Sourcing Phage-Encoded Terminators Using ONT-cappable-seq for SynBio Applications in Pseudomonas.

Efficient transcriptional terminators are essential for the performance of genetic circuitry in microbial SynBio hosts. In recent years, several libraries of characterized strong terminators have become available for model organisms such as Escherichia coli. Conversely, terminator libraries for nonmodel species remain scarce, and individual terminators are often ported over from model systems, leading to unpredictable performance in their new hosts. In this work, we mined the genomes of Pseudomonas infecting phages LUZ7 and LUZ100 for transcriptional terminators utilizing the full-length RNA sequencing technique "ONT-cappable-seq" and validated these terminators in three Gram-negative hosts using a terminator trap assay. Based on these results, we present nine terminators for E. coli, Pseudomonas putida, and Pseudomonas aeruginosa, which outperform current reference terminators. Among these, terminator LUZ7 T50 displays potent bidirectional activity. These data further support that bacteriophages, as evolutionary-adapted natural predators of the targeted bacteria, provide a valuable source of microbial SynBio parts.

Early antitermination in the atypical coliphage mEp021 mediated by the Gp17 protein.

The coliphage mEp021 belongs to a phage group with a unique immunity repressor, and its life cycle requires the host factor Nus. mEp021 has been classified as non-lambdoid based on its specific characteristics. The mEp021 genome carries a gene encoding an Nλ-like antiterminator protein, termed Gp17, and three nut sites (nutL, nutR1, and nutR2). Analysis of plasmid constructs containing these nut sites, a transcription terminator, and a GFP reporter gene showed high levels of fluorescence when Gp17 was expressed, but not in its absence. Like lambdoid N proteins, Gp17 has an arginine-rich motif (ARM), and mutations in its arginine codons inhibit its function. In infection assays using the mutant phage mEp021ΔGp17::Kan (where gp17 has been deleted), gene transcripts located downstream of transcription terminators were obtained only when Gp17 was expressed. In contrast to phage lambda, mEp021 virus particle production was partially restored (>1/3 relative to wild type) when nus mutants (nusA1, nusB5, nusC60, and nusE71) were infected with mEp021 and Gp17 was overexpressed. Our results suggest that RNA polymerase reads through the third nut site (nutR2), which is more than 7.9 kbp downstream of nutR1.

Recognition of transcription terminators during retrotransposition: How to keep a group II intron quiet.

Chung et al. recently presented the structure of a primitive group IIC intron with its DNA target, which reveals the structural requirements that this class of intron uses to recognize a transcription terminator stem loop at the DNA level for insertion during retrotransposition.

Structural basis for intrinsic transcription termination.

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.

Plant terminators: the unsung heroes of gene expression.

To be properly expressed, genes need to be accompanied by a terminator, a region downstream of the coding sequence that contains the information necessary for the maturation of the mRNA 3' end. The main event in this process is the addition of a poly(A) tail at the 3' end of the new transcript, a critical step in mRNA biology that has important consequences for the expression of genes. Here, we review the mechanism leading to cleavage and polyadenylation of newly transcribed mRNAs and how this process can affect the final levels of gene expression. We give special attention to an aspect often overlooked, the effect that different terminators can have on the expression of genes. We also discuss some exciting findings connecting the choice of terminator to the biogenesis of small RNAs, which are a central part of one of the most important mechanisms of regulation of gene expression in plants.

Intrinsic and Rho-dependent termination cooperate for efficient transcription termination at 3' untranslated regions.

The intrinsic, and the Rho-dependent mechanisms of transcription termination are conserved in bacteria. Generally, the two mechanisms have been illustrated as two independent pathways occurring in the 3' ends of different genes with contrasting requirements to halt RNA synthesis. However, a majority of intrinsic terminators terminate transcription inefficiently leading to transcriptional read-through. The unwanted transcription in the downstream region beyond the terminator would have undesired consequences. To prevent such transcriptional read-through, bacteria must have evolved ways to terminate transcription more efficiently at or near the termination sites. We describe the participation of both the mechanisms, where intrinsic terminator and Rho factor contribute to prevent transcriptional read-through. Contribution from both the termination processes is demonstrated at the downstream regions of the genes both in vitro and in vivo in mycobacteria. Distinct patterns of cooperation between the two modes of termination were observed at the 3' untranslated regions of the genes to ensure efficient termination. We demonstrate similar mode of operation between the two termination processes in Escherichia coli suggesting a likely prevalence of this cooperation across bacteria. The reporter system developed to assess the Rho - intrinsic termination collaboration in vivo for mycobacteria and E. coli can readily be applied to other bacteria.

Architecture of the yeast Pol III pre-termination complex and pausing mechanism on poly(dT) termination signals.

RNA polymerase (Pol) III is specialized to transcribe short, abundant RNAs, for which it terminates transcription on polythymine (dT) stretches on the non-template (NT) strand. When Pol III reaches the termination signal, it pauses and forms the pre-termination complex (PTC). Here, we report cryoelectron microscopy (cryo-EM) structures of the yeast Pol III PTC and complementary functional states at resolutions of 2.7-3.9 Å. Pol III recognizes the poly(dT) termination signal with subunit C128 that forms a hydrogen-bond network with the NT strand and, thereby, induces pausing. Mutating key interacting residues interferes with transcription termination in vitro, impairs yeast growth, and causes global termination defects in vivo, confirming our structural results. Additional cryo-EM analysis reveals that C53-C37, a Pol III subcomplex and key termination factor, participates indirectly in Pol III termination. We propose a mechanistic model of Pol III transcription termination and rationalize why Pol III, unlike Pol I and Pol II, terminates on poly(dT) signals.

Factor-stimulated intrinsic termination: getting by with a little help from some friends.

Transcription termination is known to occur via two mechanisms in bacteria, intrinsic termination (also frequently referred to as Rho-independent or factor-independent termination) and Rho-dependent termination. Based primarily on in vitro studies using Escherichia coli RNA polymerase, it was generally assumed that intrinsic termination and Rho-dependent termination are distinct mechanisms and that the signals required for intrinsic termination are present primarily within the nucleic acids. In this review, we detail recent findings from studies in Bacillus subtilis showing that intrinsic termination in this organism is highly stimulated by NusA, NusG, and even Rho. In NusA-stimulated intrinsic termination, NusA facilitates the formation of weak terminator hairpins and compensates for distal U-rich tract interruptions. In NusG-stimulated intrinsic termination, NusG stabilizes a sequence-dependent pause at the point of termination, which extends the time frame for RNA hairpins with weak terminal base pairs to form in either a NusA-stimulated or a NusA-independent fashion. In Rho-stimulated intrinsic termination, Rho prevents the formation of antiterminator-like RNA structures that could otherwise compete with the terminator hairpin. Combined, NusA, NusG, and Rho stimulate approximately 97% of all intrinsic terminators in B. subtilis. Thus, the general view that intrinsic termination is primarily a factor-independent process needs to be revised to account for recent findings. Moreover, the historical distinction between Rho-dependent and intrinsic termination is overly simplistic and needs to be modernized.

In vivo regulation of bacterial Rho-dependent transcription termination by the nascent RNA.

Bacterial Rho is a RNA-dependent ATPase that functions in the termination of transcription. The in vivo nature of the bacterial Rho-dependent terminators, as well as the mechanism of the Rho-dependent termination process, are not fully understood. Here, we measured the in vivo termination efficiencies of 72 Rho-dependent terminators in Escherichia coli by systematically performing qRT-PCR analyses of cDNA prepared from mid-log phase bacterial cultures. We found that these terminators exhibited a wide range of efficiencies, and many behaved differently in vivo compared to the predicted or experimentally determined efficiencies in vitro. Rho-utilization sites (rut sites) present in the RNA terminator sequences are characterized by the presence of C-rich/G-poor sequences or C > G bubbles. We found that weaker terminators exhibited a robust correlation with the properties (size, length, density, etc.) of these C > G bubbles of their respective rut sites, while stronger terminators lack this correlation, suggesting a limited role of rut sequences in controlling in vivo termination efficiencies. We also found that in vivo termination efficiencies are dependent on the rates of ATP hydrolysis as well as Rho-translocation on the nascent RNA. We demonstrate that weaker terminators, in addition to having rut sites with diminished C > G bubble sizes, are dependent on the Rho-auxiliary factor, NusG, in vivo. From these results, we concluded that in vivo Rho-dependent termination follows a nascent RNA-dependent pathway, where Rho-translocation along the RNA is essential and rut sequences may recruit Rho in vivo, but Rho-rut binding strengths do not regulate termination efficiencies.

Structural characterization of the ANTAR antiterminator domain bound to RNA.

Regulated transcription termination provides an efficient and responsive means to control gene expression. In bacteria, rho-independent termination occurs through the formation of an intrinsic RNA terminator loop, which disrupts the RNA polymerase elongation complex, resulting in its dissociation from the DNA template. Bacteria have a number of pathways for overriding termination, one of which is the formation of mutually exclusive RNA motifs. ANTAR domains are a class of antiterminator that bind and stabilize dual hexaloop RNA motifs within the nascent RNA chain to prevent terminator loop formation. We have determined the structures of the dimeric ANTAR domain protein EutV, from Enterococcus faecialis, in the absence of and in complex with the dual hexaloop RNA target. The structures illustrate conformational changes that occur upon RNA binding and reveal that the molecular interactions between the ANTAR domains and RNA are restricted to a single hexaloop of the motif. An ANTAR domain dimer must contact each hexaloop of the dual hexaloop motif individually to prevent termination in eubacteria. Our findings thereby redefine the minimal ANTAR domain binding motif to a single hexaloop and revise the current model for ANTAR-mediated antitermination. These insights will inform and facilitate the discovery of novel ANTAR domain RNA targets.

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region.

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

Massively parallel characterization of engineered transcript isoforms using direct RNA sequencing.

Transcriptional terminators signal where transcribing RNA polymerases (RNAPs) should halt and disassociate from DNA. However, because termination is stochastic, two different forms of transcript could be produced: one ending at the terminator and the other reading through. An ability to control the abundance of these transcript isoforms would offer bioengineers a mechanism to regulate multi-gene constructs at the level of transcription. Here, we explore this possibility by repurposing terminators as 'transcriptional valves' that can tune the proportion of RNAP read-through. Using one-pot combinatorial DNA assembly, we iteratively construct 1780 transcriptional valves for T7 RNAP and show how nanopore-based direct RNA sequencing (dRNA-seq) can be used to characterize entire libraries of valves simultaneously at a nucleotide resolution in vitro and unravel genetic design principles to tune and insulate termination. Finally, we engineer valves for multiplexed regulation of CRISPR guide RNAs. This work provides new avenues for controlling transcription and demonstrates the benefits of long-read sequencing for exploring complex sequence-function landscapes.

The influence of regulatory elements on Mycoplasma hyopneumoniae 7448 transcriptional response during oxidative stress and heat shock.

BACKGROUND: The comprehension of genome organization and gene modulation is essential for understanding pathogens' infection mechanisms. Mycoplasma hyopneumoniae 7448 genome is organized in transcriptional units (TUs), which are flanked by regulatory elements such as putative promoters, terminators and repetitive sequences. Yet the relationship between the presence of these elements and bacterial responses during stress conditions remains unclear. Thus, in this study, in silico and RT-qPCR analyses were associated to determine the effect of regulatory elements in gene expression regulation upon heat shock and oxidative stress conditions. METHODS AND RESULTS: Thirteen TU's organizational profiles were found based on promoters and terminators distribution. Differential expression in genes sharing the same TUs was observed, suggesting the activity of internal regulatory elements. Moreover, 88.8% of tested genes were differentially expressed under oxidative stress in comparison to the control condition, being 81.3% of them surrounded by their own regulatory elements. Similarly, under heat shock, 44.4% of the genes showed regulation when compared to control condition, being 75.0% of them surrounded by their own regulatory elements. CONCLUSIONS: Altogether, this data suggests the activity of internal regulatory elements in gene modulation of M. hyopneumoniae 7448 transcription.

aCPSF1 cooperates with terminator U-tract to dictate archaeal transcription termination efficacy.

Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea; however, the working mechanism remains elusive. This work, through analyzing transcript-3'end-sequencing data of Methanococcus maripaludis, found genome-wide positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficacies (TTEs). In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding affinity, and in vivo assays demonstrated the two elements are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract cooperatively determine high TTEs. The N-terminal KH domains equip aCPSF1 with specific-binding capacity to terminator U-tract and the aCPSF1-terminator U-tract cooperation; while the nuclease activity of aCPSF1 was also required for TTEs. aCPSF1 also guarantees the terminations of transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract cooperation in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in archaea, which may be widely employed by archaeal phyla; using one trans-action factor to recognize U-rich terminator signal and cleave transcript 3'-end, the archaeal aCPSF1-dependent transcription termination may represent a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery.

Clusters of hairpins induce intrinsic transcription termination in bacteria.

Intrinsic transcription termination (ITT) sites are currently identified by locating single and double-adjacent RNA hairpins downstream of the stop codon. ITTs for a limited number of genes/operons in only a few bacterial genomes are currently known. This lack of coverage is a lacuna in the existing ITT inference methods. We have studied the inter-operon regions of 13 genomes covering all major phyla in bacteria, for which good quality public RNA-seq data exist. We identify ITT sites in 87% of cases by predicting hairpin(s) and validate against 81% of cases for which the RNA-seq derived sites could be calculated. We identify 72% of these sites correctly, with 98% of them located ≤ 80 bases downstream of the stop codon. The predicted hairpins form a cluster (when present < 15 bases) in two-thirds of the cases, the remaining being single hairpins. The largest number of clusters is formed by two hairpins, and the occurrence decreases exponentially with an increasing number of hairpins in the cluster. Our study reveals that hairpins form an effective ITT unit when they act in concert in a cluster. Their pervasiveness along with single hairpin terminators corroborates a wider utilization of ITT mechanisms for transcription control across bacteria.

Data-Driven and in Silico-Assisted Design of Broad Host-Range Minimal Intrinsic Terminators Adapted for Bacteria.

Efficient transcription termination relying on intrinsic terminators is critical to maintain cell fitness by avoiding unwanted read-through in bacteria. Natural intrinsic terminator (NIT) typically appears in mRNA as a hairpin followed by approximately eight conserved uridines (U-tract) at the 3' terminus. Owing to their simple structure, small size, and protein independence, assorted NITs have been redesigned as robust tools to construct gene circuits. However, most NITs exert functions to adapt to their physiological requirements rather than the demand for building synthetic gene circuits, rendering uncertain working performance when they are constructed intact in synthetic gene circuits. Here, rather than modifying NITs, we established a data-driven and in silico-assisted (DISA) design framework to forward engineer minimal intrinsic terminators (MITs). By comprehensively analyzing 75 natural intrinsic terminators from Bacillus subtilis, we revealed that two pivotal features, the length of the U-tract and the thermodynamics of the terminator hairpin, were involved in the sequence-activity relationship (SAR) of termination efficiency (TE). As per the SAR, we leveraged DISA to fabricate an array of MITs composed of in silico-assisted designed minimal hairpins and fixed U-tracts. Most of these MITs exhibited high TE in diverse Gram-positive and Gram-negative bacteria. In contrast, the TEs of the NITs were highly varied in different hosts. Moreover, TEs of MITs were flexibly tuned over a wide range by modulating the length of the U-tract. Overall, these results demonstrate an efficient framework to forward design functional and broad host-range terminators independent of tedious and iterative screening of mutagenesis libraries of natural terminators.

A novel computational framework for genome-scale alternative transcription units prediction.

Alternative transcription units (ATUs) are dynamically encoded under different conditions and display overlapping patterns (sharing one or more genes) under a specific condition in bacterial genomes. Genome-scale identification of ATUs is essential for studying the emergence of human diseases caused by bacterial organisms. However, it is unrealistic to identify all ATUs using experimental techniques because of the complexity and dynamic nature of ATUs. Here, we present the first-of-its-kind computational framework, named SeqATU, for genome-scale ATU prediction based on next-generation RNA-Seq data. The framework utilizes a convex quadratic programming model to seek an optimum expression combination of all of the to-be-identified ATUs. The predicted ATUs in Escherichia coli reached a precision of 0.77/0.74 and a recall of 0.75/0.76 in the two RNA-Sequencing datasets compared with the benchmarked ATUs from third-generation RNA-Seq data. In addition, the proportion of 5'- or 3'-end genes of the predicted ATUs, having documented transcription factor binding sites and transcription termination sites, was three times greater than that of no 5'- or 3'-end genes. We further evaluated the predicted ATUs by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses. The results suggested that gene pairs frequently encoded in the same ATUs are more functionally related than those that can belong to two distinct ATUs. Overall, these results demonstrated the high reliability of predicted ATUs. We expect that the new insights derived by SeqATU will not only improve the understanding of the transcription mechanism of bacteria but also guide the reconstruction of a genome-scale transcriptional regulatory network.

Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho.

The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ∼42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides.

Control of a programmed cell death pathway in Pseudomonas aeruginosa by an antiterminator.

In Pseudomonas aeruginosa the alp system encodes a programmed cell death pathway that is switched on in a subset of cells in response to DNA damage and is linked to the virulence of the organism. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified target locus, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. AlpA thus functions in a mechanistically unusual manner to control the expression of virulence genes in this opportunistic pathogen.

Mechanism of RNA polymerase III termination-associated reinitiation-recycling conferred by the essential function of the N terminal-and-linker domain of the C11 subunit.

RNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3'-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3'-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3'-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3'-cleavage activity increases growth rate but is nonessential.