Biology subjects

Wolfe, M. B.

Publications and source records attributed to Wolfe, M. B..

7 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

RNAP-seq: in vitro genome-scale transcription reveals preferential RNA polymerase pausing on Clostridioides difficile antisense DNA

Mechanistic dissection of gene transcription and its regulation in diverse organisms remains a central challenge for molecular biology. In vivo analyses using high-throughput DNA sequencing readouts (e.g., RNA-seq, Term-seq, and NET-seq) have revealed that transcriptional pausing by RNA polymerase (RNAP) underpins the regulation of gene transcription. However, these in vivo methods are difficult to apply in non-model organisms and lack the power of bottom-up biochemical analysis that can deconvolute the confounding effects of the cellular environment. Here we report a method for unbiased transcription of entire genomes in vitro called RNAP-seq, which is capable of defining pause, arrest, and termination sites with single-nucleotide resolution and of defining the regulatory contributions of cis-acting RNA and DNA sequences and dissociable transcription factors with complete control over transcription conditions. Using RNAP-seq to compare transcription by recombinant RNAPs from Clostridioides difficile (Cdf) and Escherichia coli (Eco), we discovered that CdfRNAP exhibits pausing signatures distinct from EcoRNAP. CdfRNAP pauses more frequently and more strongly at T-rich sequences, particularly on antisense regions of genes. We also defined the action of CdfNusG at genome scale, revealing its preferential action in riboswitch control regions. These findings demonstrate lineage-specific pausing features and suggest that genome composition and RNAP co-evolve to shape gene expression. RNAP-seq is broadly adaptable to diverse bacterial species and offers a powerful framework for uncovering fundamental principles of transcription that govern gene expression. HighlightsO_LIRNAP-seq enables unbiased transcription of every base-pair in an organisms genome C_LIO_LIRNAP-seq reveals distinct E. coli (Eco) and C. difficile (Cdf) pause sequences C_LIO_LICdfNusG broadly enhances pausing and termination, notably in riboswitch-encoding DNA C_LIO_LICdfRNAP but not EcoRNAP pauses more on the T-rich antisense strand of Cdf genes C_LI

genomics↗

bioRxiv2025

ShapeME: A tool and web front-end for de novo discovery of structural motifs underpinning protein-DNA interactions

Determining where transcriptional regulators bind within a genome is paramount to understanding how gene expression is regulated. Historically, position weight matrices (PWMs) have been used to define the binding preferences of DNA binding proteins1. However, PWMs treat the identity of each base in a sequence as an independent and additive measure of binding preference, which can limit their utility2. Models that consider higher order interactions between nearby bases yield greater success in predicting proteins binding to DNA, but for many proteins there is still substantial room for improvement in predicting and understanding the determinants of proteins binding to DNA3. In addition to DNA sequence motifs, structural motifs (e.g., a narrow minor groove width) are important determinants of binding for some DNA-binding proteins4. Despite the initial success of algorithms using structural features of DNA to predict binding properties of proteins from either ChIP-seq or SELEX data5-8, there remains a need for a de novo structural motif discovery framework which can be applied to data from a variety of experimental designs. Here, we present a unified workflow, capable of utilizing virtually any type of data representing sequence coverage or enrichment (e.g. ChIP-seq, RNA-seq, SELEX, etc.), to discover short structural motifs with explanatory power for a proteins DNA binding preference. We couple the DNAshapeR algorithm9 with our own information-theoretic approach to de novo motif discovery, and wrap shape and sequence motif inference and model selection into a single tool called ShapeME. Application of our structural motif discovery algorithm to proteins with ChIP-seq data in ENCODE datasets reveals a subset of proteins where short structural motifs outperform the best PWM for that protein as determined from the JASPAR database, or as identified by the sequence motif elicitation tool STREME. Our approach offers a powerful and versatile framework for inferring structural DNA binding motifs, and will complement current sequence-based motif elicitation tools in discovery of protein-DNA interaction principles. A web-based interface to ShapeME is available at https://seq2fun.dcmb.med.umich.edu/shapeme, with full source code available at https://github.com/freddolino-lab/ShapeME.

bioinformatics↗

bioRxiv2023

The Genetics of Aerotolerant Growth in a Naturally Reduced Genome Alphaproteobacterium

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living Alphaproteobacterium, Zymomonas mobilis, has a naturally reduced genome containing fewer than 2000 protein coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model Alphaproteobacterium. Here, we use genome-scale CRISPRi-seq to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth, or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rnf respiratory complex are required for anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. ImportanceThe inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the Alphaproteobacterium, Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

genetics↗

bioRxiv2020

Hybrid analysis reveals how DNA sequence governs genomic location and DNA contacts of bacterial chromatin H-NS filaments

Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD-DNA contacts in linear and bridged filaments are uncertain. To characterize the contacts that silence transcription by RNA polymerase, we combined {middle dot}OH footprinting, molecular dynamics, statistical modeling, and DBD mapping using a chemical nuclease (Fe2+-EDTA) tethered to the DBDs (TEN-map). We find that H-NS DBDs contact DNA at indistinguishable locations in bridged or linear filaments and that the DBDs vary in orientation and position with ~10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged H-NS switching in which linear filaments able to inhibit only transcription initiation switch to bridged filaments able to inhibit both initiation and elongation using the same irregularly spaced DNA contact sites. HighlightsO_LITethered-nuclease mapping (TEN-map) of H-NS DNA-binding domains detects DNA contacts C_LIO_LIBridged and linear H-NS filaments use the same DNA contact sites C_LIO_LIH-NS-DNA contacts are unevenly spaced with ~10 bp average separation C_LIO_LIAT-steps, minor groove width, and electrostatic potential best predict contact sites C_LI

molecular biology↗

bioRxiv2020

Principles of mRNA control by human PUM proteins elucidated from multi-modal experiments and integrative data analysis

The human PUF-family proteins, PUM1 and PUM2, post-transcriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3 UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a rulebook of key contextual features that differentiate functional vs. non-functional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

molecular biology↗

bioRxiv2018

Escherichia coli Lrp regulates one-third of the genome via direct, cooperative, and indirect routes

The global regulator Lrp plays a crucial role in regulating metabolism, virulence and motility in response to environmental conditions. Lrp has previously been shown to activate or repress approximately 10% of genes in Escherichia coli. However, the full spectrum of targets, and how Lrp acts to regulate them, has stymied earlier study. We have combined matched ChIP-seq and RNA sequencing under nine physiological conditions to map the binding and regulatory activity of Lrp as it directs responses to nutrient abundance. In addition to identifying hundreds of novel Lrp targets, we observe two new global trends: first, that Lrp will often bind to promoters in a poised position under conditions when it has no regulatory activity, and second, that nutrient levels induce a global shift in the equilibrium between non-specific and sequence-specific DNA binding. The overall regulatory behavior of Lrp, which as we now show regulates 35% of E. coli genes directly or indirectly under at least one condition, thus arises from the interaction between changes in Lrp binding specificity and cooperative action with other regulators.

microbiology↗