Protein:Protein Interactions in Control of a Transcriptional Switch

Protein partner exchange plays a key role in regulating many biological switches. Although widespread, the mechanisms dictating protein partner identity and, therefore, the outcome of a switch have been determined for a limited number of systems. The Escherichia coli protein BirA undergoes a switch between posttranslational biotin attachment and transcription repression in response to cellular biotin demand. Moreover, the functional switch reflects formation of alternative mutually exclusive protein:protein interactions by BirA. Previous studies provided a set of alanine-substituted BirA variants with altered kinetic and equilibrium parameters of forming these interactions. In this work, DNase I footprinting measurements were employed to investigate the consequences of these altered properties for the outcome of the BirA functional switch. The results support a mechanism in which BirA availability for DNA binding and, therefore, transcription repression is controlled by the rate of the competing protein:protein interaction. However, occupancy of the transcriptional regulatory site on DNA by BirA is exquisitely tuned by the equilibrium constant governing its homodimerization. [Display omitted] •Protein partner swapping controls many biological switches.•Partner exchange can be subject to kinetic and/or equilibrium control.•BirA switches between enzymatic and transcription repression functions.•Direct and inhibition footprint titrations were used to investigate the switch.•The switch is subject to a hierarchy of kinetic and equilibrium control.