Structural and functional insights into transcription activation of the essential LysR-type transcriptional regulators.

The enormous LysR-type transcriptional regulators (LTTRs), which are diversely distributed amongst prokaryotes, play crucial roles in transcription regulation of genes involved in basic metabolic pathways, virulence and stress resistance. However, the precise transcription activation mechanism of these genes by LTTRs remains to be explored. Here, we determine the cryo-EM structure of a LTTR-dependent transcription activation complex comprising of Escherichia coli RNA polymerase (RNAP), an essential LTTR protein GcvA and its cognate promoter DNA. Structural analysis shows two N-terminal DNA binding domains of GcvA (GcvA_DBD) dimerize and engage the GcvA activation binding sites, presenting the -35 element for specific recognition with the conserved σ70R4. In particular, the versatile C-terminal domain of α subunit of RNAP directly interconnects with GcvA_DBD, σ70R4 and promoter DNA, providing more interfaces for stabilizing the complex. Moreover, molecular docking supports glycine as one potential inducer of GcvA, and single molecule photobleaching experiments kinetically visualize the occurrence of tetrameric GcvA-engaged transcription activation complex as suggested for the other LTTR homologs. Thus, a general model for tetrameric LTTR-dependent transcription activation is proposed. These findings will provide new structural and functional insights into transcription activation of the essential LTTRs.

How total mRNA influences cell growth.

Experimental observations tracing back to the 1960s imply that ribosome quantities play a prominent role in determining a cell's growth. Nevertheless, in biologically relevant scenarios, growth can also be influenced by the levels of mRNA and RNA polymerase. Here, we construct a quantitative model of biosynthesis providing testable scenarios for these situations. The model explores a theoretically motivated regime where RNA polymerases compete for genes and ribosomes for transcripts and gives general expressions relating growth rate, mRNA concentrations, ribosome, and RNA polymerase levels. On general grounds, the model predicts how the fraction of ribosomes in the proteome depends on total mRNA concentration and inspects an underexplored regime in which the trade-off between transcript levels and ribosome abundances sets the cellular growth rate. In particular, we show that the model predicts and clarifies three important experimental observations, in budding yeast and Escherichia coli bacteria: i) that the growth-rate cost of unneeded protein expression can be affected by mRNA levels, ii) that resource optimization leads to decreasing trends in mRNA levels at slow growth, and iii) that ribosome allocation may increase, stay constant, or decrease, in response to transcription-inhibiting antibiotics. Since the data indicate that a regime of joint limitation may apply in physiological conditions and not only to perturbations, we speculate that this regime is likely self-imposed.

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration.

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches - like most structured RNAs - are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

Structures of the mumps virus polymerase complex via cryo-electron microscopy.

The viral polymerase complex, comprising the large protein (L) and phosphoprotein (P), is crucial for both genome replication and transcription in non-segmented negative-strand RNA viruses (nsNSVs), while structures corresponding to these activities remain obscure. Here, we resolved two L-P complex conformations from the mumps virus (MuV), a typical member of nsNSVs, via cryogenic-electron microscopy. One conformation presents all five domains of L forming a continuous RNA tunnel to the methyltransferase domain (MTase), preferably as a transcription state. The other conformation has the appendage averaged out, which is inaccessible to MTase. In both conformations, parallel P tetramers are revealed around MuV L, which, together with structures of other nsNSVs, demonstrates the diverse origins of the L-binding X domain of P. Our study links varying structures of nsNSV polymerase complexes with genome replication and transcription and points to a sliding model for polymerase complexes to advance along the RNA templates.

Key interactions of RNA polymerase with 6S RNA and secondary channel factors during pRNA synthesis.

Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.

Reciprocating RNA Polymerase batters through roadblocks.

RNA polymerases must transit through protein roadblocks to produce full-length transcripts. Here we report real-time measurements of Escherichia coli RNA polymerase passing through different barriers. As intuitively expected, assisting forces facilitated, and opposing forces hindered, RNA polymerase passage through lac repressor protein bound to natural binding sites. Force-dependent differences were significant at magnitudes as low as 0.2 pN and were abolished in the presence of the transcript cleavage factor GreA, which rescues backtracked RNA polymerase. In stark contrast, opposing forces promoted passage when the rate of RNA polymerase backtracking was comparable to, or faster than the rate of dissociation of the roadblock, particularly in the presence of GreA. Our experiments and simulations indicate that RNA polymerase may transit after roadblocks dissociate, or undergo cycles of backtracking, recovery, and ramming into roadblocks to pass through. We propose that such reciprocating motion also enables RNA polymerase to break protein-DNA contacts that hold RNA polymerase back during promoter escape and RNA chain elongation. This may facilitate productive transcription in vivo.

Reclassification of Aestuariicella albida as Pseudomaricurvus albidus comb. nov. and Aestuariicella hydrocarbonica as Pseudomaricurvus hydrocarbonicus comb. nov. Based on Comparative Genomics and Molecular Synapomorphies.

The genus Aestuariicella has been recently reclassified as a member of the family Cellvibrionaceae. However, the taxonomic position of the genus as a distinct member of the family has not been clarified. In the present study, we performed multilayered analyses anchored on genome sequences to clarify the relationship between the genera Aestuariicella and Pseudomaricurvus within the family Cellvibrionaceae. Phylogenetic analyses based on 16S rRNA gene, RNA polymerase beta subunit (RpoB) protein, and core gene sequences showed a well-supported tight cluster formed by the members of the two genera. Moreover, the analysis of the average amino acid identity (AAI) revealed that the members of the two genera shared 68.16-79.48% AAI, values which were within the range of observed AAI (≥ 67.23%) among the members of the same genus within the family Cellvibrionaceae. Members of the two genera also shared several common characteristics. Furthermore, molecular synapomorphies in a form of conserved signature indels were identified in six protein sequences that were exclusively shared by the members of the two genera. Based on the phylogenetic and molecular evidence presented here, we propose the reclassification of the species Aestuariicella albida and Aestuariicella hydrocarbonica as Pseudomaricurvus albidus comb. nov. and Pseudomaricurvus hydrocarbonicus comb. nov., respectively.

Whole-genome sequencing of clinical isolates from tuberculosis patients in India: real-world data indicates a high proportion of pre-XDR cases.

Treatment decisions for tuberculosis (TB) in the absence of full drug-susceptibility data can result in amplifying resistance and may compromise treatment outcomes. Genomics of Mycobacterium tuberculosis (M.tb) from clinical samples enables detection of drug resistance to multiple drugs. We performed whole-genome sequencing (WGS) for 600 clinical samples from patients with tuberculosis to identify the drug-resistance profile and mutation spectrum. We documented the reasons reported by clinicians for referral. WGS identified a high proportion (51%) of pre-extensively drug-resistant (pre-XDR) cases followed by multidrug-resistant tuberculosis (MDR-TB) (15.5%). This correlates with the primary reason for referral, as non-response to the first-line treatment (67%) and treatment failure or rifampicin resistance (14%). Multivariate analysis indicated that all young age groups (P < 0.05), male gender (P < 0.05), and Beijing strain (P < 0.01) were significant independent predictors of MDR-TB or MDR-TB+ [pre-extensively drug-resistant tuberculosis (XDR-TB) and XDR-TB]. Ser315Thr (72.5%) in the inhA gene and Ser450Leu in the rpoB gene (65.5%) were the most prevalent mutations, as were resistance-conferring mutations to pyrazinamide (41%) and streptomycin (61.33%). Mutations outside the rifampicin resistance-determining region (RRDR), Ile491Phe and Val170Phe, were seen in 1.3% of cases; disputed mutations in rpoB (Asp435Tyr, His445Asn, His445Leu, and Leu430Pro) were seen in 6% of cases, and mutations to newer drugs such as bedaquiline and linezolid in 1.0% and 7.5% of cases, respectively. This study on clinical samples highlights that there is a high proportion of pre-XDR cases and emerging resistance to newer drugs; ongoing transmission of these strains can cause serious threat to public health; and whole-genome sequencing can effectively identify and support precision medicine for TB. IMPORTANCE: The current study is based on real-world data on the TB drug-resistance profile by whole-genome sequencing of 600 clinical samples from patients with TB in India. This study indicates the clinicians' reasons for sending samples for WGS, which is for difficult-to-treat cases and/or relapse and treatment failure. The study reports a significant proportion of cases with pre-XDR-TB strains that warrant policy makers' attention. It reflects the current iterative nature of the diagnostic tests under programmatic conditions that leads to delays in appropriate diagnosis and empirical treatment. India had an estimated burden of 2.95 million TB cases in 2020 and 135,000 multidrug-resistant cases. However, WGS profiles of M.tb from India remains disproportionately poorly represented. This study adds a significant body of data on the mutation profiles seen in M.tb isolated from patients with TB in India, mutations outside the RRDR, disputed mutations, and resistance-conferring mutations to newer drugs such as bedaquiline and linezolid.

Comparison of targeted next-generation sequencing and the Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in clinical isolates and sputum specimens.

Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.

Cryo-EM Structure of Porphyromonas gingivalis RNA Polymerase.

Porphyromonas gingivalis, an anaerobic CFB (Cytophaga, Fusobacterium, and Bacteroides) group bacterium, is the keystone pathogen of periodontitis and has been implicated in various systemic diseases. Increased antibiotic resistance and lack of effective antibiotics necessitate a search for new intervention strategies. Here we report a 3.5 Å resolution cryo-EM structure of P. gingivalis RNA polymerase (RNAP). The structure displays new structural features in its ω subunit and multiple domains in ß and ß' subunits, which differ from their counterparts in other bacterial RNAPs. Superimpositions with E. coli RNAP holoenzyme and initiation complex further suggest that its ω subunit may contact the σ4 domain, thereby possibly contributing to the assembly and stabilization of initiation complexes. In addition to revealing the unique features of P. gingivalis RNAP, our work offers a framework for future studies of transcription regulation in this important pathogen, as well as for structure-based drug development.

The effect of pseudoknot base pairing on cotranscriptional structural switching of the fluoride riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

A protective role for type I interferon signaling following infection with Mycobacterium tuberculosis carrying the rifampicin drug resistance-conferring RpoB mutation H445Y.

Interleukin-1 (IL-1) signaling is essential for controlling virulent Mycobacterium tuberculosis (Mtb) infection since antagonism of this pathway leads to exacerbated pathology and increased susceptibility. In contrast, the triggering of type I interferon (IFN) signaling is associated with the progression of tuberculosis (TB) disease and linked with negative regulation of IL-1 signaling. However, mice lacking IL-1 signaling can control Mtb infection if infected with an Mtb strain carrying the rifampin-resistance conferring mutation H445Y in its RNA polymerase ß subunit (rpoB-H445Y Mtb). The mechanisms that govern protection in the absence of IL-1 signaling during rpoB-H445Y Mtb infection are unknown. In this study, we show that in the absence of IL-1 signaling, type I IFN signaling controls rpoB-H445Y Mtb replication, lung pathology, and excessive myeloid cell infiltration. Additionally, type I IFN is produced predominantly by monocytes and recruited macrophages and acts on LysM-expressing cells to drive protection through nitric oxide (NO) production to restrict intracellular rpoB-H445Y Mtb. These findings reveal an unexpected protective role for type I IFN signaling in compensating for deficiencies in IL-1 pathways during rpoB-H445Y Mtb infection.

Concerted transformation of a hyper-paused transcription complex and its reinforcing protein.

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a ß-barrel, which binds the ribosome. Here, we report structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH, which reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life.

Comprehensive evaluation of T7 promoter for enhanced yield and quality in mRNA production.

The manufacturing of mRNA vaccines relies on cell-free based systems that are easily scalable and flexible compared with the traditional vaccine manufacturing processes. Typically, standard processes yield 2 to 5 g L-1 of mRNA, with recent process optimisations increasing yields to 12 g L-1. However, increasing yields can lead to an increase in the production of unwanted by-products, namely dsRNA. It is therefore imperative to reduce dsRNA to residual levels in order to avoid intensive purification steps, enabling cost-effective manufacturing processes. In this work, we exploit sequence modifications downstream of the T7 RNA polymerase promoter to increase mRNA yields whilst simultaneously minimising dsRNA. In particular, transcription performance was optimised by modifying the sequence downstream of the T7 promoter with additional AT-rich sequences. We have identified variants that were able to produce higher amounts of mRNA (up to 14 g L-1) in 45 min of reaction. These variants exhibited up to a 30% reduction in dsRNA byproduct levels compared to a wildtype T7 promoter, and have similar EGFP protein expression. The results show that optimising the non-coding regions can have an impact on mRNA production yields and quality, reducing overall manufacturing costs.

Determinants of mer Promoter Activity from Pseudomonas aeruginosa.

Since the MerR family is known for its special regulatory mechanism, we aimed to explore which factors determine the expression activity of the mer promoter. The Tn501/Tn21 mer promoter contains an abnormally long spacer (19 bp) between the -35 and -10 elements, which is essential for the unique DNA distortion mechanism. To further understand the role of base sequences in the mer promoter spacer, this study systematically engineered a series of mutant derivatives and used luminescent and fluorescent reporter genes to investigate the expression activity of these derivatives. The results reveal that the expression activity of the mer promoter is synergistically modulated by the spacer length (17 bp is optimal) and the region upstream of -10 (especially -13G). The spacing is regulated by MerR transcription factors through symmetrical sequences, and -13G presumably functions through interaction with the RNA polymerase sigma-70 subunit.

Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis.

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

Transcription-driven DNA supercoiling counteracts H-NS-mediated gene silencing in bacterial chromatin.

In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to "unroll" disrupting the H-NS bridges and releasing H-NS.

Architecture of an RNA polymerase ribozyme illuminates the RNA World.

'Ribo-organisms' of the primordial RNA World would have needed ribozymes that catalyze RNA replication. McRae, Wan, Kristoffersen et al. recently revealed how these RNA replicases might have functioned by solving the structure of an artificial polymerase ribozyme. This work illustrates how complex RNA structures evolve, with implications for the origins of life.

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.