T4 AsiA Research Articles

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.

Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA

The 10 kDa bacteriophage T4 antisigma protein AsiA binds the Escherichia coli RNA polymerase promoter specificity subunit, σ70, with high affinity and inhibits its transcription activity. AsiA binds to σ70 primarily through an interaction with σ70 conserved region 4.2, which has also been implicated in sequence-specific recognition of the −35 consensus promoter element. Here we show that AsiA forms a stable ternary complex with core RNA polymerase (RNAP) and σ70 and thus does not inhibit σ70 activity by preventing its binding to core RNAP. We investigated the effect of AsiA on open promoter complex formation and abortive initiation at two −10/−35 type promoters and two “extended −10” promoters. Our results indicate that the binding of AsiA to σ70 and the interaction of σ70 region 4.2 with the −35 consensus promoter element of −10/−35 promoters is mutually exclusive. In contrast, AsiA has much less effect on open promoter complex formation and abortive initiation from extended −10 promoters, which lack a −35 consensus element and do not require σ70 conserved region 4.2. From these results we conclude that T4 AsiA inhibits E. coli RNAP σ70 holoenzyme transcription at −10/−35 promoters by interfering with the required interaction between σ70 conserved region 4.2 and the −35 consensus promoter element.

Interaction of bacteriophage T4 AsiA protein with Escherichia coliσ70 and its variant

Bacteriophage T4 produces a small protein AsiA, which inhibits transcription from σ 70-dependent promoters in E. coli by tightly binding to σ 70 and is therefore termed as anti-sigma factor. We observed that there was no inhibition of single round transcription at lac UV5 promoter when AsiA was added to preformed open complex between RNA polymerase and template DNA. However, transcription was found to proceed normally at `extended −10' promoters in the presence of AsiA. It appears therefore that AsiA binds σ 70 at its 4.2-subdomain or in its close vicinity. Further experiments on immunoprecipitation of σ 70 and a mutant σ 70-V576G with AsiA seem to corroborate such conclusion.

Domain Organization of the Escherichia coliRNA Polymerase σ 70Subunit

We used limited trypsin digestion to determine the domain organization of the Escherichia coliRNA polymerase σ 70subunit. Trypsin-resistant fragments containing σ 70conserved region 2 (σ 70 2), and carboxy-terminal fragments containing conserved regions 3 and 4 (σ 70 3 – 4) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of σ 70·σ 70 2bound core RNA polymerase competitively with intact σ 70. In contrast to σ 70 2alone, the RNA polymerase holoenzyme formed with σ 70 2specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the −10 promoter element (the Pribnow box). σ 70 2also forms crystals that are suitable for X-ray analysis. σ 70 3 – 4bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on σ 70was further localized to within σ 70[551 – 608], comprising σ conserved region 4.2.

Characterization of Pre-transcription Complexes Made at a Bacteriophage T4 Middle Promoter: Involvement of the T4 MotA Activator and the T4 AsiA Protein, a σ 70Binding Protein, in the Formation of the Open Complex

Bacteriophage T4 middle promoters have excellent matches to the −10 consensus sequence for the σ 70subunit of Escherichia coliRNA polymerase, but a binding site for the T4 transcriptional activator MotA replaces the σ 70−35 consensus. E. coliRNA polymerase transcribes from middle promoters with or without the activator. In contrast, transcription by T4-modified E. coliRNA polymerase, which is present during T4 infection, requires MotA. We show that transcription by unmodified polymerase from the T4 middle promoter P uvsXis independent of the specific sequences within the −35 region, and the DNase I footprint obtained with unmodified polymerase and P uvsXresembles those seen previously with E. coli"extended −10" promoters. In contrast, although T4-modified polymerase alone binds P uvsX, promoter unwinding and detection of a DNase I footprint requires MotA. This footprint is significantly different from that obtained with unmodified polymerase, starting upstream of around position −20. Previous work has indicated that the T4 AsiA protein, which binds tightly to σ 70, is the phage modification required for MotA activation. We show that in the presence of AsiA, MotA, and otherwise unmodified polymerase, DNase I protection of P uvsXis now similar to that obtained with the fully modified polymerase and MotA up to around position −40. However, protection upstream of −40 is still similar to that seen with unmodified polymerase. Our results support the idea that MotA-dependent activation requires AsiA binding to σ 70to achieve specific protein-DNA contacts within the −20 to −40 region of a middle promoter.

Bacteriophage T4 MotA and AsiA proteins suffice to direct Escherichia coli RNA polymerase to initiate transcription at T4 middle promoters.

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 sigma 70-core RNA polymerase interactions and to inhibit some sigma 70-dependent transcription, presumably through binding to the sigma 70 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 sigma 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.