SummaryThe AsiA protein, encoded by bacteriophage T4, inhibits E 70-dependent transcription at bacterial and early-phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the ¹35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C-terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of 70 , which recognizes the ¹35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary-phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a ¹35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by E 70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of 70 with the ¹35 hexamer.

Bacteriophage T4 development in Escherichia coli is regulated mainly at the transcriptional level. A number of phageencoded transcription factors modify both the structure and the function of the host RNA polymerase (RNAP), 1 which is responsible for the transcription of all T4 genes. These RNAP modifications result in the sequential utilization of the three classes of T4 promoters: early, middle, and late (1). Early promoters are transcribed immediately upon infection by unmodified host RNAP, which recognizes their bacterial-like promoter elements situated at Ϫ10 and Ϫ35 relative to the transcription start site (2). Shortly thereafter, the ␣ subunits of the RNAP are ADPribosylated by two phage-encoded proteins, Alt and Mod, and the RNAP is tightly bound by the products of T4 genes rpbA and asiA (3). Middle promoters contain an E. coli-like Ϫ10 consensus sequence, but lack an identifiable Ϫ35 region. This is replaced by a "Mot box" ((t/a)(t/a)TGCTT(t/c)A), a 9-bp sequence centered around Ϫ30, which is the binding site for the T4 transcriptional activator MotA (3, 4). Although it is possible that ADP-ribosylation and association with the RpbA protein modulate transcription in vivo, transcription initiation from a middle promoter in vitro requires only the E. coli RNAP holoenzyme, and the purified T4 proteins MotA and AsiA (5-7).AsiA is a 10.6-kDa protein that was first identified through its tight association with the 70 subunit of RNAP, and the resulting inhibition of transcription at E. coli and T4 early promoters (6, 8 -10). The interaction between AsiA and 70 is thought to decrease the affinity of 70 for the core polymerase (8, 10) and diminish the binding of RNA polymerase to phages T4 and T7 DNA (9). Nonetheless, AsiA is a coactivator of middle transcription, which also requires the 70 subunit (6). Therefore, the activity of AsiA cannot be merely a result of 70 -core dissociation. In light of its dual role, and the relative simplicity of the prokaryotic system in which it functions, the study of AsiA should provide some insight into how the activity of a major sigma factor is regulated through specific proteinprotein interactions.Numerous kinetic and structural studies of the complexes formed between the E. coli RNAP holoenzyme (R) and several promoter sequences (P) have resulted in the following basic model for productive transcription initiation (11-13).

Development of bacteriophage T4 in Escherichia coli requires the sequential recognition of three classes of promoters: early, middle, and late. Recognition of middle promoters is known to require the motA gene product, a protein that binds specifically to the "Mot box" located at the -30 region of these promoters. In vivo, the asiA gene product is as critical for middle mode RNA synthesis as is that of the motA gene. In vitro, AsiA protein is known to loosen the r70-core RNA polymerase interactions and to inhibit some oJ70-dependent transcription, presumably through binding to the cr7O subunit. Here we show that, in vitro, purified MotA and AsiA proteins are both necessary and sufficient to activate transcription initiation at T4 middle promoters by the E. coli RNA polymerase in a o-70-dependent manner. AsiA is also shown to inhibit recognition of T4 early promoters and may play a pivotal role in the recognition of all three classes of phage promoters.In the course of phage T4 development in Escherichia coli, all phage gene transcripts are synthesized by the host RNA polymerase (RNAP), whose structure and functional properties are modified by phage-coded proteins, leading to the sequential recognition of three different classes of promoters: early, middle, and late. The early promoters are recognized immediately after infection by unmodified host RNAP even when phage protein synthesis is inhibited. They are also recognized in vitro on T4 DNA by purified E. coli RNAP holoenzyme (a2313' a-70). During this early period, the host RNAP undergoes several T4-induced changes (1). Its a subunits are ADP-ribosylated (by the T4 alt and mod gene products), and it becomes tightly associated with at least two small T4 proteins, RpbA and AsiA. By 3 min after infection (at 30WC), some, if not all, of these early promoters are turned off (2, 3), but the mechanism of this transcriptional shutoff has not yet been elucidated. At about the same time, transcription from middle promoters is turned on. These promoters have a conserved "Mot box" sequence, 5'-(a/t)(a/t)TGCTT(t/c)A-3', centered around bp -30, 11-13 bases upstream of a standard E. coli -10 consensus sequence (4-6). The T4 late promoters have simpler sequence determinants, consisting primarily of the octamer TATAAATA at the -10 region. Transcription from these late promoters requires, among other things, the phage cr factor gp55 (7). In vitro, the E. coli a-70 has been shown to be dominant over gp55 in competition for core RNAP and reduces late promoter recognition (8,9). Yet, during the late period of T4 development, a-70 and gp55 coexist (and apparently cofunction) in the infected cell (10, 11).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1451Recognition of T4 middle promoters requires the early gene product MotA. MotA protein has been purified and is known to bind specifically to the -30 Mot box seque...