The asiA gene of bacteriophage T4 codes for the anti-sigma 70 protein

Abstract

The anti-&0 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the o70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme. We have partially purified the anti-&0 factor and obtained the sequence of a C-terminal peptide of this protein. Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590. This protein has been overproduced in a phage T7 expression system and partially purified. It shows a strong inhibitory activity towards &70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on o70-independent transcription (by RNA polymerase core enzyme). At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100. Our results corroborate the initial observations on the properties of the phage T4 10K anti-o70 factor, and we therefore propose that the gene which we call asi4, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor.During infection of Escherichia coli by phage T4, a large part of the program of viral gene expression is regulated at the transcriptional level. The host's RNA polymerase transcribes the 200 or so viral genes from different classes of T4-specific promoters (23). Some of these classes are recognized only after T4-coded functions modify this enzyme's specificity. The a subunits of RNA polymerase are covalently modified by ADP-ribosylation, and the enzyme binds a series of phage-coded proteins (13). Among these, the products of genes 33, 45, and 55 are required for late transcription (12). In particular, gp 55 (16) redirects RNA polymerase transcription initiation from T4 late promoters by replacing the E. coli c70 subunit. The product of gene rpbA (45) strongly binds to RNA polymerase core. A smaller RNA polymerase-binding gene product, the 10-kDa (10K) protein, copurifies with au7 on phosphocellulose and inhibits o70-directed transcription initiation by E. coli RNA polymerase holoenzyme (36). It is believed that the interaction of the T4 10K protein with bacterial c70 weakens o&0-core interaction; this, in turn, would allow gp 55 to successfully compete for core enzyme during T4 development (12). The 10K protein found in lysates from T4-infected cells strongly binds to RNA polymerase agarose affinity columns (27). Starting from this observation, we have previously used a biochemical approach to identify the gene encoding the T4 10K protein (25) T4 (36). This approach to the partial purification of gp asiA was chosen to minimize its progressive loss, observed during the purification of RNA polymerase from T4-infected cells (35). In Fig. 1, we show the autoradiography of an SDS-polyacrylamide gel illustrating the separation of the RNA polymerase-binding proteins coded by phage T4 after Bio-Rex 70 chromatography. The gp asiA is essentially found in...

Solution structure and stability of the anti-sigma factor AsiA: Implications for novel functions

“…The cloning, overexpression, and purification of AsiA have been described (5,12,26), as has preparation of isotopically labeled AsiA using isotopically labeled rich media (16). In addition, the asiA gene was subcloned into the pET-24b expression vector and transformed into E. coli BL21(DE3) for expression.…”

“…The bacteriophage T4-encoded AsiA protein, product of the asiA gene (5), and the first anti-factor to be discovered, binds tightly to the 70 subunit of the E. coli RNA polymerase holoenzyme (6)(7)(8)(9)(10), altering the specificity of the complex toward both phage and host promoters. Following infection by bacteriophage T4, the E. coli RNA polymerase is recruited to sequentially transcribe genes from the T4 early, middle, and late promoters.…”

Studies on the ω Subunit of Escherichia Coli RNA Polymerase

“…Several other polypeptides like NusA (Greenblatt and Li, 1981) and GreA (Sparkowski and Das, 1990;Borukhov et al, 1992) proteins bind RNA polymerase and are required for efficient transcription termination/antitermination and elongation, respectively, but they play no role in simple RNA synthesis. Other proteins are found in different preparations of RNA polymerase whose functions are unknown, for example the stringent starvation protein (Ishihama and Saitoh, 1979) and a small protein associated with RNA polymerase after T4 infection now identified as anti-a (Stevens, 1972;Orsini et al, 1993). The w subunit of molecular mass 10 105 Da is another small protein detected in all E. coli RNA polymerase preparations (Burgess, 1969).…”

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

“…Typically, anti-factors interact with core binding determinants in their cognate factors, thereby preventing their association with the RNAP core enzyme (15). The first anti-factor identified was the AsiA protein of bacteriophage T4, which targets 70 (7,16,17); however, unlike most other well-characterized anti-factors, AsiA binds its cognate factor in the context of the RNAP holoenzyme (18). As a component of the 70 -containing holoenzyme, AsiA inhibits transcription from the Ϫ10/Ϫ35 class of promoters, but does not inhibit transcription from extended Ϫ10 promoters (18).…”