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Genetically identical cells exhibit diverse phenotypes, even when experiencing the same environment. This phenomenon, in part, originates from cell-to-cell variability (noise) in protein expression. While various kinetic schemes of stochastic transcription initiation are known to affect gene expression noise, how post-transcription initiation events contribute to noise at the protein level remains incompletely understood. To address this question, we developed a stochastic simulation-based model of bacterial gene expression that integrates well-known dependencies between transcription initiation, transcription elongation dynamics, mRNA degradation and translation. We identified realistic conditions under which mRNA lifetime and transcriptional pauses modulate the protein expression noise initially introduced by the promoter architecture. For instance, we found that the short lifetime of bacterial mRNAs facilitates the production of protein bursts. Conversely, RNA polymerase (RNAP) pausing at specific sites during transcription elongation can attenuate protein bursts by fluidizing the RNAP traffic to the point of erasing the effect of a bursty promoter. Pause-prone sites, if located close to the promoter, can also affect noise indirectly by reducing both transcription and translation initiation due to RNAP and ribosome congestion. Our findings highlight how the interplay between transcription initiation, transcription elongation, translation and mRNA degradation shapes the distribution in protein numbers. They also have implications for our understanding of gene evolution and suggest combinatorial strategies for modulating phenotypic variability by genetic engineering.

Bacterial cells are often erroneously regarded as a tiny sack of molecules, but growing evidence suggests surprisingly sophisticated internal organization in bacteria. Here we present how mRNA is spatially organized in a model bacterium, Caulobacter crescentus. We developed 3D super-resolution microscopy technique based on fluorescence in situ hybridization to probe chromosomally encoded mRNA of a specific gene with ∼25 nm lateral resolution and ∼50 nm axial resolution. We found that most of the mRNA molecules are localized near the gene loci on the chromosome and that the remaining mRNA molecules are distributed around cell area with interesting localization patterns in the axial direction. We investigated spatial distribution of mRNA in relation to the cellular morphology and discuss cell cycle-dependent localization of certain bacterial mRNA.

Allostery is well-documented for proteins but less recognized for DNA-protein interactions, in which DNA has been often considered as a mere template providing recognition sequences. Here we report that for two proteins bound on DNA at a separation of tens of base pairs, their DNA binding affinities can be significantly altered. This coupling effect oscillates between positive and negative cooperativity, depending on the separation distance between the two proteins on the DNA. With a DNA hairpin experiment, we provide definitive evidence for the structural basis of DNA allostery. We prove this effect is not due to protein-protein interactions but originates from the distortion of the inter-helical distance along the linker DNA. The oscillation has a periodicity of ∼10 base pairs, the helical pitch of the B-form DNA, and a characteristic decay length of ∼15 base pairs. In the theoretical analysis, we elucidate the relation between the mechanical structural distortion of DNA induced by protein-binding and the free energy coupling measured thermodynamically, providing a complete picture for the origin of DNA allostery. The allosteric coupling between two DNA-bound proteins is found to be ubiquitous, regardless of proteins’ properties, implying its general roles in gene regulation. We demonstrate such DNA allostery affects gene expression levels in live E.coli cells. Pertinent to eukaryotic gene expression, we show that the binding affinity of a transcription factor depends on its separation from nearby nucleosomes. This work provides the first comprehensive study of allostery through DNA, with the understanding of its physical underpinning and ubiquity and biological relevance.

Often in nature, a macromolecule undergoes conformational changes upon binding of a ligand in order to modify its affinity for another ligand at a distant binding site. This phenomenon, called “allostery”, is a fundamental mechanism for dynamic regulation of macromolecular properties. Although allostery is well-documented in proteins, it is less recognized for DNA-protein interactions, in which DNA has been often considered a mere template providing recognition sequences for proteins. Here we investigate the allosteric interactions through DNA both in single molecule experiments in vitro and in live cells. In the in vitro experiments, we demonstrate that when two proteins specifically bind to DNA within tens of base pairs, the binding affinity of one protein is altered by the other. We prove that this is not due to protein-protein interactions but to allostery through DNA. As the distance between the two proteins is varied, this allosteric coupling oscillates between positive and negative cooperativity with a periodicity of ∼10 base pairs, the helical pitch of the B-form DNA. The allostery through DNA is explained in terms of the free energy associated with the overall conformation of the ternary complex. We also demonstrate that such allostery affects gene expression in live E. coli cells, suggesting its physiological relevance.