POLE Mutation Spectra Are Shaped by the Mutant Allele Identity, Its Abundance, and Mismatch Repair Status.

Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis.

Combined analyses of within-host SARS-CoV-2 viral kinetics and information on past exposures to the virus in a human cohort identifies intrinsic differences of Omicron and Delta variants.

The emergence of successive Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) during 2020 to 2022, each exhibiting increased epidemic growth relative to earlier circulating variants, has created a need to understand the drivers of such growth. However, both pathogen biology and changing host characteristics-such as varying levels of immunity-can combine to influence replication and transmission of SARS-CoV-2 within and between hosts. Disentangling the role of variant and host in individual-level viral shedding of VOCs is essential to inform Coronavirus Disease 2019 (COVID-19) planning and response and interpret past epidemic trends. Using data from a prospective observational cohort study of healthy adult volunteers undergoing weekly occupational health PCR screening, we developed a Bayesian hierarchical model to reconstruct individual-level viral kinetics and estimate how different factors shaped viral dynamics, measured by PCR cycle threshold (Ct) values over time. Jointly accounting for both interindividual variation in Ct values and complex host characteristics-such as vaccination status, exposure history, and age-we found that age and number of prior exposures had a strong influence on peak viral replication. Older individuals and those who had at least 5 prior antigen exposures to vaccination and/or infection typically had much lower levels of shedding. Moreover, we found evidence of a correlation between the speed of early shedding and duration of incubation period when comparing different VOCs and age groups. Our findings illustrate the value of linking information on participant characteristics, symptom profile and infecting variant with prospective PCR sampling, and the importance of accounting for increasingly complex population exposure landscapes when analysing the viral kinetics of VOCs. Trial Registration: The Legacy study is a prospective observational cohort study of healthy adult volunteers undergoing weekly occupational health PCR screening for SARS-CoV-2 at University College London Hospitals or at the Francis Crick Institute (NCT04750356) (22,23). The Legacy study was approved by London Camden and Kings Cross Health Research Authority Research and Ethics committee (IRAS number 286469). The Legacy study was approved by London Camden and Kings Cross Health Research Authority Research and Ethics committee (IRAS number 286469) and is sponsored by University College London Hospitals. Written consent was given by all participants.

Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity.

Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5' → 3' exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3' flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3' end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.

The Adjuvant Combination of dmLT and Monophosphoryl Lipid A Activates the Canonical, Nonpyroptotic NLRP3 Inflammasome in Dendritic Cells and Significantly Interacts to Expand Antigen-Specific CD4 T Cells.

Adjuvants are often essential additions to vaccines that enhance the activation of innate immune cells, leading to more potent and protective T and B cell responses. Only a few vaccine adjuvants are currently used in approved vaccine formulations in the United States. Combinations of one or more adjuvants have the potential to increase the efficacy of existing and next-generation vaccines. In this study, we investigated how the nontoxic double mutant Escherichia coli heat-labile toxin R192G/L211A (dmLT), when combined with the TLR4 agonist monophosphoryl lipid A (MPL-A), impacted innate and adaptive immune responses to vaccination in mice. We found that the combination of dmLT and MPL-A induced an expansion of Ag-specific, multifaceted Th1/2/17 CD4 T cells higher than that explained by adding responses to either adjuvant alone. Furthermore, we observed more robust activation of primary mouse bone marrow-derived dendritic cells in the combination adjuvant-treated group via engagement of the canonical NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex. This was marked by a multiplicative increase in the secretion of active IL-1ß that was independent of classical gasdermin D-mediated pyroptosis. Moreover, the combination adjuvant increased the production of the secondary messengers cAMP and PGE2 in dendritic cells. These results demonstrate how certain adjuvant combinations could be used to potentiate better vaccine responses to combat a variety of pathogens.

SARS-CoV-2 Antibody Response Is Associated with Age and Body Mass Index in Convalescent Outpatients.

COVID-19 has had an unprecedented global impact on human health. Understanding the Ab memory responses to infection is one tool needed to effectively control the pandemic. Among 173 outpatients who had virologically confirmed SARS-CoV-2 infection, we evaluated serum Ab concentrations, microneutralization activity, and enumerated SARS-CoV-2-specific B cells in convalescent human blood specimens. Serum Ab concentrations were variable, allowing for stratification of the cohort into high and low responders. Neither participant sex, the timing of blood sampling following the onset of illness, nor the number of SARS-CoV-2 spike protein-specific B cells correlated with serum Ab concentration. Serum Ab concentration was positively associated with microneutralization activity and participant age, with participants under the age of 30 showing the lowest Ab level. These data suggest that young adult outpatients did not generate as robust Ab memory, compared with older adults. Body mass index was also positively correlated with serum Ab levels. Multivariate analyses showed that participant age and body mass index were independently associated with Ab levels. These findings have direct implications for public health policy and current vaccine efforts. Knowledge gained regarding Ab memory following infection will inform the need for vaccination in those previously infected and allow for a better approximation of population-wide protective immunity.

RNA Polymerase Pausing during Initial Transcription.

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

Interactions between Influenza A Virus Nucleoprotein and Gene Segment Untranslated Regions Facilitate Selective Modulation of Viral Gene Expression.

The influenza A virus (IAV) genome is divided into eight negative-sense, single-stranded RNA segments. Each segment exhibits a unique level and temporal pattern of expression; however, the exact mechanisms underlying the patterns of individual gene segment expression are poorly understood. We previously demonstrated that a single substitution in the viral nucleoprotein (NP:F346S) selectively modulates neuraminidase (NA) gene segment expression while leaving other segments largely unaffected. Given what is currently known about NP function, there is no obvious explanation for how changes in NP can selectively modulate the replication of individual gene segments. In this study, we found that the specificity of this effect for the NA segment is virus strain specific and depends on the untranslated region (UTR) sequences of the NA segment. While the NP:F346S substitution did not significantly alter the RNA binding or oligomerization activities of NP in vitro, it specifically decreased the ability of NP to promote NA segment viral RNA (vRNA) synthesis. In addition to NP residue F346, we identified two other adjacent aromatic residues in NP (Y385 and F479) capable of similarly regulating NA gene segment expression, suggesting a larger role for this domain in gene-segment specific regulation. Our findings reveal a novel role for NP in selective regulation of viral gene segment replication and provide a framework for understanding how the expression patterns of individual viral gene segments can be modulated during adaptation to new host environments. IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen that remains a significant source of morbidity and mortality. Escape from host immunity or emergence into new host species often requires mutations that modulate the functional activities of the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA), which are responsible for virus attachment to and release from host cells, respectively. Maintaining the functional balance between the activities of HA and NA is required for fitness across multiple host systems. Thus, selective modulation of viral gene expression patterns may be a key determinant of viral immune escape and cross-species transmission potential. We identified a novel mechanism by which the viral nucleoprotein (NP) gene can selectively modulate NA segment replication and gene expression through interactions with the segment UTRs. Our work highlights an unexpected role for NP in selective regulation of expression from the individual IAV gene segments.

Enterotoxigenic Escherichia coli Enterotoxins Regulate Epithelial to Immune Relay of IL-33 and IL-1Ra Cytokines.

Enterotoxigenic Escherichia coli (ETEC) remain a major cause of diarrheal mortality and morbidity in children in low-resource settings. Few studies have explored the consequences of simultaneous intoxication with heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) despite the increased prevalence of wild ETEC isolates expressing both toxins. We therefore used a combination of tissue culture and murine models to explore the impact of simultaneous ST + LT intoxication on epithelial and myeloid cells. We report that LT induces sustained production of interleukin 33 (IL-33) and interleukin 1 receptor antagonist (IL-1Ra) in T84 intestinal epithelial cells via cAMP production and protein kinase A activation. We demonstrate that combined ST + LT intoxication hastens epithelial transcriptional responses induced more slowly by LT alone. ST- and LT-mediated luminal fluid accumulation in vivo correlates with significant increases in IL-33 and IL-1Ra in small intestinal mucosal scrapings. Additionally, IL-33 receptor (IL-33R)-deficient mice are significantly less susceptible to ST-mediated secretion than wildtype mice. In the immune compartment, IL-33 is sensed by myeloid cells, and LT suppresses IL-33-induced tumor necrosis factor α (TNF-α) secretion from macrophages and bone marrow-derived dendritic cells (BMDCs) but amplifies IL-33-mediated induction of IL-6 from BMDCs. In conclusion, our studies suggest that enterotoxin-induced IL-33 and IL-1Ra modulate intestinal inflammation and IL-1 receptor signaling in the intestinal mucosa in response to ETEC enterotoxins.

Synergistic Effect between 3'-Terminal Noncoding and Adjacent Coding Regions of the Influenza A Virus Hemagglutinin Segment on Template Preference.

The influenza A virus genome is comprised of eight single-stranded negative-sense viral RNA (vRNA) segments. Each of the eight vRNA segments contains segment-specific nonconserved noncoding regions (NCRs) of similar sequence and length in different influenza A virus strains. However, in the subtype-determinant segments, encoding hemagglutinin (HA) and neuraminidase (NA), the segment-specific noncoding regions are subtype specific, varying significantly in sequence and length at both the 3' and 5' termini among different subtypes. The significance of these subtype-specific noncoding regions (ssNCR) in the influenza virus replication cycle is not fully understood. In this study, we show that truncations of the 3'-end H1-subtype-specific noncoding region (H1-ssNCR) resulted in recombinant viruses with decreased HA vRNA replication and attenuated growth phenotype, although the vRNA replication was not affected in single-template RNP reconstitution assays. The attenuated viruses were unstable, and point mutations at nucleotide position 76 or 56 in the adjacent coding region of HA vRNA were found after serial passage. The mutations restored the HA vRNA replication and reversed the attenuated virus growth phenotype. We propose that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multisegment environment. These results provide novel insights into the role of the 3'-end nonconserved noncoding regions and adjacent coding regions on template preference in multiple-segmented negative-strand RNA viruses. IMPORTANCE While most influenza A virus vRNA segments contain segment-specific nonconserved noncoding regions of similar length and sequence, these regions vary considerably both in length and sequence in the segments encoding HA and NA, the two major antigenic determinants of influenza A viruses. In this study, we investigated the function of the 3'-end H1-ssNCR and observed a synergistic effect between the 3'-end H1-ssNCR nucleotides and adjacent coding nucleotide(s) of the HA segment on template preference in a multisegment environment. The results unravel an additional level of complexity in the regulation of RNA replication in multiple-segmented negative-strand RNA viruses.

Salmonella Persistence and Host Immunity Are Dictated by the Anatomical Microenvironment.

The intracellular bacterial pathogen Salmonella is able to evade the immune system and persist within the host. In some cases, these persistent infections are asymptomatic for long periods and represent a significant public health hazard because the hosts are potential chronic carriers, yet the mechanisms that control persistence are incompletely understood. Using a mouse model of chronic typhoid fever combined with major histocompatibility complex (MHC) class II tetramers to interrogate endogenous, Salmonella-specific CD4+ helper T cells, we show that certain host microenvironments may favorably contribute to a pathogen's ability to persist in vivo We demonstrate that the environment in the hepatobiliary system may contribute to the persistence of Salmonella enterica subsp. enterica serovar Typhimurium through liver-resident immunoregulatory CD4+ helper T cells, alternatively activated macrophages, and impaired bactericidal activity. This contrasts with lymphoid organs, such as the spleen and mesenteric lymph nodes, where these same cells appear to have a greater capacity for bacterial killing, which may contribute to control of bacteria in these organs. We also found that, following an extended period of infection of more than 2 years, the liver appeared to be the only site that harbored Salmonella bacteria. This work establishes a potential role for nonlymphoid organ immunity in regulating chronic bacterial infections and provides further evidence for the hepatobiliary system as the site of chronic Salmonella infection.

Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device.

We show how a bird's-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratory-on-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known as well as unique structural variation. Following its mapping, a molecule of interest was rescued from the chip; amplified and localized to a chromosome by FISH; and interrogated down to 1-bp resolution with a commercial sequencer, thereby reconciling haplotype-phased chromosome substructure with sequence.

Simultaneous exposure to Escherichia coli heat-labile and heat-stable enterotoxins increases fluid secretion and alters cyclic nucleotide and cytokine production by intestinal epithelial cells.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease and death, especially in children in developing countries. ETEC causes disease by colonizing the small intestine and producing heat-labile toxin (LT), heat-stable toxin (ST), or both LT and ST (LT+ST). The majority of ETEC strains produce both ST and LT. Despite the prevalence of LT+ST-producing organisms, few studies have examined the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins. In the current report, we demonstrate that when LT and ST are both present, they increase water movement into the intestinal lumen over and above the levels observed with either toxin alone. As expected, cultured intestinal epithelial cells increased their expression of intracellular cyclic GMP (cGMP) when treated with ST and their expression of intracellular cyclic AMP (cAMP) when treated with LT. When both toxins were present, cGMP levels but not cAMP levels were synergistically elevated compared with the levels of expression caused by the corresponding single-toxin treatment. Our data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells in response to LT are significantly reduced in animals exposed to both enterotoxins. These findings suggest that there may be complex differences between the epithelial cell intoxication and, potentially, secretory outcomes induced by ETEC strains expressing LT+ST compared with strains that express LT or ST only. Our results also reveal a novel mechanism wherein ST production may reduce the hosts' ability to mount an effective innate or adaptive immune response to infecting organisms.

DNA catenation maintains structure of human metaphase chromosomes.

Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical 'X' shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIα significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation, and destroyed to complete sister chromatid disjunction. In addition to demonstrating the value of microfluidics as a tool for examining chromosome structure, these results lend support to certain models of DNA catenation organization and regulation: in particular, we conclude from our observation of centromere-concentrated catenation that spindle forces could play a driving role in decatenation and that Topoisomerase IIα is differentially regulated at the centromeres, perhaps in conjunction with cohesin.

ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication.

The ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein-1 (ARFRP1/AP-1)-dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across ß-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.

COVID-19 in non-hospitalised adults caused by either SARS-CoV-2 sub-variants Omicron BA.1, BA.2, BA.4/5 or Delta associates with similar illness duration, symptom severity and viral kinetics, irrespective of vaccination history.

BACKGROUND: SARS-CoV-2 variant Omicron rapidly evolved over 2022, causing three waves of infection due to sub-variants BA.1, BA.2 and BA.4/5. We sought to characterise symptoms and viral loads over the course of COVID-19 infection with these sub-variants in otherwise-healthy, vaccinated, non-hospitalised adults, and compared data to infections with the preceding Delta variant of concern (VOC). METHODS: In a prospective, observational cohort study, healthy vaccinated UK adults who reported a positive polymerase chain reaction (PCR) or lateral flow test, self-swabbed on alternate weekdays until day 10. We compared participant-reported symptoms and viral load trajectories between infections caused by VOCs Delta and Omicron (sub-variants BA.1, BA.2 or BA.4/5), and tested for relationships between vaccine dose, symptoms and PCR cycle threshold (Ct) as a proxy for viral load using Chi-squared (χ2) and Wilcoxon tests. RESULTS: 563 infection episodes were reported among 491 participants. Across infection episodes, there was little variation in symptom burden (4 [IQR 3-5] symptoms) and duration (8 [IQR 6-11] days). Whilst symptom profiles differed among infections caused by Delta compared to Omicron sub-variants, symptom profiles were similar between Omicron sub-variants. Anosmia was reported more frequently in Delta infections after 2 doses compared with Omicron sub-variant infections after 3 doses, for example: 42% (25/60) of participants with Delta infection compared to 9% (6/67) with Omicron BA.4/5 (χ2 P < 0.001; OR 7.3 [95% CI 2.7-19.4]). Fever was less common with Delta (20/60 participants; 33%) than Omicron BA.4/5 (39/67; 58%; χ2 P = 0.008; OR 0.4 [CI 0.2-0.7]). Amongst infections with an Omicron sub-variants, symptoms of coryza, fatigue, cough and myalgia predominated. Viral load trajectories and peaks did not differ between Delta, and Omicron, irrespective of symptom severity (including asymptomatic participants), VOC or vaccination status. PCR Ct values were negatively associated with time since vaccination in participants infected with BA.1 (ß = -0.05 (CI -0.10-0.01); P = 0.031); however, this trend was not observed in BA.2 or BA.4/5 infections. CONCLUSION: Our study emphasises both the changing symptom profile of COVID-19 infections in the Omicron era, and ongoing transmission risk of Omicron sub-variants in vaccinated adults. TRIAL REGISTRATION: NCT04750356.

Establishment of isotype-switched, antigen-specific B cells in multiple mucosal tissues using non-mucosal immunization.

Although most pathogens infect the human body via mucosal surfaces, very few injectable vaccines can specifically target immune cells to these tissues where their effector functions would be most desirable. We have previously shown that certain adjuvants can program vaccine-specific helper T cells to migrate to the gut, even when the vaccine is delivered non-mucosally. It is not known whether this is true for antigen-specific B cell responses. Here we show that a single intradermal vaccination with the adjuvant double mutant heat-labile toxin (dmLT) induces a robust endogenous, vaccine-specific, isotype-switched B cell response. When the vaccine was intradermally boosted, we detected non-circulating vaccine-specific B cell responses in the lamina propria of the large intestines, Peyer's patches, and lungs. When compared to the TLR9 ligand adjuvant CpG, only dmLT was able to drive the establishment of isotype-switched resident B cells in these mucosal tissues, even when the dmLT-adjuvanted vaccine was administered non-mucosally. Further, we found that the transcription factor Batf3 was important for the full germinal center reaction, isotype switching, and Peyer's patch migration of these B cells. Collectively, these data indicate that specific adjuvants can promote mucosal homing and the establishment of activated, antigen-specific B cells in mucosal tissues, even when these adjuvants are delivered by a non-mucosal route. These findings could fundamentally change the way future vaccines are formulated and delivered.

Transient RNA structures cause aberrant influenza virus replication and innate immune activation.

During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVGs) and mini viral RNAs (mvRNAs). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid-inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Here, we provide evidence that RNA structures, called template loops (t-loops), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are suggestive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response.

Real-Time Single-Molecule Studies of RNA Polymerase-Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway.

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-σ70 holoenzyme to the promoter DNA. This initial RNAP-promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RPO) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RPO and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RPO formation on a consensus lac promoter. We find that the promoter opening may proceed rapidly from the closed to open conformation in a single apparent step, or may instead involve a significant intermediate between these states. The formed RPO complexes are also different with respect to their transcription bubble stability. The RNAP cleft loops, and especially the ß' rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond -35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RPO.