Bacteriophage T4 early promoters are utilized immediately after infection and are abruptly turned off 2–3 min later (at 30°C) when the middle promoters are activated. The viral early protein AsiA has been suspected to bring about this transcriptional switch: not only does it activate transcription at middle promoters in vivo and in vitro but it also shows potent anti‐σ70 activity in vitro, suggesting that it is responsible for the shut‐off of early transcription. We show here that after infection with a phage deleted for the asiA gene the inhibition of early transcription occurs to the same extent and with the same kinetics as in a wild‐type infection. Thus, another AsiA‐independent circuit efficiently turns off early transcription. The association of a mutation in asiA with a mutation in mod, rpbA, motA or motB has no effect on the inhibition of early promoters, showing that none of these phage‐encoded transcriptional regulators is necessary for AsiA‐independent shut‐off. It is not known whether AsiA is able to inhibit early promoters in vivo, but host transcription is strongly inhibited in vivo upon induction of AsiA from a multicopy plasmid.
SummaryPhage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme E s s s s 70 . We studied in vitro the effects of the T4 anti-s s s s 70 factor AsiA on the activity of several T4 early promoters. In single-round transcription, promoters motB, denV, mrh.2, motA wild type and UP element-deleted motA are strongly resistant to inhibition by AsiA. The a a a a -C-terminal domain of E s s s s 70 is crucial to this resistance. DNase I footprinting of E s s s s 70 and E s s s s 70 AsiA on motA and mrh.2 shows extended contacts between the holoenzyme with or without AsiA and upstream regions of these promoters. A TG AE AE AE AE TC mutation of the extended ----10 motif in the motA UP element-deleted promoter strongly increases susceptibility to inhibition by AsiA, but has no effect on the motA wild-type promoter: either the UP element or the extended ----10 site confers resistance to AsiA. Potassium permanganate reactivity shows that the two structure elements are not equivalent: with AsiA, the motA UP element-deleted promoter opens more slowly whereas the motA TC promoter opens like the wild type. Changes in UV laser photoreactivity at position +4 on variants of motA reveal an analogous distinction in the roles of the extended ----10 and UP promoter elements.
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