Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the 70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at ؊30, and also interacts with 70 A programmed cascade of transcriptional events is initiated when bacteriophage T4 infects its host Escherichia coli (reviewed in reference 57). T4 early genes are transcribed immediately after infection by using the existing host RNA polymerase holoenzyme comprising the core (␣ 2 ␤␤Ј) and the 70 subunit. Early T4 promoters do not require T4-encoded transcription factors, since they contain excellent matches to the ideal 70 sequences in their Ϫ10 and Ϫ35 regions (61; reviewed in reference 60). In contrast, transcription from T4 middle promoters uses two T4 early gene products, the transcriptional activator MotA and the coactivator AsiA (23,39,43,44; reviewed in reference 57). Late promoter utilization requires the replacement of 70 by the T4 sigma factor, gp55, as well as other phage-encoded activators and coactivators (reviewed in reference 62).The MotA protein binds as a monomer (6, 33) to a 9-bp element (MotA box) centered at position Ϫ30 of middle promoter DNA (3,19,23). In addition, MotA forms a complex with 70 (18). Nuclear magnetic resonance and crystallographic studies indicate that the 211 amino acids of MotA are organized into an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a small flexible linker (15,33,34 (44,55,56). Binding sites for AsiA have been mapped within C-terminal amino acids (regions 4.1 and 4.2) of 70 (8,49,50,53,59). Residues within region 4.2 normally contact the Ϫ35 element of host promoter DNA (5,9,17,29,54). In the absence of MotA, AsiA binding to 70 inhibits transcription by polymerase from promoters that require recognition of the Ϫ35 canonical sequences (8,45,51), suggesting that the presence of AsiA inhibits the 70 region 4.2-DNA interaction. In this paper we show that the interaction of a MotA Nterminal peptide (amino acids 1 to 97) with 70 , like the interaction of AsiA with 70 , involves the C-terminal region of 70 . In addition, deletions of the amino acids within the far-Cterminal region of 70 (amino acids 604 to 613) impair the ability of RNA polymerase to perform MotA-dependent activation in vitro. We also show that a MotA C-terminal peptide, beginning at amino acid 102, binds DNA with an apparent dissociation constant like that of wild-type MotA. Our results support a model for MotA-dependent activation in which the interaction between the DNA-bound MotA and the C-terminal region of 70 helps to substitute functionally for an interaction between 70 and a promoter Ϫ35 element. MATERIALS AND METHODSStrains. E. coli KS1 (12) contains a chromosomal lacZ reporter gene under the control of a derivative of the lac promoter P lac that carries a lambda operator (O R 2) centered at position Ϫ62 in place of the ...

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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...