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The past decade has revolutionized our understanding of regulatory noncoding RNAs (ncRNAs). Among the most recently identified ncRNAs are downstream-of-gene (DoG)-containing transcripts that are produced by widespread transcriptional readthrough. The discovery of DoGs has set the stage for future studies to address many unanswered questions regarding the mechanisms that promote readthrough transcription, RNA processing, and the cellular functions of the unique transcripts. In this review, we summarize current findings regarding the biogenesis, function, and mechanisms regulating this exciting new class of RNA molecules. Downstream-of-gene-containing transcripts (DoGs) are noncoding RNAs (ncRNAs) that are becoming increasingly identified as hallmarks of cellular stress and disease. Their discovery has laid the foundation for the next decade to be an exciting time for unraveling the enigmas that control DoG biogenesis and function. [ABSTRACT FROM AUTHOR]

RNA polymerase II (RNAPII) transcription involves initiation from a promoter, transcriptional elongation through the gene, and termination in the terminator region. In bacteria, terminators often contain specific DNA elements provoking polymerase dissociation, but RNAPII transcription termination is thought to be driven entirely by protein co-factors. We used biochemical reconstitution, single-molecule studies, and genome-wide analysis in yeast to study RNAPII termination. Transcription into natural terminators by pure RNAPII results in spontaneous termination at specific sequences containing T-tracts. Single-molecule analysis indicates that termination involves pausing without backtracking. The "torpedo" Rat1-Rai1 exonuclease (XRN2 in humans) greatly stimulates spontaneous termination but is ineffectual on other paused RNAPIIs. By contrast, elongation factor Spt4-Spt5 (DSIF) suppresses termination. Genome-wide analysis further indicates that termination occurs by transcript cleavage at the poly(A) site exposing a new 5′ RNA-end that allows Rat1-Rai1 loading, which then catches up with destabilized RNAPII at specific termination sites to end transcription. [Display omitted] • RNAPII terminates spontaneously at terminator T-tracts in vitro • Spontaneous termination involves prolonged pausing without backtracking • Torpedo exonuclease Rat1 cooperates with T-tracts to elicit efficient termination • 3′ end sequencing confirms T-tract directed termination in vivo Han et al. use reconstitution of transcription and single-molecule analysis to show that RNAPII pauses and then spontaneously terminates at terminator sequences containing T-tracts. The "torpedo" Rat1-Rai1 exonuclease greatly stimulates spontaneous termination, whereas elongation factor Spt4-Spt5 suppresses it. Termination at T-tracts in cells is confirmed by genome-wide analysis. [ABSTRACT FROM AUTHOR]

Summary To study the function of Rli1/ABCE1 in vivo, we used ribosome profiling and biochemistry to characterize its contribution to ribosome recycling. When Rli1 levels were diminished, 80S ribosomes accumulated both at stop codons and in the adjoining 3′UTRs of most mRNAs. Frequently, these ribosomes reinitiated translation without the need for a canonical start codon, as small peptide products predicted by 3′UTR ribosome occupancy in all three reading frames were confirmed by western analysis and mass spectrometry. Eliminating the ribosome-rescue factor Dom34 dramatically increased 3′UTR ribosome occupancy in Rli1 depleted cells, indicating that Dom34 clears the bulk of unrecycled ribosomes. Thus, Rli1 is crucial for ribosome recycling in vivo and controls ribosome homeostasis. 3′UTR translation occurs in wild-type cells as well, and observations of elevated 3′UTR ribosomes during stress suggest that modulating recycling and reinitiation is involved in responding to environmental changes. [ABSTRACT FROM AUTHOR]

SPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor. [Display omitted] • Auxin-inducible degradation discriminates direct roles of human SPT6 in transcription • Acute loss of SPT6 globally impairs RNAPII processivity and speed • SPT6 is required for efficient transcription termination on protein-coding genes • Long-term loss of SPT6 ultimately results in cryptic intragenic transcription Histone chaperone SPT6 is essential for maintaining epigenetic integrity in the wake of transcription by RNAP II. By studying the effects of targeted acute depletion of SPT6 with multi-omics and mathematical modeling, Narain et al. uncovered direct roles of SPT6 in ensuring efficient transcription elongation and termination in human cells. [ABSTRACT FROM AUTHOR]

The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process. [Display omitted] • mtRF1a triggers polypeptide release at canonical stop codons UAA and UAG • ICT1 rescues the mitoribosome when stalled on aberrant, truncated mRNAs • mtRRF and mtEFG2 promote ribosome recycling by dissolution of intersubunit contacts • mtEFG2 is electrostatically and sterically optimized for ribosome recycling The mitochondrial translation system is very different from the machinery of the eukaryotic cytosolic protein synthesis. Using a combination of biochemical reconstitution and cryoelectron microscopy, Kummer et al. uncover how mitoribosomes terminate polypeptide synthesis on different mRNA substrates and how the ribosome prepares for the production of the next protein. [ABSTRACT FROM AUTHOR]

Summary NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription. Graphical Abstract Highlights • NusG triggers an allosteric relay in Rho to promote activity on nonideal substrates • Rho can be reprogrammed to bypass NusG for transcription termination • NusE directly competes with Rho for binding to NusG to promote antitermination • NusG paralogs lack Rho-binding determinants, explaining functional specificity Lawson et al. show that NusG, a member of a universally conserved transcription factor family, helps isomerize the Rho transcription termination factor from an open-ring loading state to an active, closed-ring conformation. This action overrides the innate sequence bias of Rho to terminate aberrant transcriptional events emanating from translational stress. [ABSTRACT FROM AUTHOR]

Summary The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd’s ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a “release and catch-up” mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle’s resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution. [ABSTRACT FROM AUTHOR]

Summary The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd’s ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a “release and catch-up” mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle’s resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution. [ABSTRACT FROM AUTHOR]

Summary In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E . coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E . coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD , greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis -acting RNA signals that globally control bacterial transcription. [ABSTRACT FROM AUTHOR]