Arabidopsis and maize terminator strength is determined by GC content, polyadenylation motifs and cleavage probability.

The 3' end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.

Utilizing RNA-seq Data to Infer Bacterial Transcription Termination Sites and Validate Predictions.

The transcription termination process is an important part of the gene expression process in the cell. It has been studied extensively, but many aspects of the mechanism are not well understood. The widespread availability of experimental RNA-seq data from high-throughput experiments provides a unique opportunity to infer the end of the transcription units genome wide. This data is available for both Rho-dependent and Rho-independent termination pathways that drive transcription termination in bacteria. Our book chapter gives an overview of the current knowledge of Rho-independent transcription termination mechanisms and the prediction approaches currently deployed to infer the termination sites. Thereafter, we describe our method that uses cluster hairpins to detect Rho-independent transcription termination sites. These clusters are a group of hairpins that lies at <15 bp from each other and are together capable of enforcing the termination process. The idea of a group of hairpins being extensively used for transcription termination is new, and results show that at least 52% of the total cases are of this type, while in the remaining cases, a single strong hairpin is capable of driving transcription termination. The reads derived from the RNA-seq data for corresponding bacteria have been used to validate the predicted sites. The predictions that match these RNA-seq derived sites have higher confidence, and we find almost 98% of the predicted sites, including alternate termination sites, to match the RNA-seq data. We discuss the features of predicted hairpins in detail for a better understanding of the Rho-independent transcription termination mechanism in bacteria. We also explain how users can use the tools developed by us to do transcription terminator predictions and design their experiments through genome-level visualization of the transcription termination sites from the precomputed INTERPIN database.

Characterization of the dual regulation by a c-di-GMP riboswitch Bc1 with a long expression platform from Bacillus thuringiensis.

A riboswitch generally regulates the expression of its downstream genes through conformational change in its expression platform (EP) upon ligand binding. The cyclic diguanosine monophosphate (c-di-GMP) class I riboswitch Bc1 is widespread and conserved among Bacillus cereus group species. In this study, we revealed that Bc1 has a long EP with two typical ρ-independent terminator sequences 28 bp apart. The upstream terminator T1 is dominant in vitro, while downstream terminator T2 is more efficient in vivo. Through mutation analysis, we elucidated that Bc1 exerts a rare and incoherent "transcription-translation" dual regulation with T2 playing a crucial role. However, we found that Bc1 did not respond to c-di-GMP under in vitro transcription conditions, and the expressions of downstream genes did not change with fluctuation in intracellular c-di-GMP concentration. To explore this puzzle, we conducted SHAPE-MaP and confirmed the interaction of Bc1 with c-di-GMP. This shows that as c-di-GMP concentration increases, T1 unfolds but T2 remains almost intact and functional. The presence of T2 masks the effect of T1 unwinding, resulting in no response of Bc1 to c-di-GMP. The high Shannon entropy values of EP region imply the potential alternative structures of Bc1. We also found that zinc uptake regulator can specifically bind to the dual terminator coding sequence and slightly trigger the response of Bc1 to c-di-GMP. This work will shed light on the dual-regulation riboswitch and enrich our understanding of the RNA world.IMPORTANCEIn nature, riboswitches are involved in a variety of metabolic regulation, most of which preferentially regulate transcription termination or translation initiation of downstream genes in specific ways. Alternatively, the same or different riboswitches can exist in tandem to enhance regulatory effects or respond to multiple ligands. However, many putative conserved riboswitches have not yet been experimentally validated. Here, we found that the c-di-GMP riboswitch Bc1 with a long EP could form a dual terminator and exhibit non-canonical and incoherent "transcription-translation" dual regulation. Besides, zinc uptake regulator specifically bound to the coding sequence of the Bc1 EP and slightly mediated the action of Bc1. The application of SHAPE-MaP to the dual regulation mechanism of Bc1 may establish the foundation for future studies of such complex untranslated regions in other bacterial genomes.

Compatibility of termination mechanisms in bacterial transcription with inference on eukaryotic models.

Transcription termination has evolved to proceed through diverse mechanisms. For several classes of terminators, multiple models have been debatably proposed. Recent single-molecule studies on bacterial terminators have resolved several long-standing controversies. First, termination mode or outcome is twofold rather than single. RNA is released alone before DNA or together with DNA from RNA polymerase (RNAP), i.e. with RNA release for termination, RNAP retains on or dissociates off DNA, respectively. The concomitant release, described in textbooks, results in one-step decomposition of transcription complexes, and this 'decomposing termination' prevails at ρ factor-dependent terminators. Contrastingly, the sequential release was recently discovered abundantly from RNA hairpin-dependent intrinsic terminations. RNA-only release allows RNAP to diffuse on DNA in both directions and recycle for reinitiation. This 'recycling termination' enables one-dimensional reinitiation, which would be more expeditious than three-dimensional reinitiation by RNAP dissociated at decomposing termination. Second, while both recycling and decomposing terminations occur at a hairpin-dependent terminator, four termination mechanisms compatibly operate at a ρ-dependent terminator with ρ in alternative modes and even intrinsically without ρ. RNA-bound catch-up ρ mediates recycling termination first and decomposing termination later, while RNAP-prebound stand-by ρ invokes only decomposing termination slowly. Without ρ, decomposing termination occurs slightly and sluggishly. These four mechanisms operate on distinct timescales, providing orderly fail-safes. The stand-by mechanism is benefited by terminational pause prolongation and modulated by accompanying riboswitches more greatly than the catch-up mechanisms. Conclusively, any mechanism alone is insufficient to perfect termination, and multiple mechanisms operate compatibly to achieve maximum possible efficiency under separate controls.

Extraordinary long-stem confers resistance of intrinsic terminators to processive antitermination.

Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.