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Transcriptional kinetic synergy: A complex landscape revealed by integrating modeling and synthetic biology.

ABSTRACT: Transcription factors (TFs) control gene expression, often acting synergistically. Classical thermodynamic models offer a biophysical explanation for synergy based on binding cooperativity and regulated recruitment of RNA polymerase. Because transcription requires polymerase to transition through multiple states, recent work suggests that "kinetic synergy" can arise through TFs acting on distinct steps of the transcription cycle. These types of synergy are not mutually exclusive and are difficult to disentangle conceptually and experimentally. Here, we model and build a synthetic circuit in which TFs bind to a single shared site on DNA, such that TFs cannot synergize by simultaneous binding. We model mRNA production as a function of both TF binding and regulation of the transcription cycle, revealing a complex landscape dependent on TF concentration, DNA binding affinity, and regulatory activity. We use synthetic TFs to confirm that the transcription cycle must be integrated with recruitment for a quantitative understanding of gene regulation.

SUBMITTER: Martinez-Corral R

PROVIDER: S-EPMC10472254 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Transcriptional kinetic synergy: A complex landscape revealed by integrating modeling and synthetic biology.

Martinez-Corral Rosa R Park Minhee M Biette Kelly M KM Friedrich Dhana D Scholes Clarissa C Khalil Ahmad S AS Gunawardena Jeremy J DePace Angela H AH

Cell systems 20230401 4

Transcription factors (TFs) control gene expression, often acting synergistically. Classical thermodynamic models offer a biophysical explanation for synergy based on binding cooperativity and regulated recruitment of RNA polymerase. Because transcription requires polymerase to transition through multiple states, recent work suggests that "kinetic synergy" can arise through TFs acting on distinct steps of the transcription cycle. These types of synergy are not mutually exclusive and are difficul ...[more]

PMID: 37080164

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Project description:Under mildly acidic conditions (pH 4-4.5) apomyoglobin (apoMb) adopts a partially structured equilibrium state ( M-state) that structurally resembles a kinetic intermediate encountered at a late stage of folding to the native structure at neutral pH. We have previously reported that the M-state is formed rapidly (<1 ms) via a multistate process and thus offers a unique opportunity for exploring early stages of folding by both experimental and computational techniques. In order to gain structural insight into intermediates and barriers at the residue level, we studied the folding/unfolding kinetics of 12 apoMb mutants at pH 4.2 using fluorescence-detected ultrafast mixing techniques. Global analysis of the submillisecond folding/unfolding kinetics vs urea concentration for each variant, based on a sequential four-state mechanism ( U ⇔ I ⇔ L ⇔ M), allowed us to determine elementary rate constants and their dependence on urea concentration for most transitions. Comparison of the free energy diagrams constructed from the kinetic data of the mutants with that of wild-type apoMb yielded quantitative information on the effects of mutations on the free energy (ΔΔ G) of both intermediates and the first two kinetic barriers encountered during folding. Truncation of conserved aliphatic side chains on helices A, G, and H gives rise to a stepwise increase in ΔΔ G as the protein advances from U toward M, consistent with progressive stabilization of native-like contacts within the primary core of apoMb. Helix-helix contacts in the primary core contribute little to the first folding barrier ( U ⇔ I) and thus are not required for folding initiation but are critical for the stability of the late intermediate, L, and the M-state. Alanine substitution of hydrophobic residues at more peripheral helix-helix contact sites of the native structure, which are still absent or unstable in the M-state, shows both positive (destabilizing) and negative (stabilizing) ΔΔ G, indicating that non-native contacts are formed initially and weakened or lost as a result of subsequent structural rearrangement steps.

Dataset Information

Transcriptional kinetic synergy: A complex landscape revealed by integrating modeling and synthetic biology.

Publications

Transcriptional kinetic synergy: A complex landscape revealed by integrating modeling and synthetic biology.

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