Biology subjects

Mueller, A. U.

Publications and source records attributed to Mueller, A. U..

5 recordsLinked to original sources

bioRxiv2026

ppGpp regulates transcription elongation via direct and indirect inputs to RNA polymerase pausing and nucleotide addition

The signaling molecules guanosine 5'-tri/diphosphate 3'-diphosphate, (p)ppGpp, control bacterial protein synthesis rates and cell growth by targeting transcription, translation, NTP synthesis, and other functions. In lineages like E. coli, (p)ppGpp produced in response to charged-tRNA deficiency directly targets transcribing RNAP polymerase (RNAP) to match its pace to the pioneering ribosome on the nascent RNA (transcription-translation coupling). However, the mechanism by which (p)ppGpp slows RNAP is poorly defined. (p)ppGpp may allosterically stimulate RNAP pausing, inhibit catalysis, promote backtracking, compete for substrate GTP, inhibit GTP synthesis, or uncouple transcription-translation by inhibiting translation. Using a combination of cryo-EM, biochemical assays, and quantitative nascent elongating transcript sequencing (qNET-seq), we establish that (p)ppGpp allosterically regulates pausing and nucleotide addition via distinct motions of the RNAP swivel module and both competes with and lowers GTP in vivo. (p)ppGpp stimulates swiveling at pause sites to delay escape but may also inhibit counter-swiveling required in every round of nucleotide addition. HighlightsO_LIppGpp biases RNAP toward swiveling and away from a catalytically-competent state C_LIO_LIppGpp effects on RNAP conformation explain ppGpp stimulation of RNAP pausing C_LIO_LIppGpp stimulation of RNAP pausing in vivo is mediated mainly by reduced GTP levels C_LIO_LIppGpp may allosterically slow RNAP and increases gene occupancy in vivo C_LI

molecular biology↗

bioRxiv2026

Structural principles of transcriptional collisions

RNA polymerase (RNAP) must navigate crowded genomic tracks to robustly produce transcripts. DNA-bound proteins act as transcription-impeding roadblocks, which are eventually overcome by RNAPs through unclear mechanisms. Here, we use cryo-electron microscopy to visualize actively transcribing E. coli RNAP upon collision with an inactivated restriction enzyme (EcoRI*) or with another converging RNAP. Both collisions induce RNAP backtracking into an inactive swiveled state. Swiveling is coupled to DNA deformation through a characteristic structural landscape, mediating RNAP pause stabilization across distinct collision geometries. EcoRI* roadblock bypass efficiency is impacted by factors that modulate RNAP swiveling, backtracking rescue, and roadblock stability. In comparison, head-on RNAP-RNAP collisions feature substantial heterogeneity suggestive of sustained dynamics, with variable inter-RNAP distances that are modulated by nascent transcript hairpins which position bidirectional termination sites. By resolving, to our knowledge, novel structures of actively transcribing RNAP undergoing collisions, we provide a mechanistic framework for interpreting mechanical conflicts during transcription.

biophysics↗

bioRxiv2025

Real-time capture of σN transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding

Bacterial {sigma} factors bind RNA polymerase (E) to form holoenzyme (E{sigma}), conferring promoter specificity to E and playing a key role in transcription bubble formation. {sigma}N is unique among {sigma} factors in its structure and functional mechanism, requiring activation by specialized AAA+ ATPases. E{sigma}N forms an inactive promoter complex where the N-terminal {sigma}N region I ({sigma}N-RI) threads through a small DNA bubble. On the opposite side of the DNA, the ATPase engages {sigma}N-RI within the pore of its hexameric ring. Here, we perform kinetics-guided structural analysis of de novo formed E{sigma}N initiation complexes and engineer a biochemical assay to measure ATPase-mediated {sigma}N-RI translocation during promoter melting. We show that the ATPase exerts mechanical action to translocate about 30 residues of {sigma}N-RI through the DNA bubble, disrupting inhibitory structures of {sigma}N to allow full transcription bubble formation. A local charge switch of {sigma}N-RI from positive to negative may help facilitate disengagement of the otherwise processive ATPase, allowing subsequent {sigma}N disentanglement from the DNA bubble.

biochemistry↗

bioRxiv2024

Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-electron microscopy (cryo-EM) was used to capture four intermediates populated 120 or 500 milliseconds (ms) after mixing Escherichia coli {sigma}70-RNAP and the PR promoter. Cryo-EM snapshots revealed the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As nt-strand "read-out" extends, the RNAP clamp closes, expelling an inhibitory {sigma}70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating yet unknown conformational changes load it in subsequent steps. Because these events likely describe DNA opening at many bacterial promoters, this study provides needed insights into how DNA sequence regulates steps of RPo formation.

biophysics↗

bioRxiv2019

Structure and functional implications of WYL-domain-containing transcription factor PafBC involved in the mycobacterial DNA damage response

In mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determined the crystal structure of Arthrobacter aurescens PafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA molecules. Using an in vivo mutational screen in Mycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.

molecular biology↗