A new mechanism for the control of a prokaryotic transcriptional regulator: antagonistic binding of positive and negative effectors

Abstract

SummaryMalT, the transcriptional activator of the Escherichia coli maltose regulon, self-associates, binds promoter DNA and activates initiation of transcription only in the presence of ATP and maltotriose, the inducer. In vivo studies have revealed that MalT action is negatively controlled by the MalY protein. Using a biochemical approach, we analyse here the mechanism whereby MalY represses MalT activity. We show that MalY inhibits transcription activation by MalT in a purified transcription system. In vitro, a constitutive MalT variant (which is partially active in the absence of maltotriose) is less sensitive than wild-type MalT to repression by MalY, as observed in vivo. We demonstrate that MalY forms a complex with MalT only in the absence of maltotriose and that, conversely, MalY inhibits maltotriose binding by MalT. Together, these results establish that MalY acts directly upon MalT without the help of any factor, and that MalY is a negative effector of MalT competing with the inducer for MalT binding.

MalK, the ATP-binding Cassette Component of the Escherichia coli Maltodextrin Transporter, Inhibits the Transcriptional Activator MalT by Antagonizing Inducer Binding

“…MalT via the same mechanism as MalY and Aes (19,20). Indeed, in all three cases the negative effector inhibits maltotriose binding and vice versa.…”

“…BL21( DE3) ⌬malT220 contains a deletion of the entire malT gene (19). Plasmids pOM163 and pOM163-T38R are pET28b(ϩ) (Novagen) derivatives producing DT1 or DT1-T38R with a Strep tag at their C terminus (DT1S) (20).…”

“…The chromatograms were dried and scanned on a PhosphorImager, and the amount of ApApC synthesized was quantified. The malPp DNA template is a 320-bp fragment containing the malPp promoter (from Ϫ154 to ϩ130), prepared as described (19).…”

“…In addition to MalK, two other proteins, namely MalY, a ␤ C-S lyase, and Aes, an acyl esterase, down-regulate MalT activity (17,18). Recent studies have revealed that both MalY and Aes interact directly with MalT and compete with maltotriose for MalT binding (19,20). Available data suggest that unliganded MalT is in equilibrium between an inactive monomeric form and an active monomeric form prone to self-association.…”

“…The requirement for ATP as a positive effector of MalT might reflect a role for the ATPase activity of MalT in the competition between positive and negative effectors. Indeed, both ATP and ADP can promote MalT multimerization or inhibitory complex formation, but ATP is more effective in driving MalT self-association, whereas ADP is more effective in promoting the formation of an inhibitory complex (16,19,20).…”