Substitutions in bacteriophage T4 AsiA and Escherichia coli sigma(70) that suppress T4 motA activation mutations.

Bacteriophage T4 uses three stages of transcription: early, middle, and late. This temporal progression is achieved by using three classes of promoters and by modifying the host RNA polymerase. Middle transcription requires two phage-encoded proteins, the MotA transcription factor and a coactivator, AsiA (for a review, see reference 37). AsiA binds to the ς70 subunit of host RNA polymerase and inhibits transcription from most Escherichia coli promoters. MotA binds to T4 middle promoters at the mot box, a consensus sequence centered at −30 (13, 30). The C- and N-terminal domains of MotA were solved by nuclear magnetic resonance and X-ray crystallography, respectively (9, 10). The C-terminal domain binds DNA, while the N-terminal domain is involved in transcriptional activation (10, 11). Based on the structural data, two residues (D30A and F31A) on an acidic, hydrophobic surface patch of the N-terminal domain were altered, resulting in a motA positive control mutant (motA-pc1). The mutant protein (full-length) binds normally to its DNA site but does not efficiently activate transcription (10). MotA probably interacts directly with the ς70 subunit of host RNA polymerase during activation of middle-mode transcription. MotA and ς70 form a complex with unique mobility in native polyacrylamide gels, although this was tested in the absence of DNA and the other RNA polymerase subunits (11). Furthermore, MotA binds specifically to the −30 region of middle promoters, thus acting like a class II activator (see reference 16 for a review). Other class II activators generally interact with the 4.2 region of ς70, which has a predicted helix-turn-helix motif and recognizes the −35 sequence of E. coli promoters (see reference 12 for a review). Various amino acid substitutions within the 4.2 region affect activation by transcription factors, including FNR, AraC, CRP, and PhoB (20, 21, 23, 24). The 4.2 region has also been shown to interact with phage λ cI repressor protein, which also binds at a site near the −30 region to activate transcription (22). Additionally or alternatively, MotA may interact with the AsiA coactivator. AsiA has been shown to bind to ς70 in the 4.2 region (6, 32, 34, 35), implying that AsiA is in close proximity to DNA-bound MotA. Upon binding to ς70, AsiA blocks transcription from host promoters requiring the −35 region (6, 33). Thus, AsiA blocks the 4.2 domain from recognizing the −35 region and possibly interacts directly with MotA to activate T4 middle promoters. Using native polyacrylamide gel electrophoresis, Gerber and Hinton (11) detected a ternary MotA-AsiA-ς70 complex. This complex was detected in the absence of promoter DNA and other RNA polymerase subunits but it may well reflect interactions that occur during initiation of middle-mode transcription. Since MotA and AsiA can each bind separately to ς70, the existence of this ternary complex does not clarify whether MotA and AsiA directly interact with each other. To investigate these interactions, we conducted genetic selections to try to identify substitutions in AsiA and ς70 that suppress the activation defect caused by motA-pc1. In this report, we describe two asiA mutants and five rpoD (ς70) mutants that gave moderate suppression in vivo. In vitro analysis of the mutant proteins failed to recapitulate the suppression but did reveal a novel class of ς70 mutants.