The calculation of transcript flux ratios reveals single regulatory mechanisms capable of activation and repression

Abstract

The regulation of transcription allows cells to adjust the rate of RNA polymerases (RNAPs) initiated in a promoter-specific manner. Classically, transcription factors are directed to a subset of promoters via the recognition of DNA sequence motifs. However, a unique class of regulators is recruited directly through interactions with RNAP. Surprisingly, these factors may still possess promoter specificity, and it has been postulated that the same kinetic mechanism leads to different regulatory outcomes depending on a promoter's basal rate constants. However, mechanistic studies of regulation typically report factor activity in terms of changes in the thermodynamics or kinetics of individual steps or states while qualitatively linking these observations to measured changes in transcript production. Here, I present online calculators that allow for the direct testing of mechanistic hypotheses by calculating the steady-state transcript flux in the presence and absence of a factor as a function of initiation rate constants. By evaluating how the flux ratio of a single kinetic mechanism varies across promoter space, quantitative insights into the potential of a mechanism to generate promoter-specific regulatory outcomes are obtained. Using these calculations, I predict that the mycobacterial transcription factor CarD is capable of repression in addition to its known role as an activator of ribosomal genes. In addition, a modification of the mechanism of the stringent response factors DksA/guanosine 5'-diphosphate 3'-diphosphate (ppGpp) is proposed based on their ability to differentially regulate transcription across promoter space. Overall, I conclude that a multifaceted kinetic mechanism is a requirement for differential regulation by this class of factors.

Keywords: CarD; DksA; kinetics; regulation; transcription.

Fig. 1.

Kinetic mechanism of transcription initiation. (A) RNAP (R) and promoter DNA (P) may bind to form the closed complex (RP_c). RNAP may dissociate from closed complex or isomerize to open complex (RP_o). Open complex may collapse to closed complex, or as a function of NTPs, it may escape the promoter and generate an RNA transcript. (B) The scheme in A represented as an energy landscape where transition-state barriers between the states determine the rates of interconversion according to transition-state theory. In this example, RP_o is relatively unstable compared with RP_c as might be the case for a ribosomal promoter.

Fig. 2.

Open complex stabilization alone leads to repression. Decreasing both the rates of bubble collapse and promoter escape by a factor of 10 leads to the energy landscape shown in red and a flux ratio less than one (0.72). This calculator can be found at https://egalburt.github.io/transcript-flux-calculator/fluxcalc.html.

Fig. 3.

Open complex stabilization via lowering the energy of RP_o directly leads to varying degrees of repression across promoter space. This calculator may be found at https://egalburt.github.io/transcript-flux-calculator/fluxcalc_mech.html.

Fig. 4.

Stimulating promoter opening stabilizes open complex and leads to transcription activation. Increasing the rate of unwinding fivefold leads to the energy landscape shown in green and a flux ratio of 4.2. Importantly, while this landscape stabilizes open complex relative to closed complex just as in Fig. 2, this mechanism leads to activation instead of repression.

Fig. 5.

Flux regulation by CarD. Increasing the rate of promoter opening in addition to the stabilization of RP_o leads to differential regulation across promoter space. The plots are generated with the following basal rate constants: k_on = 0.1 nM⁻¹ s⁻¹, k_off = 10 s⁻¹, k_open = 1 s⁻¹, k_close = 0.1 s⁻¹, k_escape = 0.1 s⁻¹. The regulatory mechanism is a 3-fold increase in the rate of opening coupled with a 0.3-fold change in the rate of closing and the rate of escape. (A) Relatively unstable open complexes are activated, whereas relatively stable open complexes are repressed by this mechanism ([R] = 1,000 nM). (B) The titration of polymerase concentration can lead to a switch from activation to repression that depends on the value of k_off.

Fig. 6.

Flux regulation by DksA/ppGpp. (A) Stabilization of the transition state between RP_c (RP₁) and RP_o (RP₂) leads only to activation across promoter space. The closing and opening rates have been increased threefold in the context of the basal rate constants: k_on = 0.1 nM⁻¹ s⁻¹, k_off = 10 s⁻¹, k_open = 1 s⁻¹, k_close = 0.1 s⁻¹, k_escape = 0.5 s⁻¹, [R] = 1,000 nM. (B) Increasing the closing rate by a larger factor than the opening rate leads to differential regulation. Here, the regulatory mechanism has increased the closing rate fivefold and the opening rate threefold in the context of the basal rate constants in A. Promoters with closing rates faster than escape rates are repressed, and promoters with escape rates more rapid than closing rates are activated by this mechanism.