 DNA element. Instead they have a different consensus sequence (a MotA box, 59-atTGCTTtA-39) centred at 230 (Marshall et al, 1999;Stitt & Hinton, 1994;Truncaite et al, 2003). Activation of middle promoters requires a T4 transcription activator, MotA (Hinton, 1991;Mattson et al, 1974), and a T4 transcription co-activator, AsiA (Hinton et al, 1996b;Ouhammouch et al, 1994Ouhammouch et al, , 1995Stevens, 1973).…”

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Transcriptional takeover by σ appropriation: remodelling of the σ 70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA

et al. 2005

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Activation of bacteriophage T4 middle promoters, which occurs about 1 min after infection, uses two phage-encoded factors that change the promoter specificity of the host RNA polymerase. These phage factors, the MotA activator and the AsiA co-activator, interact with the s 70 specificity subunit of Escherichia coli RNA polymerase, which normally contacts the "10 and "35 regions of host promoter DNA. Like host promoters, T4 middle promoters have a good match to the canonical s 70 DNA element located in the "10 region. However, instead of the s 70 DNA recognition element in the promoter's "35 region, they have a 9 bp sequence (a MotA box) centred at "30, which is bound by MotA. Recent work has begun to provide information about the MotA/AsiA system at a detailed molecular level. Accumulated evidence suggests that the presence of MotA and AsiA reconfigures protein-DNA contacts in the upstream promoter sequences, without significantly affecting the contacts of s 70 with the "10 region. This type of activation, which is called 's appropriation', is fundamentally different from other well-characterized models of prokaryotic activation in which an activator frequently serves to force s 70 to contact a less than ideal "35 DNA element. This review summarizes the interactions of AsiA and MotA with s 70 , and discusses how these interactions accomplish the switch to T4 middle promoters by inhibiting the typical contacts of the C-terminal region of s 70 , region 4, with the host "35 DNA element and with other subunits of polymerase. OverviewUpon infection of Escherichia coli, bacteriophage T4 establishes its own developmental cycle. Within 20 min, the phage programmes the generation of approximately 200 copies of its genome, the packaging of that DNA, and finally its escape from the host by lysis (reviewed by Miller et al., 2003). Regulation of this cycle is achieved largely by phage promoters, which sequentially express early, middle and late phage genes. Because T4 does not encode its own RNA polymerase, it must direct the host transcriptional machinery to these phage promoters at the correct time during infection. T4 encodes factors that accomplish this takeover by altering the specificity of the host E. coli RNA polymerase as infection proceeds (reviewed by Miller et al., 2003;Stitt & Hinton, 1994).E. coli RNA polymerase consists of a core of five subunits (a 2 , b, b9 and v), which contains the RNA-synthesizing activity, and a s factor that binds to a specific promoter sequence and sets the start site for transcription (reviewed by Gruber & Gross, 2003;Paget & Helmann, 2003 and a 235 element, having a consensus sequence of 59-TTGACA-39 (Campbell et al., 2002;Gardella et al., 1989;Keener & Nomura, 1993;Murakami et al., 2002b;Siegele et al., 1989;Vassylyev et al., 2002;Waldburger et al., 1990).(All sequences are given as the top, i.e. the non-template, strand of DNA.) To a first approximation, the strength of a host promoter reflects the match between its 210 and 235 sequences and the canonical sequences for these region...

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Identification of a promoter motif regulating the major DNA damage response mechanism of

Gamulin¹,

Ćetković²,

Ahel³

2004

FEMS Microbiology Letters

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The principal response of many bacteria to DNA damage is mediated by a mechanism dependent on the LexA and RecA proteins. However, Mycobacterium tuberculosis was recently reported to regulate a majority of DNA repair genes independently of RecA and LexA, suggesting that an unknown RecA/LexA-independent mechanism controls the major DNA damage response pathway in this organism. Here we have identified a motif tTGTCRgtg-8nt-TAnnnT that defines a novel RecA/LexA-independent promoter (RecA-NDp) of M. tuberculosis. Furthermore, we show that the RecA-NDp type of promoter precedes DNA repair genes in other Actinomycetales.

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Twelve New MotA-dependent Middle Promoters of Bacteriophage T4: Consensus Sequence Revised

Cited by 20 publications

References 47 publications

A Mutation within the β Subunit ofEscherichia coliRNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

A Mutation within the β Subunit ofEscherichia coliRNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

Transcriptional takeover by σ appropriation: remodelling of the σ 70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA

Identification of a promoter motif regulating the major DNA damage response mechanism of

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