Structural Basis for Control of Bacterial RNA Polymerase Pausing by a Riboswitch and its Ligand

Folding of nascent transcripts can be modulated by the proximal RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ1 riboswitch (que-ePEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination, however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-ePEC in ligand-free and ligand-bound states. In the absence of preQ1, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, upon ligand binding the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which small nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.

The chromatin organization of a chlorarachniophyte nucleomorph genome

Nucleomoprhs are remnants of secondary endosymbiotic events between two eukaryote cells wherein the endosymbiont has retained its eukaryotic nucleus. Nucleomorphs have evolved at least twice independently, in chlorarachniophytes and cryptophytes, yet they have converged on a remarkably similar genomic architecture, characterized by the most extreme compression and miniaturization among all known eukaryotic genomes. Previous computational studies have suggested that nucleomorph chromatin likely exhibits a number of divergent features. In this work, we provide the first maps of open chromatin, active transcription, and three-dimensional organization for the nucleomorph genome of the chlorarachniophyte Bigelowiella natans. We find that the B. natans nucleomorph genome exists in a highly accessible state, akin to that of ribosomal DNA in some other eukaryotes, and that it is highly transcribed over its entire length, with few signs of polymerase pausing at transcription start sites (TSSs). At the same time, most nucleomorph TSSs show very strong nucleosome positioning. Chromosome conformation (Hi-C) maps reveal that nucleomorph chromosomes interact with one other at their telomeric regions, and show the relative contact frequencies between the multiple genomic compartments of distinct origin that B. natans cells contain.

Quantitative imaging of transcription in living Drosophila embryos reveals the impact of core promoter motifs on promoter state dynamics

Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.

The Drosophila HP1 family is associated with active gene expression across chromatin contexts

Drosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a to be transcribed. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, we examined the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. We found that HP1 proteins share many of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than non-target genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared to non-target genes. Specifically, co-localization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. Our data broaden understanding of the mechanism of transcriptional activation by HP1a and highlight the need to consider particular protein-protein interactions, rather than broader chromatin context, to predict impacts of HP1 at transcription start sites.

The Drosophila HP1 family is associated with active gene expression across chromatin contexts

Drosophila Heterochromatin Protein 1a (HP1a) is essential for heterochromatin formation and is involved in transcriptional silencing. However, certain loci require HP1a to be transcribed. One model posits that HP1a acts as a transcriptional silencer within euchromatin while acting as an activator within heterochromatin. However, HP1a has been observed as an activator of a set of euchromatic genes. Therefore, it is not clear whether, or how, chromatin context informs the function of HP1 proteins. To understand the role of HP1 proteins in transcription, we examined the genome-wide binding profile of HP1a as well as two other Drosophila HP1 family members, HP1B and HP1C, to determine whether coordinated binding of these proteins is associated with specific transcriptional outcomes. We found that HP1 proteins share many of their endogenous binding targets. These genes are marked by active histone modifications and are expressed at higher levels than non-target genes in both heterochromatin and euchromatin. In addition, HP1 binding targets displayed increased RNA polymerase pausing compared to non-target genes. Specifically, co-localization of HP1B and HP1C was associated with the highest levels of polymerase pausing and gene expression. Analysis of HP1 null mutants suggests these proteins coordinate activity at transcription start sites to regulate transcription. Depletion of HP1B or HP1C alters expression of protein-coding genes bound by HP1 family members. Our data broaden understanding of the mechanism of transcriptional activation by HP1a and highlight the need to consider particular protein-protein interactions, rather than broader chromatin context, to predict impacts of HP1 at transcription start sites.

NusG is an intrinsic transcription termination factor that stimulates motility and coordinates gene expression with NusA

NusA and NusG are transcription factors that stimulate RNA polymerase pausing in Bacillus subtilis. While NusA was known to function as an intrinsic termination factor in B. subtilis, the role of NusG in this process was unknown. To examine the individual and combinatorial roles that NusA and NusG play in intrinsic termination, Term-seq was conducted in wild type, NusA depletion, ΔnusG, and NusA depletion ΔnusG strains. We determined that NusG functions as an intrinsic termination factor that works alone and cooperatively with NusA to facilitate termination at 88% of the 1400 identified intrinsic terminators. Our results indicate that NusG stimulates a sequence-specific pause that assists in the completion of suboptimal terminator hairpins with weak terminal A-U and G-U base pairs at the bottom of the stem. Loss of NusA and NusG leads to global misregulation of gene expression and loss of NusG results in flagella and swimming motility defects.

Quantitative imaging of transcription in living Drosophila embryos reveals the impact of core promoter motifs on promoter state dynamics

Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.

Basic mechanisms and kinetics of pause-interspersed transcript elongation

RNA polymerase pausing during elongation is an important mechanism in the regulation of gene expression. Pausing along DNA templates is thought to be induced by distinct signals encoded in the nucleic acid sequence and halt elongation complexes to allow time for necessary co-transcriptional events. Pausing signals have been classified as those producing short-lived elemental, long-lived backtracked, or hairpin-stabilized pauses. In recent years, structural microbiology and single-molecule studies have significantly advanced our understanding of the paused states, but the dynamics of these states are still uncertain, although several models have been proposed to explain the experimentally observed pausing behaviors. This review summarizes present knowledge about the paused states, discusses key discrepancies among the kinetic models and their basic assumptions, and highlights the importance and challenges in constructing theoretical models that may further our biochemical understanding of transcriptional pausing.