In vivo tests of thermodynamic models of transcription repressor function

Self Cite

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.

Section: Discussionsupporting

confidence: 88%

Protein:Protein Interactions in Control of a Transcriptional Switch

Adikaram

Beckett

2013

Self Cite

“…One approach for assessing correlation in this case is to linearize the data via log-transformation and apply a Pearson's test. 15,16 Using this method, we find that the correlation for both the simulated and experimental data in Fig. 3a and d is now statistically significant (P ≤ 0.01; see Supplementary Fig.…”

Section: Resultsmentioning

confidence: 88%

Glucocorticoid Receptor–DNA Interactions: Binding Energetics Are the Primary Determinant of Sequence-Specific Transcriptional Activity

Bain

Yang

Connaghan

et al. 2012

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. A long-standing question has focused on how GR and other receptors precisely control gene expression. One difficulty in addressing this is that GR function is influenced by multiple factors including ligand and coactivator levels, chromatin state, and allosteric coupling. Moreover, the receptor recognizes an array of DNA sequences that generate a range of transcriptional activities. Such complexity suggests that any single parameter-DNA binding affinity, for example-is unlikely to be a dominant contributor to function. Indeed, a number of studies have suggested that for GR and other receptors, binding affinity toward different DNA sequences is poorly correlated with transcriptional activity. As a step toward determining the factors most predictive of GR function, we rigorously examined the relationship between in vitro GR-DNA binding energetics and in vivo transcriptional activity. We first demonstrate that previous approaches for assessing affinity-function relationships are problematic due to issues of data transformation and linearization. Thus, the conclusion that binding energetics and transcriptional activity are poorly correlated is premature. Using more appropriate analyses, we find that energetics and activity are in fact highly correlated. Furthermore, this correlation can be quantitatively accounted for using simple binding models. Finally, we show that the strong relationship between energetics and transcriptional activity is recapitulated in multiple promoter contexts, cell lines, and chromatin environments. Thus, despite the complexity of GR function, DNA binding energetics are the primary determinant of sequence-specific transcriptional activity.

“…7 Simulations of the dependence of transcription repression on biotin concentration indicate that as the equilibrium dimerization constant for holoBirA becomes tighter, repression occurs at lower biotin concentrations. 24 These predictions are currently being tested.…”

Section: Discussionmentioning

confidence: 98%

Functional Versatility of a Single Protein Surface in Two Protein:Protein Interactions

Adikaram

Beckett

2012

Self Cite

The ability of the Escherichia coli protein, BirA, to function as both a metabolic enzyme and a transcription repressor relies on use of a single surface for two distinct protein:protein interactions. BirA forms a heterodimer with the biotin acceptor protein of acetyl-CoA carboxylase and catalyzes post-translational biotinylation. Alternatively, it forms a homodimer that binds sequence-specifically to DNA to repress transcription initiation at the biotin biosynthetic operon. Several surface loops on BirA, two of which exhibit sequence conservation in all biotin protein ligases and the remainder of which are highly variable, are located at the two interfaces. The function of these loops in both homodimerization and biotin transfer was investigated by characterizing alanine-substituted variants at 18 positions of one constant and three variable loops. Sedimentation equilibrium measurements reveal that 11 of the substitutions, which are distributed throughout conserved and variable loops, significantly alter homodimerization energetics. By contrast, steady-state and single turnover kinetic measurements indicate that biotin transfer to BCCP is impacted by 7 substitutions, the majority of which are in the constant loop. Furthermore, constant loop residues that function in biotin transfer also support homodimerization. The results reveal clues about the evolution of a single protein surface for use in two distinct functions.