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Cell-free genomics reveals fundamental regulatory principles of the Mycobacterium tuberculosis transcription cycle

ABSTRACT: Tiers of gene regulation govern cellular life. The intrinsic activities of RNA polymerase (RNAP) constitute a primary tier, while direct modulation by accessory transcription factors (TFs) constitutes a secondary tier. Cellular signaling cascades and feedback loops generate tertiary and higher-order tiers. Dissecting gene regulation requires distinguishing direct TF targets at genome-scale from indirect network effects. A major obstacle is the lack of tools to interrogate transcription machineries from difficult-to-culture microbes—such as pathogens, commensals, and environmental species—at genome scale. Here, we introduce cell-free genomics (CFG), an empirical approach that identifies the direct targets of RNAP and TFs and systematizes their transcriptional effects. We demonstrate the efficacy of CFG by characterizing global and essential transcription initiation (CRP and holo-WhiB1) and elongation–termination factors (NusA and NusG) from the deadly pathogen, Mycobacterium tuberculosis. CFG expands our understanding of transcription principles and is broadly extensible to other perturbations and diverse species.

ORGANISM(S): Mycobacterium tuberculosis

PROVIDER: GSE270670 | GEO | 2026/01/15

REPOSITORIES: GEO

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Regulator trafficking on bacterial transcription units in vivo

Project description:The in vivo trafficking patterns on DNA by the bacterial regulators of transcript elongation Sigma70, Rho, NusA, and NusG and the explanation for high promoter-proximal levels or peaks of RNA polymerase (RNAP) are unknown. Genome-wide ChIP-chip on E. coli revealed distinct association patterns of regulators as RNAP transcribes away from promoters (Rho first, then NusA, and then NusG). However, the interactions of elongating complexes with these regulators, including a weak interaction with Sigma70, did not differ significantly among most transcription units. A modest variation of NusG signal among genes reflected increased NusG interaction as transcription progresses, rather than functional specialization of elongating complexes. Promoter-proximal RNAP peaks were offset from Sigma70 peaks in the direction of transcription and co-occurred with NusA and Rho peaks, suggesting that the RNAP peaks reflected elongating, rather than initiating, complexes. However, inhibition of Rho did not increase RNAP levels within genes downstream of the RNAP peaks, suggesting the peaks are caused by a mechanism other than simple Rho-dependent attenuation.

2009-01-03 | GSE13938 | GEO

Regulator trafficking on bacterial transcription units in vivo

2010-05-14 | E-GEOD-13938 | biostudies-arrayexpress

In-cell architecture of an active transcription-translation complex

Project description:Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between the ribosome and a NusG-bound elongating RNAP, and propose it could mediate transcription-translation coupling. Transcription inhibition stalls and rearranges the expressome, whereas translation inhibition dissociates it, demonstrating that the elongating expressome architecture requires active translation and transcription within the cell.

2020-07-30 | PXD017711 | Pride

In-cell architecture of an active transcription-translation complex

2020-07-30 | PXD017695 | Pride

Human-specific transcriptome of ventral and dorsal midbrain dopamine neurons

Project description:Neuronal loss in the substantia nigra pars compacta (SNpc) in Parkinson disease (PD) is not uniform, as dopamine neurons from the ventral tier are lost more rapidly than those of the dorsal tier. Identifying the intrinsic differences that account for this differential vulnerability may provide a key for developing new treatments for PD. Here, we compared the RNA-sequenced transcriptomes of ~100 laser captured microdissected SNpc neurons from each tier from 7 healthy controls. Expression levels of dopaminergic markers were similar across the tiers, whereas markers specific to the neighboring ventral tegmental area were virtually undetected. After accounting for unwanted sources of variation, we identified 106 differentially expressed genes (DEGs) between the SNpc tiers. The genes higher in the dorsal/resistant SNpc tier neurons displayed coordinated patterns of expression across the human brain, their protein products had more interactions than expected by chance, and they demonstrated evidence of functional convergence. No significant shared functionality was found for genes higher in the ventral/vulnerable SNpc tier. Surprisingly but importantly, none of the identified DEGs was among the familial PD genes or genome-wide associated loci. Finally, we found some DEGs in opposite tier orientation between human and analogous mouse populations. Our results highlight functional enrichments of vesicular trafficking, ion transport/homeostasis and oxidative stress genes showing higher expression in the resistant neurons of the SNpc dorsal tier. Furthermore, the comparison of gene expression variation in human and mouse SNpc populations strongly argues for the need of human-focused omics studies

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Rho and NusG suppress pervasive antisense transcription in Escherichia coli [ChIP-chip].

Project description:Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its co-factor NusG is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global co-localization of the nucleoid-associated protein H-NS with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes. Chromatin immunoprecipitation (ChIP) experiments were performed using antibodies against RNA polymerase (RNAP; Beta subunit) in wild-type cells or cells deleted for hns, nusG, or a partial deletion of nusA. Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 12 datasets.

2012-11-01 | E-GEOD-41936 | biostudies-arrayexpress

Deciphering the Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli [ChIP-Seq]

Project description:The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism in many bacteria. However, the full regulatory potential of Fur beyond iron metabolism remains undefined. Here, we comprehensively reconstructed the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response to iron availability using genome-wide measurements (ChIP-exo and RNA-seq). Polyomic data analysis revealed that a total of 81 genes in 42 transcription units (TUs) are directly regulated by three different modes of Fur regulation, including apo- and holo-Fur activation as well as holo-Fur repression. We showed that Fur connects iron transport and utilization enzymes with negative-feedback loop pairs for iron homeostasis. In addition, direct involvement of Fur in the regulation of DNA synthesis, energy metabolism, and biofilm development was found. These results indicate that Fur exhibits a comprehensive regulatory role affecting many fundamental cellular processes linked to iron metabolism in order to coordinate E. coli responses to the availability of iron. [ChIP-exo]: A total of twelve samples were analyzed. WT and Fur-8-myc tagged cells were cultured in the presense and absence of iron with biological duplicates. To analyze static RNAP binding, rifampicin was also added to the media with biological duplicates. DPD = iron chelator.

2014-09-09 | E-GEOD-54899 | biostudies-arrayexpress

Control of a gene transfer agent cluster in Caulobacter crescentus by transcriptional activation and anti-termination

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2023-11-14 | GSE247216 | GEO

Rho and NusG suppress pervasive antisense transcription in Escherichia coli

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2012-11-01 | E-GEOD-41940 | biostudies-arrayexpress

Bacillus velezensis LoaP Promotes Antitermination of Antibiotic Synthesis Operons by Antagonizing NusA

Project description:The transcription factor NusG is essential for the bacterial transcription elongation complex (TEC). During elongation, NusG dynamically interacts with other proteins, affecting transcription-translation coupling, processivity, and termination. NusG is integral to antitermination complexes, where it associates with other factors to modify the TEC such that it bypasses termination sites. In Gammaproteobacteria, these complexes enhance the expression of ribosomal RNA operons, bacteriophage operons, and CRISPR arrays. Specialized NusG paralogs, such as LoaP, RfaH, and UpxY, are believed to form their own unique antitermination complexes. RfaH, the only well-studied paralog, binds to a paused TEC via a specific nontemplate DNA sequence. The RfaH-bound TEC then enhances transcription-translation coupling while also preventing Rho termination. However, it is unclear whether the other NusG paralogs use similar mechanisms, nor have antitermination complexes been investigated for Gram-positive bacteria. Previously, Bacillus velezensis LoaP was shown to enhance transcription of the difficidin antibiotic synthesis operon, but its antitermination determinants were unknown. In this study, we purified RNA polymerase and transcription factors, and reconstituted difficidin operon (dfn) antitermination activity in vitro. The dfn 5’ leader region contains a small RNA hairpin essential for antitermination in vivo and in vitro, and an intrinsic terminator that is highly dependent on NusA. The antitermination activity of LoaP emanates from its ability to specifically antagonize NusA; no other factors are required. These findings demonstrate the basic requirements of the LoaP class of NusG paralogs and indicate that the LoaP antitermination mechanism differs significantly from that of the RfaH paradigm.

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