T4 Middle Promoters Research Articles

Structural basis of σ appropriation.

Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite extensive biochemical and genetic analyses, puzzle remains, in part, because of a lack of precise structural information for σ appropriation complex. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact σ appropriation complex, comprising AsiA, MotA, Escherichia coli RNA polymerase (RNAP), σ70 and a T4 middle promoter. As expected, AsiA binds to and remodels σ region 4 to prevent its contact with host promoters. Unexpectedly, AsiA undergoes a large conformational change, takes over the job of σ region 4 and provides an anchor point for the upstream double-stranded DNA. Because σ region 4 is conserved among bacteria, other transcription factors may use the same strategy to alter the landscape of transcription immediately. Together, the structure provides a foundation for understanding σ appropriation and transcription activation.

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity.

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

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 σ(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 β-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 β subunit may influence interactions among MotA, AsiA, and RNA polymerase.

Direct activator/co‐activator interaction is essential for bacteriophage T4 middle gene expression

The bacteriophage T4 AsiA protein is a bifunctional regulator that inhibits transcription from the major class of bacterial promoters and also serves as an essential co-activator of transcription from T4 middle promoters. AsiA binds the primary s factor in Escherichia coli, sigma(70), and modifies the promoter recognition properties of the sigma(70)-containing RNA polymerase(RNAP) holoenzyme. In its role as co-activator, AsiA directs RNAP to T4 middle promoters in the presence of the T4-encoded activator MotA. According to the current model for T4 middle promoter activation, AsiA plays an indirect role in stabilizing the activation complex by facilitating interaction between DNA-bound MotA and sigma(70). Here we show that AsiA also plays a direct role in T4 middle promoter activation by contacting the MotA activation domain. Furthermore,we show that interaction between AsiA and the beta-flap domain of RNAP is important for co-activation. Based on our findings, we propose a revised model for T4 middle promoter activation, with AsiA organizing the activation complex via three distinct protein-protein interactions.

A basic/hydrophobic cleft of the T4 activator MotA interacts with the C‐terminus of E. coli σ70 to activate middle gene transcription

Transcriptional activation often employs a direct interaction between an activator and RNA polymerase. For activation of its middle genes, bacteriophage T4 appropriates Escherichia coli RNA polymerase through the action of two phage-encoded proteins, MotA and AsiA. Alone, AsiA inhibits transcription from a large class of host promoters by structurally remodelling region 4 of sigma(70), the primary specificity subunit of E. coli RNA polymerase. MotA interacts both with sigma(70) region 4 and with a DNA element present in T4 middle promoters. AsiA-induced remodelling is proposed to make the far C-terminus of sigma(70) region 4 accessible for MotA binding. Here, NMR chemical shift analysis indicates that MotA uses a 'basic/hydrophobic' cleft to interact with the C-terminus of AsiA-remodelled sigma(70), but MotA does not interact with AsiA itself. Mutations within this cleft, at residues K3, K28 and Q76, both impair the interaction of MotA with sigma(70) region 4 and MotA-dependent activation. Furthermore, mutations at these residues greatly decrease phage viability. Most previously described activators that target sigma(70) directly use acidic residues to engage a basic surface of region 4. Our work supports accumulated evidence indicating that 'sigma appropriation' by MotA and AsiA uses a fundamentally different mechanism to activate transcription.

Middle promoters constitute the most abundant and diverse class of promoters in bacteriophage T4

The temporally regulated transcription program of bacteriophage T4 relies upon the sequential utilization of three classes of promoters: early, middle and late. Here we show that middle promoters constitute perhaps the largest and the most diverse class of T4 promoters. In addition to 45 T4 middle promoters known to date, we mapped 13 new promoters, 10 of which deviate from the consensus MotA box, with some of them having no obvious match to it. So, 30 promoters of 58 identified now deviate from the consensus sequence deduced previously. In spite of the differences in their sequences, the in vivo activities of these T4 middle promoters were demonstrated to be dependent on both activators, MotA and AsiA. Traditionally, the MotA box was restricted to a 9 bp sequence with the highly conserved motif TGCTT. New logo based on the sequences of currently known middle promoters supports the conclusion that the consensus MotA box is comprised of 10 bp with the highly conserved central motif GCT. However, some apparently good matches to the consensus of middle promoters do not produce transcripts either in vivo or in vitro, indicating that the consensus sequence alone does not fully define a middle promoter.

Mutational Analysis of σ 70 Region 4 Needed for Appropriation by the Bacteriophage T4 Transcription Factors AsiA and MotA

Transcriptional activation of bacteriophage T4 middle promoters requires σ 70-containing Escherichia coli RNA polymerase, the T4 activator MotA, and the T4 co-activator AsiA. T4 middle promoters contain the σ 70 –10 DNA element. However, these promoters lack the σ 70 –35 element, having instead a MotA box centered at –30, which is bound by MotA. Previous work has indicated that AsiA and MotA interact with region 4 of σ 70, the C-terminal portion that normally contacts –35 DNA and the β-flap structure in core. AsiA binding prevents the σ 70/β-flap and σ 70/–35 DNA interactions, inhibiting transcription from promoters that require a –35 element. To test the importance of residues within σ 70 region 4 for MotA and AsiA function, we investigated how σ 70 region 4 mutants interact with AsiA, MotA, and the β-flap and function in transcription assays in vitro. We find that alanine substitutions at residues 584–588 (region 4.2) do not impair the interaction of region 4 with the β-flap or MotA, but they eliminate the interaction with AsiA and prevent AsiA inhibition and MotA/AsiA activation. In contrast, alanine substitutions at 551-552, 554-555 (region 4.1) eliminate the region 4/β-flap interaction, significantly impair the AsiA/σ 70 interaction, and eliminate AsiA inhibition. However, the 4.1 mutant σ 70 is still fully competent for activation if both MotA and AsiA are present. A previous NMR structure shows AsiA binding to σ 70 region 4, dramatically distorting regions 4.1 and 4.2 and indirectly changing the conformation of the MotA interaction site at the σ 70 C terminus. Our analyses provide biochemical relevance for the σ 70 residues identified in the structure, indicate that the interaction of AsiA with σ 70 region 4.2 is crucial for activation, and support the idea that AsiA binding facilitates an interaction between MotA and the far C terminus of σ 70.

The Bacteriophage T4 Inhibitor and Coactivator AsiA InhibitsEscherichia coliRNA Polymerase More Rapidly in the Absence of σ70Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70and Core

The N-terminal region (region 1.1) of sigma70, the primary sigma subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of sigma70 regions 2 and 4. Region 1.1 prevents the interaction of free sigma70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of sigma70-dependent transcription from promoters that require an interaction between sigma70 region 4 and the -35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with sigma70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that sigma70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P(uvsX). However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with sigma70 that lacks region 1.1 is less stable than polymerase with full-length sigma70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free sigma70 and then the AsiA/sigma70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free sigma70 plus core, yielding more free sigma70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free sigma70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free sigma70 arises through an indirect effect.

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

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 sigma(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 sigma(70) DNA element located in the -10 region. However, instead of the sigma(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 sigma(70) with the -10 region. This type of activation, which is called 'sigma appropriation', is fundamentally different from other well-characterized models of prokaryotic activation in which an activator frequently serves to force sigma(70) to contact a less than ideal -35 DNA element. This review summarizes the interactions of AsiA and MotA with sigma(70), and discusses how these interactions accomplish the switch to T4 middle promoters by inhibiting the typical contacts of the C-terminal region of sigma(70), region 4, with the host -35 DNA element and with other subunits of polymerase.

A Family of Anti-σ 70 Proteins in T4-type Phages and Bacteria that are Similar to AsiA, a Transcription Inhibitor and Co-activator of Bacteriophage T4

Anti-sigma70 factors interact with sigma70 proteins, the specificity subunits of prokaryotic RNA polymerase. The bacteriophage T4 anti-sigma70 protein, AsiA, binds tightly to regions 4.1 and 4.2 of the sigma70 subunit of Escherichia coli RNA polymerase and inhibits transcription from sigma70 promoters that require recognition of the canonical sigma70 -35 DNA sequence. In the presence of the T4 transcription activator MotA, AsiA also functions as a co-activator of transcription from T4 middle promoters, which retain the canonical sigma70 -10 consensus sequence but have a MotA box sequence centered at -30 rather than the sigma70 -35 sequence. The E.coli anti-sigma70 protein Rsd also interacts with region 4.2 of sigma70 and inhibits transcription from sigma70 promoters. Our sequence comparisons of T4 AsiA with Rsd, with the predicted AsiA orthologs of the T4-type phages RB69, 44RR, KVP40, and Aeh1, and with AlgQ, a regulator of alginate production in Pseudomonas aeruginosa indicate that these proteins share conserved amino acid residues at positions known to be important for the binding of T4 AsiA to sigma70 region 4. We show that, like T4 AsiA, Rsd binds to sigma70 in a native protein gel and, as with T4 AsiA, a L18S substitution in Rsd disrupts this complex. Previous work has assigned sigma70 amino acid F563, within region 4.1, as a critical determinant for AsiA binding. This residue is also involved in the binding of sigma70 to the beta-flap of core, suggesting that AsiA inhibits transcription by disrupting the interaction between sigma70 region 4.1 and the beta-flap. We find that as with T4 AsiA, the interaction of KVP40 AsiA, Rsd, or AlgQ with sigma70 region 4 is diminished by the substitution F563Y. We also demonstrate that like T4 AsiA and Rsd, KVP40 AsiA inhibits transcription from sigma70-dependent promoters. We speculate that the phage AsiA orthologs, Rsd, and AlgQ are members of a related family in T4-type phage and bacteria, which interact similarly with primary sigma factors. In addition, we show that even though a clear MotA ortholog has not been identified in the KVP40 genome and the phage genome appears to lack typical middle promoter sequences, KVP40 AsiA activates transcription from T4 middle promoters in the presence of T4 MotA. We speculate that KVP40 encodes a protein that is dissimilar in sequence, but functionally equivalent, to T4 MotA.

Thermoirreversible and Thermoreversible Promoter Opening by Two Escherichia coli RNA Polymerase Holoenzymes

Promoter opening, in which the complementary DNA strands separate around the transcriptional start site, is generally thermoreversible. An exceptional case of thermoirreversible opening of the T4 late promoter has been analyzed by KMnO4 footprinting and transcription. T4 late promoters, which consist of an 8-base pair (bp) TATA box "-10" element, are recognized by the small, phage-encoded, highly diverged sigma-family initiation subunit gp55. The T4 late promoter only opens above 15-20 degrees C, but once it has been formed remains open and transcriptionally active for days at -0.5 degrees C. The low temperature-trapped open complex and its isothermally formed state are shown to be structurally distinctive. Two "extended -10" sigma 70 promoters, which, like the T4 late promoter, lack "-35" sites, have been subjected to a comparative analysis: the T4 middle promoter PrIIB2 opens and closes thermoreversibly under conditions of basal and MotA- and AsiA-activated transcription. The open galP1 promoter complex, whose transcription bubble is very AT-rich, also closes reversibly upon shift to -0.5 degrees C, but more slowly than does the rIIB2 promoter. Formation of a trapped-open low temperature state of the promoter complex appears to be a singular property of gp55-RNA polymerase holoenzyme.

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

Bacteriophage T4 middle-mode transcription requires Escherichia coli RNA polymerase, phage-encoded transcriptional activator MotA and co-activator AsiA that form a complex at a middle promoter DNA. T4 middle promoters have been defined by a consensus sequence deduced from the list of 14 middle promoters identified in earlier studies. To date, 33 middle promoters have been mapped on the T4 genome. Of these, 12 contain differences even at the highly conserved positions of the consensus sequence. In the T4 prereplicative gene cluster between genes e and rpbA, we have identified 12 new middle promoters, most of which contain differences from the consensus sequence deduced previously. Analysis of base conservation in the different sequence positions of new middle promoters, as well as those identified previously, revealed some new features of middle T4 promoters. We propose to define these promoters by a MotA box (a/t)(a/t)(a/t)TGCTTtA centred at the position −30, the sequence TAtaAT centred at −10 relative to the transcriptional start site, and the spacer region of 12(±1) base-pairs between them.

Analysis of Regions within the Bacteriophage T4 AsiA Protein Involved in its Binding to the σ 70 Subunit of E. coli RNA Polymerase and its Role as a Transcriptional Inhibitor and Co-activator

Bacteriophage T4 AsiA, a protein of 90 amino acid residues, binds to the σ 70 subunit of Escherichia coli RNA polymerase and inhibits host or T4 early transcription or, together with the T4 MotA protein, activates T4 middle transcription. To investigate which regions within AsiA are involved in forming a complex with σ 70 and in providing transcriptional functions we generated random mutations throughout AsiA and targeted mutations within the C-terminal region. We tested mutant proteins for their ability to complement the growth of T4 asiA am phage under non-suppressing conditions, to inhibit E. coli growth, to interact with σ 70 region 4 in a two-hybrid assay, to bind to σ 70 in a native protein gel, and to inhibit or activate transcription in vitro using a T4 middle promoter that is active with RNA polymerase alone, is inhibited by AsiA, and is activated by MotA/AsiA. We find that substitutions within the N-terminal half of AsiA, at amino acid residues V14, L18, and I40, rendered the protein defective for binding to σ 70. These residues reside at the monomer–monomer interface in recent NMR structures of the AsiA dimer. In contrast, AsiA missing the C-terminal 44 amino acid residues interacted well with σ 70 region 4 in the two-hybrid assay, and AsiA missing the C-terminal 17 amino acid residues (Δ74–90) bound to σ 70 and was fully competent in standard in vitro transcription assays. However, the presence of the C-terminal region delayed formation of transcriptionally competent species when the AsiA/polymerase complex was pre-incubated with the promoter in the absence of MotA. Our results suggest that amino acid residues within the N-terminal half of AsiA are involved in forming or maintaining the AsiA/σ 70 complex. The C-terminal region of AsiA, while not absolutely required for inhibition or co-activation, aids inhibition by slowing the formation of transcription complexes between a promoter and the AsiA/polymerase complex.

The bacteriophage T4 transcription activator MotA interacts with the far-C-terminal region of the sigma70 subunit of Escherichia coli RNA polymerase.

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 sigma70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at -30, and also interacts with sigma70. We show here that the N-terminal half of MotA (MotA(NTD)), which is thought to include the activation domain, interacts with the C-terminal region of sigma70 in an E. coli two-hybrid assay. Replacement of the C-terminal 17 residues of sigma70 with comparable sigma38 residues abolishes the interaction with MotA(NTD) in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a sigma70 that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotA(CTD)) binds DNA with a K(D(app)) that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of sigma70 helps to substitute functionally for an interaction between sigma70 and a promoter -35 element.

Identification of two middle promoters upstream DNA ligase gene 30 of bacteriophage T4

Bacteriophage T4 DNA ligase gene 30 lies in the cluster of prereplicative genes located counterclockwise from map units 149 to 121. Based on the early transcription studies this gene has been considered as a typical early gene of bacteriophage T4. In agreement with this assignment, two strong T4 early promoters, P E 30.8 (128.6) and P E 30.7 (128.2), located about 3.1 and 2.7 kb upstream from gene 30 have been revealed by promoter mapping and sequence analysis. In addition, the existence of a putative early promoter just upstream of gene 30 was proposed from the sequence data. However, here we show that the putative early promoter just upstream of gene 30 is, in fact, a T4 middle promoter. Furthermore, we detected one more middle promoter located in the genomic region between early promoter P E 30.7 (128.2) and DNA ligase gene 30 in the coding region of gene 30.3. Both new middle promoters have differences from the consensus MotA box, while their −10 regions match the σ 70 consensus sequence very well. The 5′ ends of MotA-dependent transcripts directed from these promoters, as well as the kinetics of 5′ end accumulation in the cells, have been determined by primer extension analysis. The results of these analyses indicate that both MotA-dependent and MotA-independent promoters control the transcription of T4 DNA ligase gene 30 in vivo. Moreover, we show that the first transcripts for gene 30 are directed from its own middle promoter, P M 30.

The MotA transcription factor from bacteriophage T4 contains a novel DNA-binding domain: the 'double wing' motif.

MotA is a transcription factor from bacteriophage T4 that helps adapt the host Escherichia coli transcription apparatus to T4 middle promoters. We have determined the crystal structure of the C-terminal DNA-binding domain of MotA (MotCF) to 1.6 A resolution using multiwavelength, anomalous diffraction methods. The structure reveals a novel DNA-binding alpha/beta motif that contains an exposed beta-sheet surface that mediates interactions with the DNA. Independent biochemical experiments have shown that MotCF binds to one surface of a single turn of DNA through interactions in adjacent major and minor grooves. We present a model of the interaction in which beta-ribbons at opposite corners of the six-stranded beta-sheet penetrate the DNA grooves, and call the motif a 'double wing' to emphasize similarities to the 'winged-helix' motif. The model is consistent with data on how MotA functions at middle promoters, and provides an explanation for why MotA can form non-specific multimers on DNA.

Binding of the bacteriophage T4 transcriptional activator, MotA, to T4 middle promoter DNA: evidence for both major and minor groove contacts

During infection, the bacteriophage T4 transcriptional activator MotA, the co-activator AsiA, and host RNA polymerase are needed to transcribe from T4 middle promoters. Middle promoters contain a −10 region recognized by the σ 70 subunit of RNA polymerase and a MotA box centered at −30 that is bound by MotA. We have investigated how the loss or modification of base determinants within the MotA box sequence 5′TTTGCTTTA3′ (positions −34 to −26 of a middle promoter) affects MotA function. Gel retardation assays with mutant MotA boxes are consistent with the idea that MotA uses minor groove contacts upstream and major groove contacts downstream of the center GC, and does not require any specific base feature at the C · G base-pair at position −30. In particular, the 5-methyl residue on the thymine residue at position −29, a major groove contact, contributes to MotA binding, while converting the T · A at −32 to a C · I base-pair, a change that affects the major but nor the minor groove, yields a MotA box that is similar to wild-type. However, methylation interference analyses indicate that neither the binding of MotA nor the binding of polymerase/MotA/AsiA to the middle promoter P uvsX is inhibited by premethylation of guanine and adenine residues, suggesting that binding does not require minor groove contact with any specific T · A base-pair. Using gel retardation analyses, we calculate an apparent dissociation constant of 130 nM for MotA binding to the wild-type MotA box. Previous work has shown that the N-terminal region of MotA is needed for an interaction between MotA and σ 70. We suggest that this MotA-σ 70 interaction helps to stabilize the relatively weak interaction of MotA with the −30 region of middle promoter DNA.

Stimulation of bacteriophage T4 middle transcription by the T4 proteins MotA and AsiA occurs at two distinct steps in the transcription cycle.

The bacteriophage T4 encodes proteins that are responsible for tightly regulating mRNA synthesis throughout phage development in Escherichia coli. The three classes of T4 promoters (early, middle, and late) are utilized sequentially by the host RNA polymerase as a result of phage-induced modifications. One such modification is the tight binding of the T4 AsiA protein to the sigma70 subunit of the RNA polymerase. This interaction is pivotal for the transition between T4 early and middle transcription, since it both inhibits recognition of host and T4 early promoters and stimulates T4 middle mode synthesis. The activation of T4 middle transcription also requires the T4 MotA protein, bound specifically to its recognition sequence, the "Mot box," which is centered at position -30 of these promoters. Accordingly, the two T4 proteins working in concert are sufficient to effectively switch the transcription specificity of the RNA polymerase holoenzyme. Herein, we investigate the mechanism of transcription activation and report that, while the presence of MotA and AsiA increases the initial recruitment of RNA polymerase to a T4 middle promoter, it does not alter the intrinsic stability of the discrete complexes formed. In addition, we have characterized the RNA polymerase-promoter species by UV laser footprinting and followed their evolution from open into initiating complexes. These data, combined with in vitro transcription assays, indicate that AsiA and MotA facilitate promoter escape, thereby stimulating the production of full-length transcripts.

The MotA transcriptional activator of bacteriophage T4 binds to its specific DNA site as a monomer.

During bacteriophage T4 middle mode gene expression, the MotA transcription factor binds to T4 middle promoters at a -30 mot box consensus sequence to allow activation. Previous binding studies showed that MotA forms multiple gel-shifted complexes with DNA, and structural evidence suggested that MotA dimerizes upon DNA binding. We have shown that a short (13 bp) mot box DNA substrate binds MotA protein but fails to form slower migrating complexes. Therefore, the slower migrating complexes in gel shift assays are caused by DNA-mediated binding events. Competition experiments indicate that the slower migrating complexes are formed by nonspecific binding events, while the first-shifted complex is caused by specific binding to the mot box. Saturation binding experiments revealed that the stoichiometry of MotA binding to DNA is 1:1 in the first-shifted complex, while the slower complexes apparently contain MotA multimers. Gel shift assays using mixtures of MotA and a GST-MotA fusion protein supported the conclusion that the first-shifted complex contains one protein molecule bound to DNA. Furthermore, MotA monomers were cross-linked by glutaraldehyde under conditions where slower complexes exist, but not under conditions that lead to only the first-shifted complex. We conclude that MotA binds specifically to the mot box as a monomer and that additional nonspecific binding events require flanking DNA.

The Interaction between the AsiA Protein of Bacteriophage T4 and the ς70 Subunit of Escherichia coli RNA Polymerase

The AsiA protein of bacteriophage T4 binds to the sigma70 subunit of Escherichia coli RNA polymerase and plays a dual regulatory role during T4 development: (i) inhibition of host and phage early transcription, and (ii) coactivation of phage middle-mode transcription, which also requires the T4 DNA binding transcriptional activator, MotA. We report that the interaction between AsiA and sigma70 occurs with a 1:1 stoichiometry. When preincubated with RNA polymerase, AsiA is a potent inhibitor of open complex formation at the lac UV5 promoter, whereas it does not perturb preformed open or intermediate promoter complexes. DNase I footprinting and electrophoretic mobility shift analyses of RNA polymerase-DNA complexes formed at the T4 early promoter P15.0 show that AsiA blocks the initial RNA polymerase binding step that leads to the formation of specific closed promoter complexes. A contrasting result is obtained on the T4 middle promoter PrIIB2, where AsiA stimulates the formation of both closed complexes and open complexes. Therefore, we propose that AsiA modulates initial DNA binding by the RNA polymerase, switching promoter usage at the level of closed complex formation.