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.

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.

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.

Engineering RNA polymerase to construct biotechnological host strains of cyanobacteria.

Application of cyanobacteria for bioproduction, bioremediation and biotransformation is being increasingly explored. Photoautotrophs are carbon-negative by default, offering a direct pathway to reducing emissions in production systems. More robust and versatile host strains are needed for constructing production strains that would function as efficient and carbon-neutral cyanofactories. We have tested if the engineering of sigma factors, regulatory units of the bacterial RNA polymerase, could be used to generate better host strains of the model cyanobacterium Synechocystis sp. PCC 6803. Overexpressing the stress-responsive sigB gene under the strong psbA2 promoter (SigB-oe) led to improved tolerance against heat, oxidative stress and toxic end-products. By targeting transcription initiation in the SigB-oe strain, we could simultaneously activate a wide spectrum of cellular protective mechanisms, including carotenoids, the HspA heat shock protein, and highly activated non-photochemical quenching. Yellow fluorescent protein was used to test the capacity of the SigB-oe strain to produce heterologous proteins. In standard conditions, the SigB-oe strain reached a similar production as the control strain, but when cultures were challenged with oxidative stress, the production capacity of SigB-oe surpassed the control strain. We also tested the production of growth-rate-controlled host strains via manipulation of RNA polymerase, but post-transcriptional regulation prevented excessive overexpression of the primary sigma factor SigA, and overproduction of the growth-restricting SigC factor was lethal. Thus, more research is needed before cyanobacteria growth can be manipulated by engineering RNA polymerase.

(p)ppGpp modifies RNAP function to confer ß-lactam resistance in a peptidoglycan-independent manner.

(p)ppGpp is a nucleotide alarmone that controls bacterial response to nutrient deprivation. Since elevated (p)ppGpp levels confer mecillinam resistance and are essential for broad-spectrum ß-lactam resistance as mediated by the ß-lactam-insensitive transpeptidase YcbB (LdtD), we hypothesized that (p)ppGpp might affect cell wall peptidoglycan metabolism. Here we report that (p)ppGpp-dependent ß-lactam resistance does not rely on any modification of peptidoglycan metabolism, as established by analysis of Escherichia coli peptidoglycan structure using high-resolution mass spectrometry. Amino acid substitutions in the ß or ß' RNA polymerase (RNAP) subunits, alone or in combination with the CRISPR interference-mediated downregulation of three of seven ribosomal RNA operons, were sufficient for resistance, although ß-lactams have no known impact on the RNAP or ribosomes. This implies that modifications of RNAP and ribosome functions are critical to prevent downstream effects of the inactivation of peptidoglycan transpeptidases by ß-lactams.

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.

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.

Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system.

We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3'UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5-3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy.

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.

Cryo-EM structures of the plant plastid-encoded RNA polymerase.

Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.

Structure of the multi-subunit chloroplast RNA polymerase.

Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.

Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination.

Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.

Hexasomal particles: consequence or also consequential?

It is long known that an RNA polymerase transcribing through a nucleosome can generate subnucleosomal particles called hexasomes. These particles lack an H2A-H2B dimer, breaking the symmetry of a nucleosome and revealing new interfaces. Whether hexasomes are simply a consequence of RNA polymerase action or they also have a regulatory impact remains an open question. Recent biochemical and structural studies of RNA polymerases and chromatin remodelers with hexasomes motivated us to revisit this question. Here, we build on previous models to discuss how formation of hexasomes can allow sophisticated regulation of transcription and also significantly impact chromatin folding. We anticipate that further cellular and biochemical analysis of these subnucleosomal particles will uncover additional regulatory roles.

Structure of the recombinant RNA polymerase from African Swine Fever Virus.

African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2´-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment.

Compensatory mutations are associated with increased in vitro growth in resistant clinical samples of Mycobacterium tuberculosis.

Mutations in Mycobacterium tuberculosis associated with resistance to antibiotics often come with a fitness cost for the bacteria. Resistance to the first-line drug rifampicin leads to lower competitive fitness of M. tuberculosis populations when compared to susceptible populations. This fitness cost, introduced by resistance mutations in the RNA polymerase, can be alleviated by compensatory mutations (CMs) in other regions of the affected protein. CMs are of particular interest clinically since they could lock in resistance mutations, encouraging the spread of resistant strains worldwide. Here, we report the statistical inference of a comprehensive set of CMs in the RNA polymerase of M. tuberculosis, using over 70 000 M. tuberculosis genomes that were collated as part of the CRyPTIC project. The unprecedented size of this data set gave the statistical tests more power to investigate the association of putative CMs with resistance-conferring mutations. Overall, we propose 51 high-confidence CMs by means of statistical association testing and suggest hypotheses for how they exert their compensatory mechanism by mapping them onto the protein structure. In addition, we were able to show an association of CMs with higher in vitro growth densities, and hence presumably with higher fitness, in resistant samples in the more virulent M. tuberculosis lineage 2. Our results suggest the association of CM presence with significantly higher in vitro growth than for wild-type samples, although this association is confounded with lineage and sub-lineage affiliation. Our findings emphasize the integral role of CMs and lineage affiliation in resistance spread and increases the urgency of antibiotic stewardship, which implies accurate, cheap and widely accessible diagnostics for M. tuberculosis infections to not only improve patient outcomes but also prevent the spread of resistant strains.

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts.

The current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.

Archaeal histone-based chromatin structures regulate transcription elongation rates.

Many archaea encode and express histone proteins to compact their genomes. Archaeal and eukaryotic histones share a near-identical fold that permits DNA wrapping through select histone-DNA contacts to generate chromatin-structures that must be traversed by RNA polymerase (RNAP) to generate transcripts. As archaeal histones can spontaneously assemble with a single histone isoform, single-histone chromatin variants provide an idealized platform to detail the impacts of distinct histone-DNA contacts on transcription efficiencies and to detail the role of the conserved cleavage stimulatory factor, Transcription Factor S (TFS), in assisting RNAP through chromatin landscapes. We demonstrate that substitution of histone residues that modify histone-DNA contacts or the three-dimensional chromatin structure result in radically altered transcription elongation rates and pausing patterns. Chromatin-barriers slow and pause RNAP, providing regulatory potential. The modest impacts of TFS on elongation rates through chromatin landscapes is correlated with TFS-dispensability from the archaeon Thermococcus kodakarensis. Our results detail the importance of distinct chromatin structures for archaeal gene expression and provide a unique perspective on the evolution of, and regulatory strategies imposed by, eukaryotic chromatin.

Towards a rational approach to promoter engineering: understanding the complexity of transcription initiation in prokaryotes.

Promoter sequences are important genetic control elements. Through their interaction with RNA polymerase they determine transcription strength and specificity, thereby regulating the first step in gene expression. Consequently, they can be targeted as elements to control predictability and tuneability of a genetic circuit, which is essential in applications such as the development of robust microbial cell factories. This review considers the promoter elements implicated in the three stages of transcription initiation, detailing the complex interplay of sequence-specific interactions that are involved, and highlighting that DNA sequence features beyond the core promoter elements work in a combinatorial manner to determine transcriptional strength. In particular, we emphasize that, aside from promoter recognition, transcription initiation is also defined by the kinetics of open complex formation and promoter escape, which are also known to be highly sequence specific. Significantly, we focus on how insights into these interactions can be manipulated to lay the foundation for a more rational approach to promoter engineering.

Single-molecule tracking reveals the functional allocation, in vivo interactions, and spatial organization of universal transcription factor NusG.

During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only ∼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging.

Persistence of backtracking by human RNA polymerase II.

RNA polymerase II (RNA Pol II) can backtrack during transcription elongation, exposing the 3' end of nascent RNA. Nascent RNA sequencing can approximate the location of backtracking events that are quickly resolved; however, the extent and genome-wide distribution of more persistent backtracking are unknown. Consequently, we developed a method to directly sequence the extruded, "backtracked" 3' RNA. Our data show that RNA Pol II slides backward more than 20 nt in human cells and can persist in this backtracked state. Persistent backtracking mainly occurs where RNA Pol II pauses near promoters and intron-exon junctions and is enriched in genes involved in translation, replication, and development, where gene expression is decreased if these events are unresolved. Histone genes are highly prone to persistent backtracking, and the resolution of such events is likely required for timely expression during cell division. These results demonstrate that persistent backtracking can potentially affect diverse gene expression programs.