Life is governed by tiers of gene regulation, driven by modulation of RNA polymerase (RNAP) by transcription factors. The second tier is composed of cell signaling cascades and feedback loops. Dissecting gene regulation requires distinguishing transcription factor targets from indirect network effects.
A new study by The Rockefeller University published in Molecular Cell titled, “Cell-free genomics reveals fundamental regulatory principles of the Mycobacterium tuberculosis transcription cycle,” has revealed fundamental features of the transcription cycle in the bacteria that causes tuberculosis. The study informs the development of new drugs.
“Gaining a deep understanding of how transcription works reveals central principles in biology that have huge significance for human health,” said Elizabeth Campbell, PhD, head of the Laboratory of Molecular Pathogenesis at The Rockefeller and corresponding author of the paper. “We can more precisely design therapeutics to target a process if we truly understand it.”
The Campbell lab sought to build a controlled, cell-free system to reveal the direct effects of a transcription factor across diverse species, from Mycobacterium tuberculosis (Mtb) to organisms that cannot be cultured at all.
Led by Ruby Froom, PhD, graduate student in the Campbell lab at the time, the study reconstructed a cell-free genomic system using purified components from Mtb. The team combined the fragmented DNA with with RNAP, its primary sigma factor, transcription factors, and specific regulators including CRP, WhiB1, NusA, and NusG.
The authors designed sequencing approaches to identify the exact genomic positions where transcription begins and ends to map the activity of RNAP. The study determined the efficacy of cell free genomics by characterizing global and essential transcription initiation and elongation-termination factors from Mtb.
Notably, the bacterium’s transcription machinery relied on classic DNA start signals that had appeared weak or absent in living cells, suggesting signal masking. The team also mapped the activity of well-known regulator, such as CRP, and revealed dozens of genes that CRP governs independently, without help from other cellular factors.
In some cases, the method showed that global regulators in living cells exhibit a more precise role in signaling. As an example, the team found that the transcription factor, WhiB1, directly controls only a small set of critical genes. Yet, WhiB1 disruption leads to wide network effects.
This cell-free approach also resolved a long-standing debate about transcription termination, showing that sequence-driven termination operates across the Mtb genome with distinct roles for NusA and NusG. As NusG is the only transcription factor conserved across all domains of life, from bacteria to humans, Campbell says these findings position Mtb as a powerful system for uncovering universal principles of gene regulation.
The new method can also improve understanding of existing tuberculosis drugs, such as rifampicin which targets RNAP, to address drug resistance. Beyond tuberculosis, the study challenges the long-standing reliance on classic model organisms to define the basic rules of gene regulation, which has limitations in translation.
“With this method, we are not just trying to see how Mtb compares to standard models. We are establishing new principles of gene expression, and uncovering ones that haven’t been addressed in other organisms,” Campbell says. “There is no one ‘model’ anymore. Just as there is no model human or model culture, bacteria are all different. We should study it all.”