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

Abstract

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...

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

“…In contrast, our results indicate that AsiA interacts with 1 structural domain of 70 (region 4) and 1 structural domain of RNAP core enzyme (the ␤-flap). This difference may reflect the fact that AsiA has an additional function that requires its presence as a stable component of the RNAP holoenzyme: to serve as a coactivator of T4 middle gene transcription (33). T4 middle promoters, which are recognized by the 70 -containing RNAP holoenzyme, bear a near-consensus Ϫ10 element and a binding site for the T4-encoded transcription activator MotA, centered at position Ϫ30 (34-36).…”

“…Activation of T4 middle gene transcription, which depends on the AsiA-containing RNAP holoenzyme, requires an interaction between DNA-bound MotA and 70 region 4 (37). Genetic analysis of the MotA/ 70 region 4 interaction suggests that MotA interacts with determinants of 70 that would be occluded when 70 region 4 is bound to the ␤-flap (37); thus it has been proposed that the role of AsiA as a coactivator of middle gene transcription is to expose this otherwise occluded surface of 70 region 4 so it is accessible to MotA (33,38).…”

Testing hypotheses for the presence of tRNA genes in mycobacteriophage genomes

“…2,6 The products of some phage genes modify the bacterial RNA polymerases to increase transcription of early phage genes. [7][8] In addition, phages can encode their own tRNA genes, and the occasional presence of the tRNA genes has been called intriguing. [9][10] The complement of genes found in a phage is understood to represent a dynamic between gene acquisition via recombination events involving horizontal (or lateral) gene transfer from the genomes of their hosts and/or of co-infecting phages and retention through positive selection.…”

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

“…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 .…”

“…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).…”