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1. LIET model: capturing the kinetics of RNA polymerase from loading to termination

   https://doi.org/10.1093/nar/gkaf246  

   Stanley, Jacob T; Barone, Georgia E F; Townsend, Hope A; Sigauke, Rutendo F; Allen, Mary A; Dowell, Robin D (April 2025, Nucleic Acids Research)

   Abstract Transcription by RNA polymerases is an exquisitely regulated step of the central dogma. Transcription is the primary determinant of cell-state, and most cellular perturbations impact transcription by altering polymerase activity. Thus, detecting changes in polymerase activity yields insight into most cellular processes. Nascent run-on sequencing provides a direct readout of polymerase activity, but no tools exist to model all aspects of this activity at genes. We focus on RNA polymerase II—responsible for transcribing protein-coding genes. We present the first model to capture the complete process of gene transcription. For individual genes, this model parameterizes each distinct stage of transcription—loading, initiation, elongation, and termination, hence LIET—in a biologically interpretable Bayesian mixture, which is applied to nascent run-on data. Our improved modeling of loading/initiation demonstrates these stages are characteristically different between sense and antisense strands. Applying LIET to 24 human cell-types, our analysis indicates the position of dissociation (the last step of termination) appears to be highly consistent, indicative of a tightly regulated process. Furthermore, by applying LIET to perturbation experiments, we demonstrate its ability to detect specific changes in pausing (5′ end), strand-bias, and dissociation location (3′ end)—opening the door to differential assessment of transcription at individual stages of individual genes. 

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2. Mechanisms and Functions of the RNA Polymerase II General Transcription Machinery during the Transcription Cycle

   https://doi.org/10.3390/biom14020176  

   Archuleta, Stephen R.; Goodrich, James A.; Kugel, Jennifer F. (February 2024, Biomolecules)

   Central to the development and survival of all organisms is the regulation of gene expression, which begins with the process of transcription catalyzed by RNA polymerases. During transcription of protein-coding genes, the general transcription factors (GTFs) work alongside RNA polymerase II (Pol II) to assemble the preinitiation complex at the transcription start site, open the promoter DNA, initiate synthesis of the nascent messenger RNA, transition to productive elongation, and ultimately terminate transcription. Through these different stages of transcription, Pol II is dynamically phosphorylated at the C-terminal tail of its largest subunit, serving as a control mechanism for Pol II elongation and a signaling/binding platform for co-transcriptional factors. The large number of core protein factors participating in the fundamental steps of transcription add dense layers of regulation that contribute to the complexity of temporal and spatial control of gene expression within any given cell type. The Pol II transcription system is highly conserved across different levels of eukaryotes; however, most of the information here will focus on the human Pol II system. This review walks through various stages of transcription, from preinitiation complex assembly to termination, highlighting the functions and mechanisms of the core machinery that participates in each stage. 

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3. Pre-mRNA processing factors differentially impact coordination between co-transcriptional cleavage and transcription termination

   https://doi.org/10.1038/s41467-025-62555-7  

   Jin, Xianhao; Li, Juzuo; Lu, Wenqin; Deng, Xian; Wei, Yanping; Shu, Yi; Liu, Bo; Liu, Zhijian; Long, Yanping; Zhu, Xinlong; et al (December 2025, Nature Communications)

   Co-transcriptional cleavage and transcription termination are closely related processes during mRNA maturation, yet their coordination remains poorly understood due to difficulties in detecting these transient events. Here, we applied single-molecule nascent RNA sequencing to simultaneously capture the cleavage status and readthrough distance on the same nascent RNA molecules and characterize 14 mutants of various pre-mRNA processing factors in Arabidopsis.Our results reveal diverse roles for these processing factors in coordinating cleavage and termination: core components of CPSF and CstF complex stimulate both cleavage and termination, facilitating access to exoribonuclease AtXRN3; mutations in nuclear poly(A) polymerase PAPS1 and AtXRN3 caused delayed termination with minimal effects on cleavage, suggesting their roles are further downstream; BORDER proteins facilitate termination while simultaneously inhibiting cleavage, suggesting a complex interplay between these two actions; the phosphatase SSU72 specifically promotes efficient termination without affecting cleavage. Our method also enables us to distinguish cleaved readthrough transcripts from full-length readthrough, and we found termination factor FPA specifically promotes termination of cleaved readthrough, suggesting FPA facilitates access of AtXRN3 to the 3’ cleavage product. Our comprehensive datasets reveal cleavage and termination are highly coordinated during pre-mRNA processing. 

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4. Single-cell nascent RNA sequencing unveils coordinated global transcription

   https://doi.org/10.1038/s41586-024-07517-7  

   Mahat, Dig B; Tippens, Nathaniel D; Martin-Rufino, Jorge D; Waterton, Sean K; Fu, Jiayu; Blatt, Sarah E; Sharp, Phillip A (July 2024, Nature)

   Abstract Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers^1,2. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations³. However, fundamental questions about the temporal regulation of transcription and enhancer–gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO–seq—a new single-cell nascent RNA sequencing assay that uses click chemistry—and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO–seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO–seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO–seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression. 

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5. Gene expression cycles drive non-exponential bacterial growth

   https://doi.org/10.1103/24rx-sxcw  

   Cylke, Arianna; Banerjee, Shiladitya (August 2025, Physical Review Research)

   Bacterial populations typically exhibit exponential growth under resource-rich conditions, yet individual cells often deviate from this pattern. Recent work has shown that the elongation rates of and increase throughout the cell cycle (super-exponential growth), while displays a midcycle minimum (convex growth), and grows linearly. Here, we develop a single-cell model linking gene expression, proteome allocation, and mass growth to explain these diverse growth trajectories. By calibrating model parameters with experimental data, we show that DNA-proportional mRNA transcription produces near-exponential growth, whereas deviations from this proportionality yield the observed non-exponential growth patterns. Analysis of gene expression perturbations reveals that ribosome expression primarily controls dry mass growth rate, whereas cell envelope protein expression more strongly affects cell elongation rate. We show that cell-cycle-dependent transcription dynamics give rise to convex, super-exponential, and linear modes of cell elongation observed experimentally, demonstrating how the timing of cell envelope and ribosomal protein expressions drive cell-cycle-specific behaviors. These findings provide a mechanistic basis for non-exponential single-cell growth and offer insights into how bacterial cells dynamically regulate elongation rates within each generation. 

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