Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing.

TEXT CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nils Walter ( nwalter@umich.edu ).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Bacterial strains, Plasmids and Growth Conditions E. coli JM109 competent cells were used for maintenance of all transcription template plasmids. E. coli XL10 gold ultracompetent cells were used for maintenance of biotinylated RNAP expression plasmids. E. coli BLR (DE3) competent cells were used for overexpression of biotinylation-tagged RNAP, and XJb (DE3) cells were used for overexpression of RNAP variants. All strains were grown at 37 °C in Luria-Bertani media or on Luria-Bertani media-agar plates supplemented with 100 μg/mL carbenicillin. Plasmids used in this study are listed in the Key Resources Table .

METHOD DETAILS Single-molecule FRET Design and preparation of RNA constructs

It is widely known that transcription initiation can be bypassed by incubating RNAP with a pre-assembled scaffold consisting of a partially complementary DNA bubble and an RNA primer ( Daube and Hippel, 1992 ; Hein et al., 2014 ; Kolb et al., 2014 ). Our DNA bubble was designed based on a sequence from ( Kolb et al., 2014 ), with minor changes so that it was predicted to not interfere with formation of the native riboswitch structure ( Table S1 ). “EC RNAs” 0, 3 and 10 were then designed to contain 10 nucleotides that were complementary to the template strand of the bubble, as well as the desired number of bases from the expression platform. The sequence of the capture probe (CP) was then designed to sequester in a duplex the same nucleotides that form the RNA:DNA hybrid in the bubble complex, and LNA bases were included to achieve a T m of ~80 °C. The RNA:LNA or RNA:DNA hybrid sequence was not based on the expression platform, and was identical in the three RNA constructs with the exception of two bases that had to be changed to accommodate the portion of the aptamer oligonucleotide that became part of the hybrid in the shortest construct, RNA0p. The riboswitch aptamer sequence included a G-to-C mutation at the second position to suppress formation of alternate structures containing helix P1a ( Kang et al., 2009 ). Aptamer labeling locations were chosen to match those used in previous work on the isolated aptamer ( Suddala et al., 2013 ; 2015 ). Ligation enabled smFRET constructs of multiple lengths to be prepared from one aptamer construct. All oligonucleotides used in smFRET experiments were purchased HPLC-purified from Dharmacon (in the case of fluorophore-labeled RNA) or Integrated DNA Technologies (in the case of DNA and unlabeled RNA). The riboswitch aptamer construct contained a 2’ Dy547 label at G37 and a 5-aminoallyl-uridine (5-N-U) at position 14. To label the 5-N-U, 4 nmol RNA was incubated for 4 hours at room temperature in a 50 μL reaction containing 30 μL of DMSO, one Cy5 mono-reactive dye pack (GE Healthcare Life Sciences) and 0.1 M sodium bicarbonate (pH 9). The RNA was isolated by ethanol precipitation. The labeled aptamer (200 pmol) and 5’ phosphorylated EC RNA (400 pmol) were annealed with a DNA or LNA splint (400 pmol-1 nmol) in a 100 μL mixture containing 150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. This annealing mixture was diluted to 500 μL in T4 RNA ligase 2 buffer, and 40 units of T4 RNA ligase 2 (New England Biolabs) were added. The reaction mixture was incubated at 37 °C for 2 hours. The reaction mixture was extracted with an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol, then concentrated down to ~20 μL in a vacuum centrifuge at 45° C and purified by denaturing, 8 M urea, 8% polyacrylamide gel electrophoresis (PAGE). The product band was extracted and the RNA isolated by ethanol precipitation.

Protein preparation

E. coli core RNAP bearing an “AviTag” biotinylation tag (GLNDIFEAQKIEWH) on the C-terminus of the β’ subunit was expressed from plasmid pIA999 in BLR(DE3) cells ( Kay et al., 2009 ). The cells were grown in LB media supplemented with 20 μM biotin and 100 μg/mL carbenicillin, induced with 1 mM IPTG at an OD600 of 0.7 and harvested 3 hours post-induction. The protein was purified essentially as described ( Svetlov and Artsimovitch, 2015 ). Briefly, the his -tagged protein was purified on a nickel-NTA agarose column, followed by a heparin column, and finally by a Mono Q ion-exchange column. The purified protein was dialyzed into storage buffer (10 mM Tris-HCl, pH 7.5, 50% glycerol, 100 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT) and stored at −80° C. Biotinylation of the β’ subunit by endogenous BirA was verified by incubating biotin-tagged and wild-type proteins with streptavidin, followed by native PAGE analysis. Further in vitro biotinylation was not performed. RNAP mutants were prepared by the same protocol, except that a Resource Q ion-exchange column was used instead of Mono Q. smFRET experiments The RNA of interest was combined at a final concentration of 0.5 μM with either capture probe (CP) or template DNA (tDNA) and nontemplate DNA (ntDNA) in buffer A (50 mM Tris-HCl, pH 7.5, 100 mM KCl). When CP was used, its concentration was 5 μM; DNA concentrations were 10 μM when immobilizing through 3’-biotinylated ntDNA and 1 μM when immobilizing through biotinylated RNAP. The mixture was annealed by incubating at 90 °C for 2 min, then 37 °C for 10 min, then RT for 10 min. For experiments in the absence of RNAP, the annealing mixture was diluted in buffer A to a concentration of 15-50 μM RNA and flowed onto a slide that had previously been passivated with a 10:1 ratio of PEG to biotinylated PEG, then incubated for 10-15 min with a 0.2 mg/mL solution of streptavidin. For experiments performed in the presence of RNAP, the annealed nucleic acid scaffold was diluted to 50 nM RNA in buffer B (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM MgCl 2 ), and E. coli RNAP was added to 250 nM. This mixture was incubated for 15 min at 37 °C, then diluted in buffer B to 50 pM RNA for immobilization. Imaging was performed at 62 ms time resolution in buffer A or B (where indicated, supplemented with preQ 1 or additional KCl), containing 44 mM glucose, 165 U/mL glucose oxidase from Aspergillus niger, 2170 U/mL catalase from Corynebacterium glutamicum and 5 mM trolox as an oxygen scavenger ( Aitken et al., 2008 ). This oxygen scavenger was chosen based on measurements of RNAP elongation activity in buffers containing various OSS components ( Figure S4 ). The slide was incubated in each imaging buffer for 10 min prior to data collection. A laser power of 10-15 mW at 532 nm was used to excite the sample in a prism-based total internal reflection geometry, and emission from DY547 and Cy5 were recorded simultaneously using an intensified CCD camera (I-Pentamax, Princeton Instruments). The sample was additionally excited by a 640 nm laser at the beginning and end of each data set to verify the presence of Cy5. For on-slide elongation assays, ECs were prepared, immobilized and imaged as described above. Imaging buffer containing 1 mM GTP was then added, and imaging was repeated after a 10-minute incubation. smFRET data analysis Locations of molecules and fluorophore intensity traces for each molecule were extracted from raw movie files using IDL (Research Systems) and analyzed using Matlab (The Math Works). Traces were manually selected for further analysis using the following criteria: single-step photobleaching, a Cy5 fluorescence intensity of >200 (arbitrary units) when excited at 640 nm, a total (DY547+Cy5) intensity of >200 when excited at 532 nm, and a fluorescence duration (prior to photobleaching) of >3 s and, if transitions were observed in the trace, anticorrelation between donor and acceptor signals. The FRET efficiency ( E FRET ) was calculated as I A /(I A +I D ) , where I A and I D represent the background corrected fluorescence intensities of the acceptor (Cy5) and donor (DY547) fluorophores, respectively. FRET histograms were created by sorting the E FRET observed for the first 100 frames of a large number of traces (>200 for main text data, >100 for supplementary data ) into 22 bins with centers ranging from E FRET = −0.05 to E FRET = 1.05. Plotting and fitting of FRET efficiency histograms was performed in OriginLab 8.1. Uncertainties in the histogram fit parameters were computed by bootstrapping, splitting the entire set of data points into three batches and fitting them independently.

Hidden Markov Modeling

(HMM) analysis was performed using the segmental k-means algorithm in the QuB software suite as previously described ( Blanco and Walter, 2010 ). To select the number of states to model our data with, HMM was performed on a selection of data sets using 1-, 2-, and 3-state models. We used fits to the corresponding histograms to select the initial values for optimization of the average E FRET and relative populations of the states. For each trace, we computed the Bayesian information criterion (BIC) and modified Bayesian information criterion (BIC'), as defined by Lerner et al. ( Lerner et al., 2018 ), according to Equations 1 - 3 : (1) BIC ( q ) = − 2 L + K ln ( n ) (2) Δ BIC ( q ) = BIC ( q ) − min p = 1 3 { BIC ( p ) } (3) BIC ′ ( q ) = Δ BIC ( q ) ∕ ( n − K ) where q is the number of states in the model being tested, K is the number of parameters in that model, L is the log-likelihood of the HMM fit, n is the number of data points in a given trace, and the number of states p varies over all of the models being considered. We then computed the mean BIC or BIC' over all of the traces in that data set. We found that for our data, BIC overvalued long traces while BIC' overvalued short traces. Unsurprisingly, then, BIC' suggested models with less than or equal to the number of states suggested by BIC. In none of the cases we tested did either metric suggest more than two states, and BIC suggested 2 states in all cases tested. We therefore used a 2-state model for the fitting and interpretation of all of our smFRET data.

Transcription Assays

Bubble-initiated transcription assays

Annealed nucleic acid scaffolds were prepared using RNA10 as described above for smFRET. Additional buffer components such as oxygen scavengers were added either immediately after annealing the nucleic acid scaffold, or after incubating the scaffold with RNAP (noted as "after RNAP" in Figure S4 ). Scaffolds were diluted to 50 nM in buffer B, E. coli RNAP core enzyme (New England Biolabs) was added to 200 nM, and the mixture was incubated at 37 °C for 15 min. The reaction mixture was then treated with 200 μM GTP and incubated for 10 min, after which an aliquot was removed and quenched into loading buffer (1x = 4 M urea, 25 mM EDTA, 45 mM Tris-borate, pH 8.3). The remaining reaction was treated with 200 μM ATP, CTP and UTP and incubated for an additional 10 min, after which another aliquot was removed and quenched. The reaction aliquots were analyzed by denaturing, 8 M urea, 15% PAGE and visualized by detecting the Cy5 fluorescence using a Typhoon 9410 Variable Mode Imager (GE Healthcare Life Sciences). Gel images for these and all following experiments were analyzed in ImageQuant (Molecular Dynamics). For RNA degradation assays ( Figure S4 ), a 10 nM solution of RNA10 was prepared in buffer B, including the specified OSS components and RNase inhibitors at the same concentrations used for smFRET. The mixture was incubated at 37 °C for 30 min and analyzed by denaturing, 8 M urea, 15% PAGE. We found through these assays that a number of common OSS components interfered with transcription in various ways. The OSS substrate protocatechuic acid (PCA) was found to interfere with transcription, as was the combined glucose/glucose oxidase/catalase (GOD/CAT) OSS, indicating inhibition by its product, D-gluconic acid. PCA and D-gluconic acid are both carboxylic acids, as are many known inhibitors of bacterial RNAP ( Elgaher et al., 2014 ), and are therefore negatively charged under our imaging conditions. Inhibition was relieved by adding the OSS after incubating the bubble scaffold with RNAP, suggesting that PCA and D-gluconic acid interfere with binding rather than elongation. It is therefore likely that at the millimolar concentrations used for oxygen scavenging, they bind to and neutralize the electropositive cleft of RNAP where the transcription bubble resides. The maximum elongation efficiency was obtained by adding the GOD/CAT OSS after incubation with RNAP; therefore, this protocol was used for all experiments reported herein. Certain OSS enzymes, particularly catalase from bovine liver, were found to have RNase contamination and were therefore avoided, echoing reports of DNase contamination in OSS enzymes ( Senavirathne et al., 2015 ). Promoter-initiated transcription assays A 151-nucleotide portion of the 5’ UTR of the queCDEF operon from B. subtilis, including the preQ 1 riboswitch, was cloned into the pUC18 plasmid between the KpnI and EcoRI restriction sites. The E. coli RecA promoter was inserted immediately upstream of the riboswitch between the BamHI and KpnI sites. In addition, 12 nucleotides not found in the wild-type sequence were inserted after the promoter in order to generate a 19-nucleotide stretch in which the RNA transcript lacks any adenosine residues. Mutant plasmids were generated by site-directed mutagenesis using the primers in Table S2 . Transcription templates for all experiments except those utilizing ΔFT RNAP were prepared by PCR using the "Forward PCR primer" and "Reverse PCR primer" indicated in Table S2 . Transcription templates for ΔFT RNAP and associated WT controls contained the Gal1 promoter instead of RecA, and were generated in two PCR steps using first "Forward Gal1 primer 1" and "Reverse PCR primer", then "Forward Gal1 primer 2" and "Reverse PCR primer". For transcription assays, halted complexes (HCs) were prepared in buffer C (20 mM Tris-acetate, pH 8.0, 20 mM sodium acetate, 2 mM magnesium acetate, 5% glycerol, 14 mM 2-mercaptoethanol, 0.1 mM EDTA) containing 1 μM labeling NTP, 5 μM of two other NTPs (omitting ATP), 56 nM α 32 P-GTP or CTP (3000 Ci/mmol), 100 μM ApC dinucleotide primer, and 40 nM DNA template. E. coli RNAP holoenzyme (New England Biolabs) was added to 80 nM, and the mixture was incubated for 15 min at 37 °C. 0.9 reaction volumes of HC was treated with 0.1 reaction volumes of pre-warmed chase solution in buffer C, which contained 200 μg/mL rifampicin and 10x the desired final concentrations of all four NTPs and preQ 1 . The mixture was incubated at 37 °C, and reaction aliquots were quenched at the desired times into an equal volume of loading buffer (8 M urea, 0.8× TBE, 0.2% bromophenol blue, 0.2% xylene cyanol, 1 mM EDTA). For most experiments, additional NTPs were then added to 200 μM, and a final aliquot was removed 3-5 minutes later. For experiments with RNAP variants (point mutants in the β' subunit and ΔFT RNAP), the concentration of RNAP was 50 nM instead of 80 nM. Sequencing ladders were prepared by combining 9 μL of HC with 1 μL of chase solution containing 250 μM of each NTP, in addition to one 3’-OMe NTP (at 25 μM for 3'-OMe GTP and 15 μM for 3’-OMe ATP, UTP and CTP). Reactions were incubated for 15 min at 37 °C before being quenched with 10 μL of loading buffer. Reaction aliquots were denatured before loading 1.5-2.5 μL of each onto a denaturing 8 M urea, 10% polyacrylamide sequencing gel. The gel was dried and exposed to a phosphor screen (typically overnight), which was then scanned on a Typhoon 9410 Variable Mode Imager. For reactions including transcription factors, cell extract or RNA hairpin and the associated control reactions, the template was purified by spermine precipitation ( Hoopes and McClure, 1981 ). These reactions were performed by preparing HCs at twice the concentration described above, adding an equal volume of 2x the desired final concentration of factor, extract or hairpin, and 30 seconds later adding the chase solution. The final concentration of all transcription factors was 100 nM. In the case of Eco NusA, it was verified that concentrations as high as 500 nM had no effect beyond that seen at 100 nM.

Transcription data analysis

Volumes of the pause band following the aptamer domain (U46), the terminator in the expression platform (U70), the terminator following the expression platform (U108), and the two bands resulting from runoff were determined using ImageQuant (Molecular Dynamics). For each lane, these volumes were background corrected by determining the volume above background of all points that had a higher intensity than the average intensity of a background box positioned in the same lane. Two background boxes were used in each lane: one to correct band U46 (positioned just below that band), and one to correct bands U70, U108 and the two runoff bands (positioned between U70 and U108). The relative intensity of each band in a given lane was determined by dividing the background-corrected volume of that band by the sum of the volumes of all five bands mentioned above. Two pause bands were observed within the aptamer domain, but these were omitted from the analysis due to their low intensity and lack of variation in response to preQ 1 and mutations. Error bars in transcription quantifications represent the standard deviation of the intensity fraction of each band across three independent experiments. To determine the K 1/2 for regulation of pausing and termination by preQ 1 , the relative intensities of U46 and U70 at 45 seconds and 90 seconds after chasing, and 5 min after adding a further chase to 200 μM of each NTP were plotted as a function of preQ 1 concentration. These six data sets were globally fit with equation 4 : (4) F ( [ pre Q 1 ] ) = y 0 + A ∗ [ pre Q 1 ] K 1 ∕ 2 + [ pre Q 1 ] where F is the intensity fraction in a given band, y 0 is the intensity fraction at zero preQ 1 , and A is the amplitude of the change that occurs upon addition of preQ 1 . A global fit was performed in which y 0 and A were allowed to vary between data sets, but all were required to have the same value of K 1/2 . To determine the pause efficiency and lifetime from detailed time courses, the natural logarithm of the fraction paused was calculated at each time point t (seconds) and the following equation was fit to the resulting data: (5) F ( t ) = Ln [ a 1 ⋅ e m 1 t + a 2 ⋅ e m 2 t ] Fitting to the logarithm of the data rather than the raw data allowed the time points with low paused fraction to still contribute to the fit. a 1 and a 2 correspond to the fraction of elongation complexes that pause with half-lives - Ln [ 2 ]/ m 1 and - Ln [ 2 ]/ m 2 , respectively. The constraint of a 1 + a 2 ≤ 1 was used in all fits. Cross-linking Assays 100 nM DNA template and 20 nM RNAP holoenzyme were incubated in buffer D (20 mM Tris-HCl, 120 mM KCl, 5 mM MgCl 2 , 5% glycerol (w/v), 1 mM 2-mercaptoethanol, pH 7.9) at 37°C for 15 min. ApU (100 μM) and starting NTPs (30 μM CTP, GTP, and ATP) were added to the promoter bound RNAP, and the reaction was incubated for 30 min at 37 °C. The EC at position 6 was immobilized on 10 μL Ni 2+ -NTA agarose beads at 37°C for 15 min. Each following walk (indicated in Figure S5 ) was completed with 5 intermediate washes with buffer D to eliminate unbound NTPs. Each wash involved addition of 1 mL of buffer D followed by a 5 second spin on a tabletop centrifuge to pellet the Ni 2+ beads with attached ECs. The supernatant was then removed by pipette. The cross-linkable analogue 4-thio-UTP or 6-thio-GTP (10 μM) was introduced into the RNA at the desired position. Upon reaching A36, 10 μCi [α- 32 P-GTP, 3000 Ci/mmol was added for 2 min. 5 μM ATP, CTP, and GTP were then added to reach C43, then 5 μM UTP and GTP to extend to U46. ECs C43 and U46 were incubated with 40 μM preQ 1 at 37°C for 5 min and exposed to 365 nm UV light (UVP, 8W Model UVLMS-38, Upland, CA) for 10 min on ice. The reactions were either quenched with stop buffer (9M Urea, 20 mM EDTA, 1XTBE, 0.5 % Brilliant Blue R and 0.5% Xylene Cyanol FF) and the sample resolved on denaturing 7M urea 15% polyacrylamide-gel, or in LDS loading dye and separated on a 4-12% Bis-Tris Novex gel (Invitrogen). The gels were dried and exposed to phosphor screens.

Simulations

Molecular dynamics simulations were performed to explore the folding landscape of the preQ 1 riboswitch bound to the RNA polymerase elongation complex. Structure-based or Gō-type simulations are particularly useful for exploring slow processes like folding that are computationally expensive to probe with more precise explicit solvent simulations. Structure-based models were originally used in protein folding ( Shea et al., 1998 ) and are based on energy landscape theory and the principle of minimal frustration ( Onuchic and Wolynes, 2004 ). In order to fold on biological time scales, native interactions must be more stabilizing than nonnative interactions, so structure-based models only include interactions between native contacts. We chose to use an all heavy atom structure-based model that has been extensively calibrated, and has been used for RNA folding ( Lutz et al., 2014 ; Whitford et al., 2009 ) and functional studies of the ribosome, a mixed protein and RNA system ( Whitford et al., 2010 ). To generate a structure-based model, a native structure is needed to specify the interactions that ought to be stabilized. Atoms in close proximity within a known structure are chosen to be contact pairs that interact within the model; the shadow algorithm was used to calculate the contact map ( Noel et al., 2012 ). Since no structure of the preQ 1 riboswitch docked to RNAP existed, a model was created from structures of various components. Run input files for various structural elements were prepared using SMOG 2 ( Noel et al., 2010 ; 2016 ), and then stitched together as described. The preQ 1 riboswitch was modeled using contacts present in its crystal structure (PDB ID: 3FU2) ( Klein et al., 2009 ). Its NMR structure (PDB ID: 2L1V) ( Kang et al., 2009 ) was used to fill in 5’ and L2 gaps from the crystal structure: any contacts and interactions with missing residues were used to supplement the interactions defined by the crystal structure. An E. coli RNAP model was adopted from a cryo-EM structure of an EC (PDB ID: 6ALH) ( Kang et al., 2017 ). The transcription bubble was modeled by creating single- and double-stranded sections of RNA and DNA using the nucleic acid builder ( http://structure.usc.edu/make-na/server.html ), and then stitching the resulting topologies together appropriately. Interactions of residues in either the riboswitch or the polymerase overrode those interactions in the DNA bubble model, so the bubble was only used to supplement interactions for missing residues. Since several topologies were being combined, and contact and dihedral interactions are scaled slightly differently in each, the dihedral and contact energies were both set to typical values of 0.7 energy units. We refer to the supplemental information of ( Hayes et al., 2014 ) for a discussion of units in the model. Models with 0 to 3 nucleotides inserted between the riboswitch and RNAP (RNA0p, RNA1, RNA2 and RNA3) were made by modifying the sequences generated with the nucleic acid builder (sequences are reported in Table S1 ). The resulting model essentially stitches together models of the riboswitch and the RNAP. Since the riboswitch and RNAP fold independently, the model is valid, but only accounts for steric interactions between the riboswitch and RNAP, and neglects any specific interactions that could be obtained from a structure of the complex. Consequently, some questions are beyond the scope of the model, while others can be robustly answered. The average number of base pairs formed in the model should be fairly robust, as the model identifies structures of the riboswitch that lead to a minimal disruption of RNAP structure. Likewise, the model should be able to elucidate the sterically allowed binding poses for the folded riboswitch, whereas identifying the dominant pose is beyond the scope of the model, as the populations of competing poses can be shifted by specific interactions between the riboswitch and RNAP. Equilibrium simulations of riboswitch folding were run using umbrella sampling on the Q (number of formed contacts) reaction coordinate with replica exchange between umbrellas to allow trapped high-energy folded poses to relax. 11 replicas were used with an exchange attempts every 5000 steps, an umbrella k constant of 0.005 energy units per contact squared, and umbrella equilibria Q 0 evenly spaced among replicas from 0 to 282 contacts. The weighted histogram analysis method (WHAM) ( Kumar et al., 1992 ) was then used to reweight each frame of each replica to an unbiased simulation. Simulations started from an unfolded preQ 1 riboswitch structure, and allowed the riboswitch to fold at a temperature of T = 85 units. (The folding temperature of the riboswitch is approximately 90 units in the presence of the polymerase.) Simulations were run for 50 million steps with the first 12.5 million steps discarded for equilibration. Simulations with the preQ 1 ligand included 5 ligands in a cubic box of 20 nm side length. The binding affinity of preQ 1 was not calibrated. Simulations were run with and without preQ 1 and varying numbers of inserted nucleotides to determine whether steric constraints allowed P2 to fold ( Table S3 ). A base pair was considered formed if the central hydrogen bond between purine N1 and pyrimidine N3 was within a factor of 1.5 of its native distance. (In the A•C mismatch, the only hydrogen bond was used instead.) The number of formed base pairs was counted for each frame and averaged using the weight given by WHAM over all replicas in the simulation. The average number of base pairs was largely unchanged in the absence or presence of preQ 1 for RNA1, RNA2 and RNA3 (1.8-2.4 versus 2.0-2.4, respectively), and depends weakly on number of inserts, but changes substantially in the zero insert case (RNA0p), where it is lower by about half a base pair with preQ 1 and a whole base pair without preQ 1 . To check the robustness of the results, the contacts in the RNA linker (all residues between the 3’ end of P2 and the 5’ end of the RNA-DNA duplex) were removed. This allows the RNA to pull slightly out of the exit tunnel, and makes the RNA more flexible so it can twist more. While about half an additional base pair could form with the more flexible model, the trends remained the same: one to three inserts formed roughly the same number of base pairs as each other, and no inserts formed half a base pair less than them. Finally, a model of the flap tip deletion mutant was made in which residues K890-K914 of the beta subunit were removed. Simulations of the flap tip deletion mutant achieved results intermediate between the other two models, but showed less sensitivity to preQ 1 in the RNA0p system. To connect with cross-linking studies and to elucidate the folded pose, the frequency with which the cross-linking residue U14 was within 8 Å of each protein residue was calculated. Two poses of the riboswitch were identified that involved interactions primarily between the riboswitch and β’ (collectively called "mode 1"), along with a third pose where U14 is wedged between the clamp and flap tip ("mode 2"). Within mode 1, simulations with the default parameters (rigid linker) yielded a pose where U14 was positioned near K76-K79, while with the floppy linker, an additional pose was observed in which it is positioned near T393. These results are in agreement with cross-linking results that show that U14 interacts with the β’ subunit.

DATA AND SOFTWARE AVAILABILITY

Custom scripts used for this work are available upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Bacterial strains, Plasmids and Growth Conditions E. coli JM109 competent cells were used for maintenance of all transcription template plasmids. E. coli XL10 gold ultracompetent cells were used for maintenance of biotinylated RNAP expression plasmids. E. coli BLR (DE3) competent cells were used for overexpression of biotinylation-tagged RNAP, and XJb (DE3) cells were used for overexpression of RNAP variants. All strains were grown at 37 °C in Luria-Bertani media or on Luria-Bertani media-agar plates supplemented with 100 μg/mL carbenicillin. Plasmids used in this study are listed in the Key Resources Table .

METHOD DETAILS Single-molecule FRET Design and preparation of RNA constructs

It is widely known that transcription initiation can be bypassed by incubating RNAP with a pre-assembled scaffold consisting of a partially complementary DNA bubble and an RNA primer ( Daube and Hippel, 1992 ; Hein et al., 2014 ; Kolb et al., 2014 ). Our DNA bubble was designed based on a sequence from ( Kolb et al., 2014 ), with minor changes so that it was predicted to not interfere with formation of the native riboswitch structure ( Table S1 ). “EC RNAs” 0, 3 and 10 were then designed to contain 10 nucleotides that were complementary to the template strand of the bubble, as well as the desired number of bases from the expression platform. The sequence of the capture probe (CP) was then designed to sequester in a duplex the same nucleotides that form the RNA:DNA hybrid in the bubble complex, and LNA bases were included to achieve a T m of ~80 °C. The RNA:LNA or RNA:DNA hybrid sequence was not based on the expression platform, and was identical in the three RNA constructs with the exception of two bases that had to be changed to accommodate the portion of the aptamer oligonucleotide that became part of the hybrid in the shortest construct, RNA0p. The riboswitch aptamer sequence included a G-to-C mutation at the second position to suppress formation of alternate structures containing helix P1a ( Kang et al., 2009 ). Aptamer labeling locations were chosen to match those used in previous work on the isolated aptamer ( Suddala et al., 2013 ; 2015 ). Ligation enabled smFRET constructs of multiple lengths to be prepared from one aptamer construct. All oligonucleotides used in smFRET experiments were purchased HPLC-purified from Dharmacon (in the case of fluorophore-labeled RNA) or Integrated DNA Technologies (in the case of DNA and unlabeled RNA). The riboswitch aptamer construct contained a 2’ Dy547 label at G37 and a 5-aminoallyl-uridine (5-N-U) at position 14. To label the 5-N-U, 4 nmol RNA was incubated for 4 hours at room temperature in a 50 μL reaction containing 30 μL of DMSO, one Cy5 mono-reactive dye pack (GE Healthcare Life Sciences) and 0.1 M sodium bicarbonate (pH 9). The RNA was isolated by ethanol precipitation. The labeled aptamer (200 pmol) and 5’ phosphorylated EC RNA (400 pmol) were annealed with a DNA or LNA splint (400 pmol-1 nmol) in a 100 μL mixture containing 150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. This annealing mixture was diluted to 500 μL in T4 RNA ligase 2 buffer, and 40 units of T4 RNA ligase 2 (New England Biolabs) were added. The reaction mixture was incubated at 37 °C for 2 hours. The reaction mixture was extracted with an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol, then concentrated down to ~20 μL in a vacuum centrifuge at 45° C and purified by denaturing, 8 M urea, 8% polyacrylamide gel electrophoresis (PAGE). The product band was extracted and the RNA isolated by ethanol precipitation.

Protein preparation

E. coli core RNAP bearing an “AviTag” biotinylation tag (GLNDIFEAQKIEWH) on the C-terminus of the β’ subunit was expressed from plasmid pIA999 in BLR(DE3) cells ( Kay et al., 2009 ). The cells were grown in LB media supplemented with 20 μM biotin and 100 μg/mL carbenicillin, induced with 1 mM IPTG at an OD600 of 0.7 and harvested 3 hours post-induction. The protein was purified essentially as described ( Svetlov and Artsimovitch, 2015 ). Briefly, the his -tagged protein was purified on a nickel-NTA agarose column, followed by a heparin column, and finally by a Mono Q ion-exchange column. The purified protein was dialyzed into storage buffer (10 mM Tris-HCl, pH 7.5, 50% glycerol, 100 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT) and stored at −80° C. Biotinylation of the β’ subunit by endogenous BirA was verified by incubating biotin-tagged and wild-type proteins with streptavidin, followed by native PAGE analysis. Further in vitro biotinylation was not performed. RNAP mutants were prepared by the same protocol, except that a Resource Q ion-exchange column was used instead of Mono Q. smFRET experiments The RNA of interest was combined at a final concentration of 0.5 μM with either capture probe (CP) or template DNA (tDNA) and nontemplate DNA (ntDNA) in buffer A (50 mM Tris-HCl, pH 7.5, 100 mM KCl). When CP was used, its concentration was 5 μM; DNA concentrations were 10 μM when immobilizing through 3’-biotinylated ntDNA and 1 μM when immobilizing through biotinylated RNAP. The mixture was annealed by incubating at 90 °C for 2 min, then 37 °C for 10 min, then RT for 10 min. For experiments in the absence of RNAP, the annealing mixture was diluted in buffer A to a concentration of 15-50 μM RNA and flowed onto a slide that had previously been passivated with a 10:1 ratio of PEG to biotinylated PEG, then incubated for 10-15 min with a 0.2 mg/mL solution of streptavidin. For experiments performed in the presence of RNAP, the annealed nucleic acid scaffold was diluted to 50 nM RNA in buffer B (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM MgCl 2 ), and E. coli RNAP was added to 250 nM. This mixture was incubated for 15 min at 37 °C, then diluted in buffer B to 50 pM RNA for immobilization. Imaging was performed at 62 ms time resolution in buffer A or B (where indicated, supplemented with preQ 1 or additional KCl), containing 44 mM glucose, 165 U/mL glucose oxidase from Aspergillus niger, 2170 U/mL catalase from Corynebacterium glutamicum and 5 mM trolox as an oxygen scavenger ( Aitken et al., 2008 ). This oxygen scavenger was chosen based on measurements of RNAP elongation activity in buffers containing various OSS components ( Figure S4 ). The slide was incubated in each imaging buffer for 10 min prior to data collection. A laser power of 10-15 mW at 532 nm was used to excite the sample in a prism-based total internal reflection geometry, and emission from DY547 and Cy5 were recorded simultaneously using an intensified CCD camera (I-Pentamax, Princeton Instruments). The sample was additionally excited by a 640 nm laser at the beginning and end of each data set to verify the presence of Cy5. For on-slide elongation assays, ECs were prepared, immobilized and imaged as described above. Imaging buffer containing 1 mM GTP was then added, and imaging was repeated after a 10-minute incubation. smFRET data analysis Locations of molecules and fluorophore intensity traces for each molecule were extracted from raw movie files using IDL (Research Systems) and analyzed using Matlab (The Math Works). Traces were manually selected for further analysis using the following criteria: single-step photobleaching, a Cy5 fluorescence intensity of >200 (arbitrary units) when excited at 640 nm, a total (DY547+Cy5) intensity of >200 when excited at 532 nm, and a fluorescence duration (prior to photobleaching) of >3 s and, if transitions were observed in the trace, anticorrelation between donor and acceptor signals. The FRET efficiency ( E FRET ) was calculated as I A /(I A +I D ) , where I A and I D represent the background corrected fluorescence intensities of the acceptor (Cy5) and donor (DY547) fluorophores, respectively. FRET histograms were created by sorting the E FRET observed for the first 100 frames of a large number of traces (>200 for main text data, >100 for supplementary data ) into 22 bins with centers ranging from E FRET = −0.05 to E FRET = 1.05. Plotting and fitting of FRET efficiency histograms was performed in OriginLab 8.1. Uncertainties in the histogram fit parameters were computed by bootstrapping, splitting the entire set of data points into three batches and fitting them independently.

Hidden Markov Modeling

(HMM) analysis was performed using the segmental k-means algorithm in the QuB software suite as previously described ( Blanco and Walter, 2010 ). To select the number of states to model our data with, HMM was performed on a selection of data sets using 1-, 2-, and 3-state models. We used fits to the corresponding histograms to select the initial values for optimization of the average E FRET and relative populations of the states. For each trace, we computed the Bayesian information criterion (BIC) and modified Bayesian information criterion (BIC'), as defined by Lerner et al. ( Lerner et al., 2018 ), according to Equations 1 - 3 : (1) BIC ( q ) = − 2 L + K ln ( n ) (2) Δ BIC ( q ) = BIC ( q ) − min p = 1 3 { BIC ( p ) } (3) BIC ′ ( q ) = Δ BIC ( q ) ∕ ( n − K ) where q is the number of states in the model being tested, K is the number of parameters in that model, L is the log-likelihood of the HMM fit, n is the number of data points in a given trace, and the number of states p varies over all of the models being considered. We then computed the mean BIC or BIC' over all of the traces in that data set. We found that for our data, BIC overvalued long traces while BIC' overvalued short traces. Unsurprisingly, then, BIC' suggested models with less than or equal to the number of states suggested by BIC. In none of the cases we tested did either metric suggest more than two states, and BIC suggested 2 states in all cases tested. We therefore used a 2-state model for the fitting and interpretation of all of our smFRET data.

Transcription Assays

Bubble-initiated transcription assays

Annealed nucleic acid scaffolds were prepared using RNA10 as described above for smFRET. Additional buffer components such as oxygen scavengers were added either immediately after annealing the nucleic acid scaffold, or after incubating the scaffold with RNAP (noted as "after RNAP" in Figure S4 ). Scaffolds were diluted to 50 nM in buffer B, E. coli RNAP core enzyme (New England Biolabs) was added to 200 nM, and the mixture was incubated at 37 °C for 15 min. The reaction mixture was then treated with 200 μM GTP and incubated for 10 min, after which an aliquot was removed and quenched into loading buffer (1x = 4 M urea, 25 mM EDTA, 45 mM Tris-borate, pH 8.3). The remaining reaction was treated with 200 μM ATP, CTP and UTP and incubated for an additional 10 min, after which another aliquot was removed and quenched. The reaction aliquots were analyzed by denaturing, 8 M urea, 15% PAGE and visualized by detecting the Cy5 fluorescence using a Typhoon 9410 Variable Mode Imager (GE Healthcare Life Sciences). Gel images for these and all following experiments were analyzed in ImageQuant (Molecular Dynamics). For RNA degradation assays ( Figure S4 ), a 10 nM solution of RNA10 was prepared in buffer B, including the specified OSS components and RNase inhibitors at the same concentrations used for smFRET. The mixture was incubated at 37 °C for 30 min and analyzed by denaturing, 8 M urea, 15% PAGE. We found through these assays that a number of common OSS components interfered with transcription in various ways. The OSS substrate protocatechuic acid (PCA) was found to interfere with transcription, as was the combined glucose/glucose oxidase/catalase (GOD/CAT) OSS, indicating inhibition by its product, D-gluconic acid. PCA and D-gluconic acid are both carboxylic acids, as are many known inhibitors of bacterial RNAP ( Elgaher et al., 2014 ), and are therefore negatively charged under our imaging conditions. Inhibition was relieved by adding the OSS after incubating the bubble scaffold with RNAP, suggesting that PCA and D-gluconic acid interfere with binding rather than elongation. It is therefore likely that at the millimolar concentrations used for oxygen scavenging, they bind to and neutralize the electropositive cleft of RNAP where the transcription bubble resides. The maximum elongation efficiency was obtained by adding the GOD/CAT OSS after incubation with RNAP; therefore, this protocol was used for all experiments reported herein. Certain OSS enzymes, particularly catalase from bovine liver, were found to have RNase contamination and were therefore avoided, echoing reports of DNase contamination in OSS enzymes ( Senavirathne et al., 2015 ). Promoter-initiated transcription assays A 151-nucleotide portion of the 5’ UTR of the queCDEF operon from B. subtilis, including the preQ 1 riboswitch, was cloned into the pUC18 plasmid between the KpnI and EcoRI restriction sites. The E. coli RecA promoter was inserted immediately upstream of the riboswitch between the BamHI and KpnI sites. In addition, 12 nucleotides not found in the wild-type sequence were inserted after the promoter in order to generate a 19-nucleotide stretch in which the RNA transcript lacks any adenosine residues. Mutant plasmids were generated by site-directed mutagenesis using the primers in Table S2 . Transcription templates for all experiments except those utilizing ΔFT RNAP were prepared by PCR using the "Forward PCR primer" and "Reverse PCR primer" indicated in Table S2 . Transcription templates for ΔFT RNAP and associated WT controls contained the Gal1 promoter instead of RecA, and were generated in two PCR steps using first "Forward Gal1 primer 1" and "Reverse PCR primer", then "Forward Gal1 primer 2" and "Reverse PCR primer". For transcription assays, halted complexes (HCs) were prepared in buffer C (20 mM Tris-acetate, pH 8.0, 20 mM sodium acetate, 2 mM magnesium acetate, 5% glycerol, 14 mM 2-mercaptoethanol, 0.1 mM EDTA) containing 1 μM labeling NTP, 5 μM of two other NTPs (omitting ATP), 56 nM α 32 P-GTP or CTP (3000 Ci/mmol), 100 μM ApC dinucleotide primer, and 40 nM DNA template. E. coli RNAP holoenzyme (New England Biolabs) was added to 80 nM, and the mixture was incubated for 15 min at 37 °C. 0.9 reaction volumes of HC was treated with 0.1 reaction volumes of pre-warmed chase solution in buffer C, which contained 200 μg/mL rifampicin and 10x the desired final concentrations of all four NTPs and preQ 1 . The mixture was incubated at 37 °C, and reaction aliquots were quenched at the desired times into an equal volume of loading buffer (8 M urea, 0.8× TBE, 0.2% bromophenol blue, 0.2% xylene cyanol, 1 mM EDTA). For most experiments, additional NTPs were then added to 200 μM, and a final aliquot was removed 3-5 minutes later. For experiments with RNAP variants (point mutants in the β' subunit and ΔFT RNAP), the concentration of RNAP was 50 nM instead of 80 nM. Sequencing ladders were prepared by combining 9 μL of HC with 1 μL of chase solution containing 250 μM of each NTP, in addition to one 3’-OMe NTP (at 25 μM for 3'-OMe GTP and 15 μM for 3’-OMe ATP, UTP and CTP). Reactions were incubated for 15 min at 37 °C before being quenched with 10 μL of loading buffer. Reaction aliquots were denatured before loading 1.5-2.5 μL of each onto a denaturing 8 M urea, 10% polyacrylamide sequencing gel. The gel was dried and exposed to a phosphor screen (typically overnight), which was then scanned on a Typhoon 9410 Variable Mode Imager. For reactions including transcription factors, cell extract or RNA hairpin and the associated control reactions, the template was purified by spermine precipitation ( Hoopes and McClure, 1981 ). These reactions were performed by preparing HCs at twice the concentration described above, adding an equal volume of 2x the desired final concentration of factor, extract or hairpin, and 30 seconds later adding the chase solution. The final concentration of all transcription factors was 100 nM. In the case of Eco NusA, it was verified that concentrations as high as 500 nM had no effect beyond that seen at 100 nM.

Transcription data analysis

Volumes of the pause band following the aptamer domain (U46), the terminator in the expression platform (U70), the terminator following the expression platform (U108), and the two bands resulting from runoff were determined using ImageQuant (Molecular Dynamics). For each lane, these volumes were background corrected by determining the volume above background of all points that had a higher intensity than the average intensity of a background box positioned in the same lane. Two background boxes were used in each lane: one to correct band U46 (positioned just below that band), and one to correct bands U70, U108 and the two runoff bands (positioned between U70 and U108). The relative intensity of each band in a given lane was determined by dividing the background-corrected volume of that band by the sum of the volumes of all five bands mentioned above. Two pause bands were observed within the aptamer domain, but these were omitted from the analysis due to their low intensity and lack of variation in response to preQ 1 and mutations. Error bars in transcription quantifications represent the standard deviation of the intensity fraction of each band across three independent experiments. To determine the K 1/2 for regulation of pausing and termination by preQ 1 , the relative intensities of U46 and U70 at 45 seconds and 90 seconds after chasing, and 5 min after adding a further chase to 200 μM of each NTP were plotted as a function of preQ 1 concentration. These six data sets were globally fit with equation 4 : (4) F ( [ pre Q 1 ] ) = y 0 + A ∗ [ pre Q 1 ] K 1 ∕ 2 + [ pre Q 1 ] where F is the intensity fraction in a given band, y 0 is the intensity fraction at zero preQ 1 , and A is the amplitude of the change that occurs upon addition of preQ 1 . A global fit was performed in which y 0 and A were allowed to vary between data sets, but all were required to have the same value of K 1/2 . To determine the pause efficiency and lifetime from detailed time courses, the natural logarithm of the fraction paused was calculated at each time point t (seconds) and the following equation was fit to the resulting data: (5) F ( t ) = Ln [ a 1 ⋅ e m 1 t + a 2 ⋅ e m 2 t ] Fitting to the logarithm of the data rather than the raw data allowed the time points with low paused fraction to still contribute to the fit. a 1 and a 2 correspond to the fraction of elongation complexes that pause with half-lives - Ln [ 2 ]/ m 1 and - Ln [ 2 ]/ m 2 , respectively. The constraint of a 1 + a 2 ≤ 1 was used in all fits. Cross-linking Assays 100 nM DNA template and 20 nM RNAP holoenzyme were incubated in buffer D (20 mM Tris-HCl, 120 mM KCl, 5 mM MgCl 2 , 5% glycerol (w/v), 1 mM 2-mercaptoethanol, pH 7.9) at 37°C for 15 min. ApU (100 μM) and starting NTPs (30 μM CTP, GTP, and ATP) were added to the promoter bound RNAP, and the reaction was incubated for 30 min at 37 °C. The EC at position 6 was immobilized on 10 μL Ni 2+ -NTA agarose beads at 37°C for 15 min. Each following walk (indicated in Figure S5 ) was completed with 5 intermediate washes with buffer D to eliminate unbound NTPs. Each wash involved addition of 1 mL of buffer D followed by a 5 second spin on a tabletop centrifuge to pellet the Ni 2+ beads with attached ECs. The supernatant was then removed by pipette. The cross-linkable analogue 4-thio-UTP or 6-thio-GTP (10 μM) was introduced into the RNA at the desired position. Upon reaching A36, 10 μCi [α- 32 P-GTP, 3000 Ci/mmol was added for 2 min. 5 μM ATP, CTP, and GTP were then added to reach C43, then 5 μM UTP and GTP to extend to U46. ECs C43 and U46 were incubated with 40 μM preQ 1 at 37°C for 5 min and exposed to 365 nm UV light (UVP, 8W Model UVLMS-38, Upland, CA) for 10 min on ice. The reactions were either quenched with stop buffer (9M Urea, 20 mM EDTA, 1XTBE, 0.5 % Brilliant Blue R and 0.5% Xylene Cyanol FF) and the sample resolved on denaturing 7M urea 15% polyacrylamide-gel, or in LDS loading dye and separated on a 4-12% Bis-Tris Novex gel (Invitrogen). The gels were dried and exposed to phosphor screens.

Simulations

Molecular dynamics simulations were performed to explore the folding landscape of the preQ 1 riboswitch bound to the RNA polymerase elongation complex. Structure-based or Gō-type simulations are particularly useful for exploring slow processes like folding that are computationally expensive to probe with more precise explicit solvent simulations. Structure-based models were originally used in protein folding ( Shea et al., 1998 ) and are based on energy landscape theory and the principle of minimal frustration ( Onuchic and Wolynes, 2004 ). In order to fold on biological time scales, native interactions must be more stabilizing than nonnative interactions, so structure-based models only include interactions between native contacts. We chose to use an all heavy atom structure-based model that has been extensively calibrated, and has been used for RNA folding ( Lutz et al., 2014 ; Whitford et al., 2009 ) and functional studies of the ribosome, a mixed protein and RNA system ( Whitford et al., 2010 ). To generate a structure-based model, a native structure is needed to specify the interactions that ought to be stabilized. Atoms in close proximity within a known structure are chosen to be contact pairs that interact within the model; the shadow algorithm was used to calculate the contact map ( Noel et al., 2012 ). Since no structure of the preQ 1 riboswitch docked to RNAP existed, a model was created from structures of various components. Run input files for various structural elements were prepared using SMOG 2 ( Noel et al., 2010 ; 2016 ), and then stitched together as described. The preQ 1 riboswitch was modeled using contacts present in its crystal structure (PDB ID: 3FU2) ( Klein et al., 2009 ). Its NMR structure (PDB ID: 2L1V) ( Kang et al., 2009 ) was used to fill in 5’ and L2 gaps from the crystal structure: any contacts and interactions with missing residues were used to supplement the interactions defined by the crystal structure. An E. coli RNAP model was adopted from a cryo-EM structure of an EC (PDB ID: 6ALH) ( Kang et al., 2017 ). The transcription bubble was modeled by creating single- and double-stranded sections of RNA and DNA using the nucleic acid builder ( http://structure.usc.edu/make-na/server.html ), and then stitching the resulting topologies together appropriately. Interactions of residues in either the riboswitch or the polymerase overrode those interactions in the DNA bubble model, so the bubble was only used to supplement interactions for missing residues. Since several topologies were being combined, and contact and dihedral interactions are scaled slightly differently in each, the dihedral and contact energies were both set to typical values of 0.7 energy units. We refer to the supplemental information of ( Hayes et al., 2014 ) for a discussion of units in the model. Models with 0 to 3 nucleotides inserted between the riboswitch and RNAP (RNA0p, RNA1, RNA2 and RNA3) were made by modifying the sequences generated with the nucleic acid builder (sequences are reported in Table S1 ). The resulting model essentially stitches together models of the riboswitch and the RNAP. Since the riboswitch and RNAP fold independently, the model is valid, but only accounts for steric interactions between the riboswitch and RNAP, and neglects any specific interactions that could be obtained from a structure of the complex. Consequently, some questions are beyond the scope of the model, while others can be robustly answered. The average number of base pairs formed in the model should be fairly robust, as the model identifies structures of the riboswitch that lead to a minimal disruption of RNAP structure. Likewise, the model should be able to elucidate the sterically allowed binding poses for the folded riboswitch, whereas identifying the dominant pose is beyond the scope of the model, as the populations of competing poses can be shifted by specific interactions between the riboswitch and RNAP. Equilibrium simulations of riboswitch folding were run using umbrella sampling on the Q (number of formed contacts) reaction coordinate with replica exchange between umbrellas to allow trapped high-energy folded poses to relax. 11 replicas were used with an exchange attempts every 5000 steps, an umbrella k constant of 0.005 energy units per contact squared, and umbrella equilibria Q 0 evenly spaced among replicas from 0 to 282 contacts. The weighted histogram analysis method (WHAM) ( Kumar et al., 1992 ) was then used to reweight each frame of each replica to an unbiased simulation. Simulations started from an unfolded preQ 1 riboswitch structure, and allowed the riboswitch to fold at a temperature of T = 85 units. (The folding temperature of the riboswitch is approximately 90 units in the presence of the polymerase.) Simulations were run for 50 million steps with the first 12.5 million steps discarded for equilibration. Simulations with the preQ 1 ligand included 5 ligands in a cubic box of 20 nm side length. The binding affinity of preQ 1 was not calibrated. Simulations were run with and without preQ 1 and varying numbers of inserted nucleotides to determine whether steric constraints allowed P2 to fold ( Table S3 ). A base pair was considered formed if the central hydrogen bond between purine N1 and pyrimidine N3 was within a factor of 1.5 of its native distance. (In the A•C mismatch, the only hydrogen bond was used instead.) The number of formed base pairs was counted for each frame and averaged using the weight given by WHAM over all replicas in the simulation. The average number of base pairs was largely unchanged in the absence or presence of preQ 1 for RNA1, RNA2 and RNA3 (1.8-2.4 versus 2.0-2.4, respectively), and depends weakly on number of inserts, but changes substantially in the zero insert case (RNA0p), where it is lower by about half a base pair with preQ 1 and a whole base pair without preQ 1 . To check the robustness of the results, the contacts in the RNA linker (all residues between the 3’ end of P2 and the 5’ end of the RNA-DNA duplex) were removed. This allows the RNA to pull slightly out of the exit tunnel, and makes the RNA more flexible so it can twist more. While about half an additional base pair could form with the more flexible model, the trends remained the same: one to three inserts formed roughly the same number of base pairs as each other, and no inserts formed half a base pair less than them. Finally, a model of the flap tip deletion mutant was made in which residues K890-K914 of the beta subunit were removed. Simulations of the flap tip deletion mutant achieved results intermediate between the other two models, but showed less sensitivity to preQ 1 in the RNA0p system. To connect with cross-linking studies and to elucidate the folded pose, the frequency with which the cross-linking residue U14 was within 8 Å of each protein residue was calculated. Two poses of the riboswitch were identified that involved interactions primarily between the riboswitch and β’ (collectively called "mode 1"), along with a third pose where U14 is wedged between the clamp and flap tip ("mode 2"). Within mode 1, simulations with the default parameters (rigid linker) yielded a pose where U14 was positioned near K76-K79, while with the floppy linker, an additional pose was observed in which it is positioned near T393. These results are in agreement with cross-linking results that show that U14 interacts with the β’ subunit.

Supplementary Material 1