A novel regulatory interplay between atypical B12 riboswitches and uORF translation in Mycobacterium tuberculosis

Abstract Vitamin B12 is an essential cofactor in all domains of life and B12-sensing riboswitches are some of the most widely distributed riboswitches. Mycobacterium tuberculosis, the causative agent of tuberculosis, harbours two B12-sensing riboswitches. One controls expression of metE, encoding a B12-independent methionine synthase, the other controls expression of ppe2 of uncertain function. Here, we analysed ligand sensing, secondary structure and gene expression control of the metE and ppe2 riboswitches. Our results provide the first evidence of B12 binding by these riboswitches and show that they exhibit different preferences for individual isoforms of B12, use distinct regulatory and structural elements and act as translational OFF switches. Based on our results, we propose that the ppe2 switch represents a new variant of Class IIb B12-sensing riboswitches. Moreover, we have identified short translated open reading frames (uORFs) upstream of metE and ppe2, which modulate the expression of their downstream genes. Translation of the metE uORF suppresses MetE expression, while translation of the ppe2 uORF is essential for PPE2 expression. Our findings reveal an unexpected regulatory interplay between B12-sensing riboswitches and the translational machinery, highlighting a new level of cis-regulatory complexity in M. tuberculosis. Attention to such mechanisms will be critical in designing next-level intervention strategies.


Introduction
RNA leaders preceding coding sequences in mRNAs have gained interest as hubs for gene expression control (1)(2)(3)(4).These include riboswitches, which are highly structured cisregulatory RNAs that sense and bind specific metabolites such as enzyme cofactors, amino acids, or nucleotides, to affect the expression of genes under their control (5)(6)(7).Typically, the regulated genes have a direct relationship with the corresponding riboswitch ligand, whereby the encoded gene product is involved in the de novo biosynthesis of the ligand or its transport ( 8 ,9 ).Riboswitches regulate gene expression via the interaction between two RNA domains: the aptamer and the expression platform.Aptamers are highly conserved and form a unique 3D structure for ligand-binding.Expression platforms execute gene regulation by adopting mutually exclusive secondary structures depending on the ligand binding status of the aptamer ( 3 ,4 ).Although the mechanisms of individual riboswitches can vary, the gene expression outcome is either permissive ('ON' switch) or non-permissive ('OFF' switch), primarily due to changes in transcription termination or translation initiation or both (10)(11)(12).Termination of transcription can be intrinsic or Rho-dependent, the latter being the dominant mechanism genome-wide in mycobacteria ( 13 ).Riboswitch-mediated translational control typically involves ligand-dependent occlusion of the translation initiation region (TIR) by a complementary anti-TIR ( αTIR) sequence, which, in turn, may be sequestered by an anti-anti-TIR ( ααTIR) sequence ( 12 ).Inhibition of translation may in turn facilitate downstream Rho-dependent transcription termination, as Rho-binding (RUT) sites become exposed on the RNA (14)(15)(16)(17).As intrinsic terminators are rare in mycobacteria, mycobacterial riboswitches are presumably mostly translational and / or Rho-dependent ( 4 ,13 ).
Mycobacterium tuberculosis , the aetiological agent of tuberculosis ( 33 ,34 ), as well as M. africanum and animal adapted lineages, lack the ability to synthesize B 12 ( 35 ,36 ), linked to the deletion of cobF , encoding a precorrin-6A synthase, and N-terminus truncation of cobL , encoding a decarboxylating precorrin-6Y C (5,15)-methyltransferase, as both are involved in the de novo B 12 biosynthesis pathway (37)(38)(39).Recent evidence suggests that the ablation of de novo B 12 biosynthesis in these organisms shaped their evolution as pathogens capable of systemic infections ( 40 ).Still, M. tuberculosis encodes several B 12 -dependent enzymes, suggesting that the pathogen utilizes host-derived B 12 , although these enzymes and their pathways are found alongside alternative B 12 -independent counterparts in M. tuberculosis .This is true for the conversion of ribonucleotides to deoxyribonucleotides ( 41 ,42 ), for the degradation of propionate ( 41 ,42 ) and for the biosynthesis of methionine from homocysteine ( 41 ,42 ).Methionine synthesis and propionate degradation are both B 12dependent pathways in humans ( 43 ).In addition, a role for the host-derived B 12 in virulence and as a signalling molecule in the cross-talk between the host and M. tuberculosis has been suggested (40)(41)(42).
B 12 has been shown to regulate expression of metE via B 12 -sensing riboswitches in M. tuberculosis and in the saprophytic mycobacterial model, Mycobacterium smegmatis , by suppressing metE mRNA levels ( 44 ,45 ).A second, homologous B 12 riboswitch occurs in M. tuberculosis upstream of a potential tricistronic operon comprising ppe2 , cobQ and cobU .PPE2 is a member of the large, Mycobacterium -specific PE / PPE protein family associated with host-pathogen interactions and virulence (46)(47)(48) and is suspected to be involved in cobalt transport ( 49 ).PPE2 has also been suggested to suppress nitric oxide production in host macrophages and hence affect the host immune response (50)(51)(52).CobQ and CobU are relics of the disrupted B 12 biosynthesis pathway ( 49 ).
Biochemical and structural studies of B 12 riboswitches in different bacteria have provided detailed insights into conserved features such as the central, ligand-binding four-way junction, the B 12 box and the 'kissing loop' (KL).Despite the conservation of these features, B 12 riboswitches exhibit differential selectivity to individual B 12 isoforms and variations in their peripheral elements ( 53 ,54 ).These characteristics form the basis for their division into Class I, IIa & IIb switches.Class I and IIb riboswitches selectively bind AdoB 12 , whereas Class IIa riboswitches show preferential binding to the slightly smaller MeB 12 and HyB 12 .Other B 12 riboswitches displaying promiscuous binding to a broad range of corrinoids have also been reported ( 49 ).
Recently, it has been proposed that codons and / or peptides arising from the translation of upstream open reading frames (uORFs) occurring within gene leaders can either positively or negatively impact the expression level of the downstream ORF ( 13 ,55-57 ).For example, translating ribosomes present on uORFs can modulate the expression of the downstream gene, which may involve translation coupling between ORFs ( 58 ,59 ).How riboswitch-associated uORFs might affect gene expression has not yet been addressed.
In the current study, we analyse ligand binding, riboswitch architecture, and control mechanisms of the metE and ppe2 riboswitches from M. tuberculosis .We present the first evidence of binding of B 12 to these elements and functional validation of the ppe2 riboswitch as an 'OFF switch.' W e found that the two riboswitches exhibit differential binding of B 12 isoforms and involve distinct structures in their expression platforms to execute B 12 -dependent control.On this basis, we propose that the ppe2 switch represents a new variant of Class IIb switches whereas the metE switch presents as a Class I member.Moreover, we show that translation of uORFs in the leaders of metE and ppe2 substantially alters the expression of their respective downstream coding regions.Interestingly, translation of the metE uORF suppresses MetE expression whereas translation of the ppe2 uORF is essential for PPE2 expression.In the latter case, we found evidence of termination-reinitiation (TeRe) ( 59 ) in LacZ reporter constructs, resulting in separate uORF and LacZ proteins as well as uORF stop codon suppression, leading to a frameshifted uORF-PPE2 fusion protein.
The unexpected variation and complexity of M. tuberculosis B 12 riboswitch-regulation expands our current understanding of such elements and reveals new intricacies of translational control in this pathogen.

Oligonucleotides, plasmids and cloning
Oligos and plasmids used in this study are listed in Supplementary Table S1 .DNA oligos longer than 100 bp were purchased as geneBlocks fragments from Integrated DNA Technologies.Other oligos and primers were purchased from Thermo Fisher Scientific.Reporter constructs were generated by inserting target sequences using either Gibson assembly or restriction cloning between the HindIII and NcoI sites of pIRaTE2020 ( 13 ), to produce in-frame translational fusions with LacZ.The 5 edges of umetE'-lacZ and umetE4'-lacZ fusions were at +87 nt and +159 nt, respectively, relative to the TSS (+1 nt) of the metE leader; the 5 boundaries of other lacZ fusions are specified in the relevant sections of the manuscript.Nucleotide substitution or deletion mutations were designed on the NEBaseChanger online tool ( https:// nebasechanger.neb.com/ ) and TOPO-cloned using the Q5 site-directed mutagenesis kit (New England Biolabs) according to the standard protocol.

RNA extraction and northern blot analysis
M. tuberculosis H37Rv cultures were rapidly chilled by directly mixing with ice and pelleted by centrifugation.RNA was isolated using the RNAPro Blue kit (MP Biomedicals) according to the manufacturer's protocol.RNA concentration and quality were evaluated on a Nanodrop 2000 spectrophotometer (Thermo Scientific).For northern blot analysis, 10 μg total RNA was separated on denaturing 8% polyacrylamide gel and transferred on a blotting paper for detection, as previously described ( 60 ).For horizontal agarose northerns, RNA was separated on 1% agarose and transferred to blotting paper according to the NorthernMax™ Gly kit (Invitrogen).RNA probes were synthesized using the mirVana miRNA probe synthesis kit (Ambion) and radiolabelled using 133 nM 32 P α-UTP (3000 Ci / mmol; Hartmann Analytic GmbH), with unlabelled UTP added to achieve a final concentration of 3 μM.RNA fragment signals were developed on radiosensitive screens and visualized on a Typhoon FLA 9500 phosphorimager (GE Healthcare).

Quantitative real-time PCR
The RNA used for quantitative real-time PCR (qRT-PCR) was isolated from cultures supplemented with 10 μg / ml exogenous AdoB 12 , as reported by others ( 35 ,44 ).qRT-PCR was performed on the QuantStudio 6 real-time PCR system (Applied Biosystems) using cDNA synthesized from 0.5 μg DNase-treated RNA using the Superscript IV Reverse Transcriptase kit (Invitrogen) and random hexamers.Each 20μl PCR reaction contained 1 × Fast SYBR Green Master Mix (Applied Biosystems), 200 nM forward and reverse primers and 5 μl of 100 × diluted cDNA or M. tuberculosis genomic DNA standards.The TSS-proximal amplicon (5 amplicon) in the metE leader covered the section between + 5 nt and + 117 nt relative to the metE TSS (genomic coordinate 1261711) ( 61 ), while the further downstream leader amplicon extended from +245 nt to +364 nt, relative to the metE TSS.The primers for the metE coding amplicon amplified +720 nt to +825 nt relative to the metE TSS.The ppe2 leader amplicon extended from +200 nt to +314 nt relative to the ppe2 TSS (genomic coordinate 309839) ( 61 ), while the ppe2 coding amplicon extended from +1007 nt to +1117 nt relative to the ppe2 TSS.The ppe2-cobQ junction amplicon extended from +1861 nt to +2118 nt relative to the ppe2 TSS.Relative gene expression was determined as a ratio of the level of target mRNA to that of 16S rRNA.All data were graphed and analysed using GraphPad Prism software for Mac OS, version 10.0 ( www.graphpad.com).

Beta-g alactosidase ( β-g al) activity assay
Protein expression levels were assessed using the betagalactosidase ( β-gal) activity assay as previously described ( 60 ).Briefly, 10-ml cell cultures were centrifuged, and the cell pellet washed thrice in Z-buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1mM MgSO 4 ) prior to one round of lysis in a FastPrep bio-pulveriser (MP Biomedicals) at speed = 6.5 and time = 30 s.The protein concentration in the cell lysate was determined using the BCA kit (Thermo Scientific) according to the manufacturer's instructions.The level of lacZ expression was calculated in Miller units (M.U.) per milligram of protein.All data were graphed and analysed using GraphPad Prism software, version 10.0 ( www.graphpad.com).

FLAG-tagging, pulldown and western blot analysis
A triple FLAG-tag was inserted to the N-terminus of uPPE2 in the ppe2'-lacZ reporter construct containing the AUGA overlap or the N-terminus of the uPPE2 nostop construct in which the AUGA overlap is eliminated using Q5 site-directed mutagenesis (New England Biolabs).The constructs were transformed into M. smegmatis wildtype in parallel with ppe2'-lacZ or uPPE2 nostop constructs without FLAG tags and 60 ml of log-phase (OD 600 ∼0.6) cultures were prepared for western blot analysis.Cells were pelleted, resuspended and washed three times in 1 ml PBS (pH 7.9), and finally resuspended in 1 ml B-PER™ Bacterial protein extraction reagent (Thermo Fisher Scientific) before disrupting with lysing matrix B in a FastPrep machine (settings: speed = 6.5, time = 40 s, three times with cooling intervals on ice).Pulldowns were carried out with Pierce™ Anti-FLAG Magnetic agarose (Thermo Fisher Scientific) largely according to manufacturer's instructions.Briefly, extracts were cleared by centrifugation and 500 μl of the supernatant was added to 50 μl of the equilibrated beads and incubated at RT for 1 h.Extracts were washed twice with PBS containing 200 mM NaCl, eluted in 100 μl 0.1 M glycine, pH 2.8 and neutralised with Tris pH 8.5.SDS loading buffer was added to all extracts before boiling and loading onto a 4-20% Mini-PROTEAN Tris-glycine gradient gel (Bio-Rad).Proteins were separated by SDS-PAGE and transferred to a PVDF membrane.The FLAG-tagged protein was detected by incubating the milk powder-blocked membrane with mouse monoclonal anti-FLAG primary antibodies (Sigma-Aldrich) diluted at 1:1000, followed by incubation with peroxidase-conjugated polyclonal goat anti-mouse IgG (Jackson ImmunoResearch) diluted at 1:1000.β-galactosidase was detected with anti-LacZ antibodies (Thermo Fisher) diluted at 1:1000, followed by incubation with peroxidase-conjugated polyclonal goat anti-rabbit IgG (Jackson ImmunoResearch) diluted at 1:1000.Signals were visualized on a Li-Cor Odyssey Fc Imager (Licor).

In-line probing
In-line probing was performed as described by Regulski and Breaker ( 62 ).Riboswitch RNA was transcribed using the Megascript T7 High Yield Transcription Kit (Invitrogen) from amplicons generated by PCR using the primers listed in Supplementary Table S1 .Transcribed RNA was extracted from denaturing 8% polyacrylamide gel in 500 μl crush-andsoak buffer (0.5 mM sodium acetate pH 5.2, 0.1% SDS, 1 mM EDTA pH 8.0, 30 μl acid phenol:chloroform) and precipitated in ethanol.The yield and purity of the transcribed product were analysed on a Nanodrop 2000.Dephosphorylation was done using calf intestinal alkaline phosphatase (1 U / μl) (Life Technologies).Each 25μl 5 end-labelling reaction contained 10 pmol RNA, 133 nM 32 P γ-ATP (6000 Ci / mmol; Hartmann Analytic GmbH), 1 μM unlabelled ATP, and 25U T4 polynucleotide kinase (10 U / μl) (New England Biolabs).The radiolabelled RNA was purified by PAGE and resuspended in 40 μl nuclease-free water.For in-line probing, reactions contained 2 μl radiolabelled RNA ( ∼25 nM), 1 × in-line reaction buffer (50 mM Tris-HCl pH 8.3, 20 mM MgCl 2 , 100 mM KCl), and the desired concentration of AdoB 12 , MeB 12 , HyB 12 or CNB 12 in 20μl total volume.Reactions were incubated on a heat block maintained at 30 ºC for 20 h and quenched with an equivalent volume of 2 × colourless gel-loading solution (10 M urea, 1.5 mM EDTA pH 8.0).In-line reaction products were separated by denaturing 6-10% polyacrylamide gel electrophoresis (PAGE) at 45W.The gels were dried and exposed to radiosensitive screens and the data were collected on a Typhoon FLA 9500 (GE Healthcare).The dissociation constants ( K d ) of the riboswitches were calculated from the in-line probing data in Figures 3 A and 5 A, by plotting the fraction of RNA cleaved at ligand-sensitive (cleaved) sites against the logarithm of AdoB 12 concentration, using the formula described in ( 62 ).In Graphpad prism (version 10.0), the data were fitted using the Richards equation to obtain K d values ( metE riboswitch:

Sequence alignments
The DNA sequences of M. tuberculosis metE and ppe2 leaders stretching from ∼40 nt upstream of the TSS to the first codon of the downstream annotated ORF were used as the input query for nucleotide alignment on the NCBI BLAST tool ( 63 ).Matches of > 95% identity in representative mycobacteria were extracted and their TSS located by examining their respective -10 elements.The 5 ends of the shortlisted sequences were trimmed to only -5 nt relative to the TSS.The DNA sequence was converted to RNA prior to alignment using t-coffee with default settings ( 64 ).Amino acid sequences of uPPE2 were similarly aligned using t-coffee default set-tings for protein alignment ( 64 ).The resulting clustalw format alignment files were downloaded and edited using the desktop version of Jalview (version 2.11.2.5) ( 65 ).

RNA secondary structure prediction and visualization
Target RNA sequences and matching folding constraints were loaded on the RNAstructure web servers ( 66 ), and the structure prediction software ran using default RNAstructure tools (version 6.4).A MaxExpect file containing a CT-formatted structure was downloaded and converted to a dot-bracketformatted file, which was used to render the 2D secondary structure using the web-based RNA2drawer app ( 67 ).

Premature transcription termination in metE and ppe2 leaders
We recently mapped multiple premature transcription termination sites (TTS) associated with RNA leaders including those of metE and ppe2 in M. tuberculosis cultures grown in standard conditions ( 13 ).The TTS patterns within these two leaders indicated multiple TTS thoughout the metE leader, compared to two, closely spaced TTS in the ppe2 leader, suggesting differences in the regulation of the switches (Figure 2 A and B) ( 13 ).To explore how B 12 might affect growth and metE and ppe2 transcription, we grew cultures of M. tuberculosis to OD 600 ∼0.6 before adding 10 μg / ml AdoB 12 ; notably, this did not affect the growth rate ( Supplementary Figure S1 A).RNA was isolated before and 1 hour after the addition of AdoB 12 , and analysed by quantitative R T-PCR (qR T-PCR) targeting the leader and the coding regions of metE and ppe2 (Figure 2 A and B).Using qRT-PCR primers targeting the metE TSS-proximal region, we observed no significant change in the RNA levels upon the addition of AdoB 12 (5 amplicon, Figure 2 C).However, the addition of AdoB 12 had a profound effect further downstream with a 19-fold decrease in the level of leader amplicon and a 60-fold decrease in metE amplicon, reflecting a reduction in transcript levels (Figure 2 C).Combined with the mapped TTS, these data suggest that AdoB 12 induces transcription termination at multiple sites in the metE mRNA.By comparison, the changes in ppe2 transcript levels were more modest, with leader RNA decreasing ∼2-fold and ppe2 RNA reducing ∼2.5-fold following AdoB 12 addition (Figure 2 D).
The cobQU genes downstream of ppe2 are associated with cobalamin synthesis ( 31 ,49 ), making them likely targets of B 12 -dependent control.Therefore, to determine if cobQ is cotranscribed with ppe2 and thus regulated by the B 12 switch, we performed qRT-PCR across the ppe2-cobQ junction.The results indicated that ppe2 and cobQ are co-transcribed and that RNA levels decrease 10-fold after AdoB 12 addition, suggesting that cobQ is also regulated by the riboswitch (Figure 2 D).RT-PCR did not amplify across the cobQ-cobU junction, suggesting that either cobU is not part of the operon or its expression was below the detection limit, which we consider more plausible ( Supplementary Figure S1 B).In summary, the results suggest transcriptional polarity in both loci , albeit to a lesser degree in ppe2 than in metE .
To confirm the notion of premature termination, we performed northern blotting of RNA at times 0, 1, 3, 6 and 24 hours post AdoB 12 -addition using a probe that hybridized to the 5 end of each leader.The transcript pattern before AdoB 12 -addition reflected the TTS mapping with multiple signals for metE and only a few for ppe2 ; moreover, some of the signals on the blots corresponded to the dominant TTS signals indicated in panels A and B (indicated by arrows in Figure 2 E  and F).The addition of AdoB 12 led to one primary but opposite B 12 -dependent change within each leader: a signal corresponding approximately to the TTS at +326 nucleotide (nt) position within the metE leader increased substantially upon AdoB 12 addition, suggesting premature termination of transcription (Figure 2 E), while a signal assumed to correspond to the +178-nt TTS within the ppe2 leader disappeared (Figure 2 F).To validate that the metE signal around +326 nt was due to premature termination of transcription, we repeated the northern blot with horizontal agarose gels, allowing for detection of larger transcripts including the full-length ( ∼3 kb) mRNA using the same probe.The image in Figure 2 G indicates the presence of a ∼3 kb transcript (at time 0), which upon the addition of AdoB 12 is replaced by a transcript < 500 nt in agreement with the 326-nt transcript observed in Figure 2 E. Together, these results strongly support the notion of B 12 -dependent termination of transcription within the metE leader.Nevertheless, we cannot rule out that the actual termination site is located downstream of +326 nt, in which case the 3 end is a result of post-termination trimming.The ppe2 results do not suggest extensive premature termination of transcription, which corroborates substantial differences between the two riboswitches.

The metE and ppe2 aptamers display variable selectivity for B 12 isoforms
To ascertain direct interactions between B 12 and the metE and ppe2 aptamers and to determine the ligand binding properties of each, we generated transcripts for in-line probing analysis ( 62 ) by in vitro transcription.Both transcripts covered the region between the TSS and a few bases downstream of their respective most distal leader TTS.Thus, the size of the metE riboswitch transcript was 345 nt, while the ppe2 transcript was 191 nt.First, we analysed the interactions between the two riboswitches and four common B 12 isoforms: AdoB 12 , MeB 12 , HyB 12 and CNB 12 .In-line probing reactions contained at least a 4-log excess concentration of ligand (1 mM) over that of RNA.In the case of metE , AdoB 12 induced the strongest modulation signals, while MeB 12 , HyB 12 or CNB 12 resulted in little to no change ( Supplementary Figure S2 ).In contrast, all four B 12 isoforms resulted in similar cleavage patterns and signal intensities in the ppe2 transcript with the possible exception of C48-C50, where only AdoB 12 caused a reduction in the cleavage signal ( Supplementary Figure S2 ).In summary, our results indicate that the regions flanked by the TSS and the distal TTS are sufficient for ligand binding in both riboswitches and while the metE riboswitch is apparently selective for AdoB 12 , the ppe2 riboswitch seems able to accommodate all four B 12 isoforms equally well.

B 12 -binding leads to occlusion of the metE translation initiation region
To interrogate the ligand sensitivity of the metE riboswitch, we performed in-line probing using a range of AdoB 12 concentrations from 0.1 to 2 mM (Figure 3 A), which suggested an approximate dissociation constant ( K d ) = 20.6 ± 7.2 μM ( Supplementary Figure S3 ).The in-line probing data were used  to apply constraints to a predicted structure of the ligandbound switch on the RNAstructure web server ( 66 ).The resulting structure indicated that the aptamer domain of the metE riboswitch is contained within the first 220 nt of the leader largely in agreement with the prediction in Rfam ( 68 ).Hence, the region downstream of this position was considered part of the expression platform.Several regions of hypercleavage, including G240 / C245 & C255 / G265, occur in the proposed expression platform in the presence of AdoB 12 (Figure 3 A).These positions are paired in the predicted unbound structure (Apo-form) but adopt single-stranded conformations in the ligand-bound state (Figure 3 B and C).An additional structure not reported in Rfam ( 68) was predicted at positions 1-14, comprising a short hairpin (P0) that is highly conserved in mycobacteria (Figure 3 C; Supplementary Figure S4 ).
According to the predicted structure, the B 12 binding pocket in the metE switch is enclosed in a four-way junction formed by P3-P6.The conserved 'B 12 box' ( 26 ) in this switch stretches from G199 to C215, and is followed immediately downstream by hypercleaved positions at G231-C233 (Figure 3 A, C).Issuing from the B 12 pocket are peripheral elements comprising a bipartite P2 arm and a large P6 extension featuring a second four-way junction formed by P7, P8, P10 and P11 (Figure 3 C).Moreover, a stereotypical KL is potentially formed by pseudoknot interactions between three conserved cytidines in L5 and a variable partner loop located in the expression platform.To validate the assignment of the KL and the regions involved, we substituted 92-94CCC in L5 with AAA and 258-259GG in L13 with UU.Separately, both of these mutations led to an increase in expression, potentially indicating loss of regulation.However, expression in the double mutant was restored to near wildtype levels (Figure 3 D), confirming our assignment of L5 as part of the KL.
The typical expression platform of B 12 riboswitches is translational ( 26 ), and although there were indications of premature termination of transcription, we found no evidence of an intrinsic terminator within the metE switch.Comparing the ligand-bound and Apo-structures in Figure 3 B and C we identified a potential expression platform, in which a broad translation initiation region (TIR) including a stretch of 25 nt upstream of the MetE start codon and a likely SD were effectively occluded by a complementary α-TIR stem in the ligandbound structure (Figure 3 C).In the Apo-form, the same α-TIR was fully sequestered by extensively pairing with an αα-TIR, thereby unmasking the TIR (Figure 3 B).Finally, the αα-TIR formed a hairpin in the ligand-bound structure, in which the apical loop (C255-U262) presented an ideal pairing partner for L5, thereby linking the KL directly to elements of the expression platform (Figure 3 B, C).This structure and the ligand preference for AdoB 12 suggest that the metE switch is a Class I switch.
To validate the predicted expression platform and its elements, we made a series of reporter constructs, in which the second codon of metE was fused in frame to lacZ ( metE'-lacZ ).The upstream edge consisted of gradual 5 extensions of the metE leader to include the predicted TIR, αTIR and the ααTIR, respectively (Figure 4 A, B).Predicted secondary structures of these partial leader-constructs are shown in Supplementary Figure S5 .To avoid B 12 -dependent folding potentially affecting β-galactosidase ( β-gal) activities, all constructs were transformed into a M. smegmatis cobK mutant incapable of de novo B 12 synthesis ( 45 ).The results indicated that the level of MetE'-LacZ expression in the minimal construct (TIR only) was much higher than that of the full-length metE leader ( ∼3-fold increase), suggesting the presence of inhibitory sequences upstream of the basic TIR (Figure 4 B).Extending the construct to include the predicted αTIR sequence reduced MetE'-LacZ expression substantially relative to full length leader ( > 50-fold reduction) (Figure 4 B), supporting the notion of translation inhibition via TIR occlusion.A further extension to include the potential ααTIR sequence massively reversed this phenotype yielding only ∼2-fold reduction relative to full length leader (Figure 4 B).Put differently, the expression levels in the TIR-only construct were > 130fold higher than when αTIR was included, but only ∼6-fold higher when both αTIR and ααTIR were included (Figure 4 B).These data further support the predicted structure-function relationship (Figures 3 and 4B ).Finally, an extension to +90 nt restored MetE'-LacZ expression to a similar level as that of the full-length switch (Figure 4 B).These findings suggest that the metE riboswitch is an 'OFF' switch that employs TIR occlusion (i.e. a translational expression platform) via the suggested elements for gene expression control.The formation of the KL directly blocks the proposed ααTIR and reinforce the αTIR-TIR pairing.As previously noted, we also observed a B 12 -enhanced TTS within the metE leader associated with a significant reduction in metE RNA levels, suggesting that the translational expression platform is augmented by Rhodependent termination of transcription and / or rapid mRNA degradation following reduced translation.

A short translated ORF within the metE aptamer suppresses MetE expression
M. tuberculosis encodes numerous short, translated ORFs (uORFs) upstream of annotated genes including genes controlled by riboswitches ( 13 , 61 , 69 ).The metE riboswitch encodes four potentially translated uORFs ( umetE1 -4 ) based on credible SD motifs and associated start codons at +88 nt (CUG), +123 nt (UUG), +140 nt (CUG) and +187 nt (GUG) (Figure 3 B, C).Ribosome binding and translation within a riboswitch aptamer will likely impact its folding and hence, the function of the switch.To investigate potential functions of metE uORFs, we first made in-frame reporter gene fusions to assess their expression.As translation in mycobacteria rarely initiates with CUG start codons ( 13 , 61 , 69 , 70 ), we pursued only umetE 2 (+123 nt to + 170 nt) and the larger umetE4 (+187 nt to +336 nt) as potentially translated uORFs (Figure 3 B, C).The fusions, covering the region upstream of the putative SD motifs to 8 codons of uMetE2 (uMetE2'-LacZ) or 5 codons of uMetE4 (uMetE4'-LacZ), respectively, were expressed in a wildtype (i.e.B 12 proficient) M. smegmatis strain.The resulting β-gal assays indicated low levels of uMetE2'-LacZ expression compared to the much more highly expressed MetE'-LacZ, whereas the expression level of uMetE4'-LacZ was no higher than background (Figure 4 C).This suggests uMetE2 may be translated in vivo thereby potentially affecting riboswitch function.
To investigate whether translation of uMetE2 had implications for MetE expression and B 12 -sensing, we mutated the start codon of uMetE2 to a non-start codon (UUG −→ UCG) within the context of the full-length MetE'-LacZ fusion (Figure 4 B; U124C).The construct was transformed into wildtype and cobK (B 12 -deficient) M. smegmatis backgrounds to assess if B 12 -sensing was intact in the mutant.To our sur- prise, this mutation led to a > 10-fold increase in MetE'-LacZ expression in both wildtype and cobK backgrounds, while the B 12 -dependent change was maintained (Figure 4 C).This finding suggested that although the mutation dramatically altered overall expression, it did not impair B 12 sensing.Conversely, deletion of the entire umetE2 (segment spanning +115 nt to +188 nt) led to complete loss of B 12 -sensing, while also increasing expression of MetE'-LacZ (Figure 4 C).In summary, these results suggest that translation of the aptamer-encoded uMetE2 is likely to contribute to the overall control of MetE expression.At this stage, we are unable to clarify whether this involves the uMetE2 peptide.

ppe2 is preceded by a riboswitch-controlled translated leader
The results presented so far suggested multiple functional differences between the metE and ppe2 switches.Therefore, to investigate in more detail the structure-function relationship of the ppe2 switch, we performed in-line probing of the ppe2 leader (from +1 nt to +191 nt) using a range of AdoB 12 concentrations from 1 to 4 mM (Figure 5 A).The results indicate that cleavage of the ppe2 riboswitch was modulated by AdoB 12 in a dose-dependent manner, with strongly protected regions at G24-U29, G61-G64 and A108-G128 and sections of hypercleavage at A131-C134 and U164 / G165 (Figure 5 A).This in turn suggests that the sequences required for ligand binding, i.e. the aptamer, are contained within the probed fragment.However, the affinity towards AdoB 12 was found to be considerably lower than that of the metE aptamer ( ppe2 switch K d = 446.1 ± 46.0 μM versus metE switch K d = 20.6 ± 7.2 μM) ( Supplementary Figure S3 ).We predicted the secondary structure of the ligand-bound ppe2 riboswitch using probingderived folding constraints and a previously proposed KL interaction between L5 and L13 for this switch ( 26 ) (Figure 5 C).Similar to that of the metE riboswitch, the B 12 binding pocket of the ppe2 riboswitch was also enclosed in a four-way junction formed by the paired segments P3-P6.However, unlike the metE switch, the ppe2 riboswitch contained a fused P1-P3 arm and a truncated P6 extension (Figure 5 C).The predicted structure suggests that the AdoB 12 -induced masking of positions A108-G128 are likely due to a combination of base pairing and ligand contacts (Figure 5 A, B).Moreover, this structure mirrors that of the metE switch where strong cleavage signals are observed immediately downstream of the B 12 box (Figure 5 A; positions A131-C134).
The region immediately upstream of the annotated ppe2 ORF is devoid of any obvious SD motifs, but a potentially translated ( 13 ) and partially conserved uORF (uPPE2) located between + 189 nt and the start of ppe2 appears to have a SD motif (Figure 5 and Supplementary Figure S6 ).In the predicted ligand-bound state, the uPPE2 TIR including this SD motif is partially masked by an αTIR stem, and together these elements form a hairpin that harbours a likely L5 pairing partner (L13, Figure 5 C).
In the predicted Apo-structure, the αTIR is sequestered by a purine-rich ααTIR sequence, while the SD motif and its downstream region are rendered more accessible for ribosome binding (Figure 5 B).Notably, the ααTIR precedes and partially overlaps the B 12 box (Figure 5 C).In summary, this structures suggests that the probed 191-nt fragment contains both aptamer and expression platform, and we conclude that the ppe2 switch presents as a very compact, translational 'OFF' switch, potentially controlling the expression of uPPE2 in addition to ppe2 and cobQU .
To assess whether uPPE2 is the first regulatory target in the ppe2 operon, we made separate in-frame LacZ fusions of PPE2 and uPPE2, which included the upstream riboswitch and 3 codons of uPPE2 for the uPPE2'-lacZ construct and 13 codons of PPE2 for PPE2'-lacZ , to incorporate the PPE motif located at residues 10-12 of PPE2 (Figure 6 A, B).The constructs were transformed into M. smegmatis wildtype and cobK and subsequent β-gal assays indicated that uPPE2'-LacZ expression was higher than that of PPE2'-LacZ (Figure 6 D).The partial conservation together with the high level of expression suggested that uPPE2 was functional and the first target in the ppe2 operon.

Expression of PPE2 depends on translation of the upstream ORF
Hundreds of adjacent ORFs in M. tuberculosis have been found to share a 4-nt N UGA overlap between stop and start codons, which may affect translation of the downstream ORF ( 13 ,59 ).As mentioned above, the annotated PPE2 ORF lacks a SD motif, but it shares an A UGA stop / start overlap with the out-of-frame uPPE2 uORF (Figure 6 A).We therefore hypothesised that translation of PPE2 might depend on translation of this uORF.To ascertain if this were the case, we introduced mutations that prevented translation initiation of uPPE2 in the fully-leadered PPE2'-LacZ fusion.In one construct, we eliminated the uPPE2 SD motif by replacing the purines with their complementary pyrimidines (uPPE2 noSD ); in the other, we changed the uPPE2 AUG start codon to the non-start CUG (uPPE2 A189C ) (Figure 6 B).Both uPPE2 noSD and uPPE2 A189C mutants exhibited significantly severely diminished PPE2'-LacZ expression (Figure 6 D), suggesting that expression of PPE2 is strictly dependent on uORF translation.This may be due to Rho-dependent termination of transcription, as rho-binding sites become exposed in the absence of uPPE2 translation.Alternatively, it could be the result of translational coupling between the uPPE2 and PPE2 ORFs or a combination of the two.
To dissect the mechanism underlying this translational dependence, we first eliminated the native stop codon in the uPPE2-PPE2'-LacZ reporter construct, such that the uORF was extended with 63 codons until the next stop, located in lacZ (uPPE2 (nostop) -PPE2'-LacZ).We subsequently introduced new stop codons 3 and 7 codons upstream (early stop) or 3 and 6 codons downstream (late stop) of the PPE2 start codon (Figure 6 C).All constructs were expressed in M. smegmatis (wildtype and cobK ) and β-gal activity determined.The results indicated that early / premature stops led to a gradual reduction in PPE2'-LacZ expression and B 12 -sensing (Figure 6 D).Conversely, introducing the new stop codons downstream of the PPE2 start codon led to a more dramatic reduction in PPE2'-LacZ expression and B 12 sensing with the uPPE2 (nostop) mutant exhibiting activity in the same range as the uPPE2 noSD and uPPE2 A189C mutants (Figure 6 D).This suggests that Rho-dependent termination did not account for the reduced expression and moreover, that a tight stop / start overlap and a forward movement of the ribosome are necessary for efficient expression of the downstream ORF.

Translation of PPE2 proceeds primarily via termination-reinitiation
Translational coupling between overlapping ORFs could proceed via a T ermination-Reinitiation (T eRe) mechanism leading to production of two separate polypeptides ( 13 ,59 ), or via stop codon suppression combined with a frameshift without peptide release, which should generate a large fusion protein encoded by the two ORFs ( 71 ,72 ).To determine which of these scenarios applied, we added an N-terminal FLAGtag to uPPE2 in the uPPE2-PPE2'-LacZ reporter fusion (Figure 7 A).If TeRe were taking place, we would expect a small, FLAG-tagged uORF of 6.3 kD and an un-tagged PPE2'-LacZ fusion of 114 kD.However, if stop codon suppression were taking place, we would expect a large, FLAG-tagged uPPE2-PPE2'-LacZ fusion of 120 kD.In addition, we included an N-terminal FLAG-tag of the uPPE2 nostop -PPE2'-LacZ mutant, which should produce a larger ( ∼13 kDa) FLAG-uORF product, while the fate and nature of PPE2 and LacZ remained unknown.
All constructs were expressed in wildtype M. smegmatis and cell extracts from FLAG-tagged and isogenic, non-FLAG-tagged reporter strains were prepared.The cell extracts were first enriched using anti-FLAG pulldowns (see Materials and Methods), and the purified extracts were separated by SDS-PAGE followed by either Coomassie staining or western blotting.
Coomassie staining of gels suggested a high degree of purification in eluates with few, faint signals mostly between 25 and 70 kD compared to input, flowthrough and bead retained fractions (Figure 7 B and Supplementary Figure S7 ).Western blotting using anti-FLAG antibodies indicated the FLAG-specific enrichment of two main signals outside this range > 100 kDa and < 15 kDa (Figure 7 B, lanes E1 and E3).These correspond roughly in size to the FLAG-uPPE2-PPE2'-LacZ fusion and the FLAG-uORF, although the potential FLAG-uORF exhibited aberrant mobility, which we ascribe to its C-terminal poly-proline stretch (Figure 7 B, lane E1; Supplementary Figure S7 ).This suggests that the uORF is mainly expressed as a separate peptide, supporting the notion of a TeRe mechanism.In addition, we observed a slightly fainter signal of ∼120 kDa, corresponding in size to the FLAG-uPPE2-PPE2'-LacZ fusion, suggesting that in a few cases, a frameshift occurred and the fusion was made.
Eliminating the native uORF stop codon (FLAG-uPPE2 nostop -PPE2'-LacZ) resulted in further reduced mobility of the FLAG-uORF signal, which had an expected size of ∼13 kDa and harboured an additional polyproline stretch compared to the wildtype uORF.In this case, we observed two signals corresponding to ∼18 kD and ∼13 kD, respectively (Figure 7 B, lane E3).Given the aberrant mobility of the wildtype uORF and the additional poly-proline stretch in the uPPE2 nostop construct, we assume that the ∼18 kD signal corresponds to the tagged uORF, while the 13 kD signal is likely a degradation product.Together, the two signals were substantially fainter than that of the wildtype FLAG-uORF, suggesting that either less was made or more was degraded, or both (Figure 7 B, lanes E1 versus E3).A signal corresponding in size to FLAG-uPPE2-PPE2'-LacZ was marginally stronger in the uPPE2 nostop construct (Figure 7 B, lane E3), suggesting that eliminating the stop codon resulted in similar or perhaps slightly increased amounts of a large, FLAG-tagged fusion protein in line with the reduced amounts of uORF.The β-gal assays (Figure 6 D) had indicated very little functional LacZ was made in the uPPE2 nostop strain; yet, of the three possible readings frames, only the one encoding lacZ was large enough to produce a protein of this size.
To resolve this conundrum, we repeated the western blot on raw and FLAG-purified cell extracts using anti-LacZ antibodies.The result, shown in Figure 7 C, indicated that LacZ could only be detected in the raw extracts, while there was no detectable signal in the FLAG-purified fractions.Given that the FLAG-enriched fractions corresponded to approximately five times as much cell extract as the input fractions, we conclude that the vast majority of LacZ produced was un-tagged, i.e. generated from TeRe.This is in agreement with the finding that the FLAG-uORF displayed the strongest signal (Figure 7 B), and further supporting the notion that TeRe is the predominant mechanism behind PPE2'-LacZ expression.The uPPE2 nostop mutant did not result in a LacZ-specific signal in the unpurified fractions, suggesting that although there seems to be more of a FLAG-tagged fusion of the anticipated size (Figure 7 B, lane E3), substantially less LacZ is made overall, which is in agreement with the β-gal activity shown in Figure 6 D.
As a final control, we compared the β-gal activities of the fusions with and without the FLAG-tags.The results supported the finding that regardless of the tag, the wildtype AUGA context led to higher levels of LacZ, seen as higher β-gal activity (Figure 7 D).However, to our surprise, we found that in both wildtype (AUGA) and uPPE2 nostop context, addition of the FLAG-tag significantly enhanced β-gal activity (Figure 7 D), suggesting that the tag had a stabilizing effect on either RNA or protein, or both.
In summary, PPE2 expression depends on translation of uPPE2, which acts as a landing pad for the ribosomes.Translation of PPE2 proceeds mainly via a TeRe mechanism, but in a few cases the UGA stop codon is suppressed and a fusion protein is made.Adding a FLAG-tag led to increased β-gal activity, possibly due to the stabilization of RNA, protein or both.

Discussion
In the current study, we have combined in-line probing, structure prediction, and biochemical and genetic approaches to compare the gene expression control mechanisms by two riboswitches in M. tuberculosis .Both switches are B 12 -sensing 'OFF' switches, in agreement with previous observations for the metE switch and on B 12 riboswitches in general ( 21 , 26 , 30 , 44 , 45 , 53 , 54 , 73 , 74 ).Both riboswitches operate via B 12 -dependent masking and unmasking of the SD sequence, and in the case of metE this is accompanied by a massive reduction in mRNA levels, which is likely a result of Rho-dependent termination potentially in combination with mRNA degradation ( 13 ).The downregulation of ppe2 mRNA was less pronounced.However, it is worth noting that the ppe2 Apo-form starts with a stem −loop, which may increase mRNA stability compared to the ligand-bound form, which has several unpaired 5 nucleotides ( 60 ).
Both riboswitches displayed features typical of B 12 riboswitches such as the B 12 box and kissing loop (KL), but there were also some unique features in both switches.For example, while the L5 half of the KL was identical in the two riboswitches, the interacting L13 differed slightly, although still providing similar base pairing.Moreover, the metE L13 overlapped with the proposed ααTIR, while ppe2 L13 was flanked by the αTIR and TIR (Figures 3 and 5 ).Finally, the metE aptamer domain and the expression platform presented as two distinct domains, where the ααTIR was located well downstream of the B 12 box and separated from the αTIR by ∼60 nt (Figure 3 ).This contrasted with the ppe2 switch, where the overall size and distance between individual elements were much smaller.The ααTIR of the ppe2 switch was located earlier in the switch in the P6 / P7 extension, i.e. upstream of the B 12 box, indicating a substantial overlap between the aptamer domain and the expression platform.Moreover, the αTIR was located only ∼20 nt further downstream of the ααTIR (Figure 5 ).
In addition to the proposed structures, we demonstrate the presence of atypical and distinct regulatory uORF-related features that contribute to downstream gene expression in the two operons.uMetE2 is encoded within the P9-P10 extension of the aptamer upstream of the conserved B 12 box.Mutating the uMetE2 start codon to eliminate translation significantly increased MetE expression, suggesting that uMetE2 is translated in situ , leading to suppression of MetE expression.Presumably, translation of uMetE2 and ligand-induced folding are mutually exclusive, but our results suggested that B 12 -sensing remains.Whether the suppression is a cis or trans effect, resulting from the peptide, remains unknown, but the fact that uMetE2 shows limited conservation ( Supplementary Figure S4 ) suggests that its function may be related to specific lifestyles of some members of the M. tuberculosis complex.
Another rather unexpected feature was the indispensable relay linking the B 12 aptamer and the ppe2-cobQ(U) operon via uPPE2, i.e. that PPE2 was expressed in a uPPE2-dependent manner.In the majority of cases, this happened via TeRe at the uPPE2-PPE2 AUGA overlap as evidenced by (i) a large amount of a FLAG-uORF protein and a much smaller amount of a tagged fusion protein; (ii) no detectable LacZ in the FLAGpurified fraction using anti-LacZ antibodies; and (iii) significantly reduced β−gal activity when this AUGA overlap is disrupted.The uPPE2 nostop mutant (FLAG-uPPE2 nostop -PPE2'-LacZ) also produced a fusion protein, which again did not give rise to a detectable signal using anti-LacZ antibodies.Scrutiny of the region revealed that the lacZ -encoding frame is the only frame that could generate a product of that size.Moreover, the next natural stop codon in the uPPE2 nostop mutant (63 codons downstream) was in an AUGA context.While this stop codon led to termination, as judged by the anti-FLAG western blot, it remains to be seen whether re-initiation and / or frameshiftfusion are enabled in this context.The absence of a LacZspecific signal in this mutant suggests that re-initiation may not be as efficient as seen in the wildtype context.Additionally, if this AUGA does allow for frameshift-fusion, the resulting LacZ protein would be devoid of over 50 N-terminal residues, which are critical for function ( 75 ,76 ).The increased activity of FLAG-tagged constructs versus their un-tagged counterparts is likely due to stabilization of RNA and / or protein.
To our knowledge, this is the first demonstration of stop codon suppression and translational coupling of a gene pair with overlapping stop and start codons in M. tuberculosis .We are currently investigating how the sequence context surrounding the AUGA stop-start overlap might influence this mechanism and how the N-terminal extension might influence PPE2 function.
We argue that the ppe2 switch qualifies as a new exceptional member of Class IIb B 12 riboswitches based on the following considerations.Firstly, the ppe2 switch does not conform to Class I since it has no P8-P12 extension.Secondly, even though it has the GGAA junctional motif at J3 / 4 similar to previously reported Class IIb switches, it lacks a corresponding UCU motif in the opposite J6 / 3 junction ( 54 ).Moreover, the ppe2 switch is neither unable to bind AdoB 12 , similar to Class IIa switches, nor AdoB 12 selective, similar to other Class IIb switches.Interestingly, positions C48 −G50, which form the L5 of the ppe2 switch, were protected from cleavage only in the presence of AdoB 12 ( Supplementary Figure S2 ), implying that in this switch, the KL might be stabilized only by binding AdoB 12 and not MeB 12 , HyB 12 or CNB 12 .Further structural analysis, such as crystallization is required to obtain the full and true images of these switches in their ligand-bound and -unbound constellations ( 53 , 73 , 77 ).
It has been suggested that PE / PPE genes inserted and expanded at different genomic loci ( 48 ).Therefore, one can speculate that ppe2 'invaded' the locus of the cobalamin biosynthetic genes cobQ / U, which were initially under the regulatory control of an ancestral B 12 -riboswitch thereby giving rise to this extra and unconventional regulatory mechanism.This notion is supported to some extent by similar scenarios in the Mbox-pe20 -mgtC locus and the recently identified PEcontaining uORF in the glyA2 locus ( 13 ).
The existence of B 12 riboswitch classes with varying ligand selectivities within the same cell raises some interesting questions.It is assumed that M. tuberculosis relies on host-derived B 12 , but whether this is obtained by the pathogen as AdoB 12 or MeB 12 remains unknown.Moreover, nothing is known about potential pathways involved in converting the scavenged B 12 isoform or precursor to the relevant riboswitch ligand or cofactor type.Recent evidence suggests that host B 12 acted as a key factor in shaping the evolution of human-and animaladapted M tuberculosis lineages, which promotes the notion of B 12 involvement in virulence and host-pathogen cross-talk ( 40 ).Therefore, understanding when and how M. tuberculosis utilizes its multi-layered riboswitch complexity to sense and adapt to a range of host niches will not only shed light on how habitats shape genomes, but also provide a deeper understanding of general pathogen adaptation during the course of infection.These unusual molecular mechanisms are yet another example of how M. tuberculosis challenges our understanding of gene expression control.

Figure 1 .
Figure 1.Chemical str uct ure of cobalamin.The different functional groups ('R' groups) that can occupy the β-axial positions are shown to the right of the str uct ure.Dimeth ylbenzimidaz ole (DMB) is highlighted on the str uct ure.

Figure 2 .
Figure 2. B 12 -dependent changes in metE and ppe2 transcripts.( A , B ) Schematic of metE and ppe2 operons.The full-length of metE and ppe2 leaders are 365 and 292 nt, respectively; ORF lengths (not drawn to scale) are metE : 2280 nt, ppe2 : 1671 nt, cobQ : 1485, cobU : 525 nt.The precise locations of dominant transcription termination sites (TTS) and their relative coverage according to ( 13 ) are indicated.Approximate locations of qRT-PCR amplicons for metE / ppe2 leaders, coding regions and ppe2-cobQ junction are indicated as double-ended arrows; exact locations are listed in Materials and Methods.The metE 5 amplicon covers a region upstream of the observed dominant TTS peaks.( C , D ) qRT-PCR of amplicons indicated in (A) and (B) before and 1 h post AdoB 12 addition; note different scales.Data represents mean ± standard deviation of at least three biological replicates, P -values t -test: * P < 0.05; ** P < 0.01.(E, F) Northern blots (vertical, 8% acrylamide) showing changes in short transcripts derived from metE ( E ) and ppe2 ( F ) leaders before and after AdoB 12 incubation with probes hybridizing to 5 end of mRNA (orange boxes labelled NP in A and B, above).Arrows right of blots indicate signals corresponding to dominant TTS peaks in A and B, abo v e. G. Horiz ontal, 1% agarose northern blot using the same metE probe as in (E).Arrow indicates a 326-nt transcript corresponding to the dominant, terminated signal in (E).

Figure 3 .
Figure 3. In-line probing of the metE riboswitch.( A ) Clea v age pattern of the metE riboswitch RNA o v er a concentration gradient of AdoB 12 (lanes, 1: 2 mM; 2: 1 mM; 3: 0.5 mM; 4: 0.1 mM; 6: 10 μM; 6: 1 μM; 7: 0.1 μM; 8: 10 nM; 9: 1 nM; 10: 0.1 nM.T1 -RNase T1; OH − -alkaline digest; NLno-ligand control; NR -no-reaction.Strongly modified positions are indicated on the right side of the gel (cropped where there was no difference ± ligand).T1-derived G positions indicated on the left.( B ) Predicted str uct ure of the metE switch without ligand.The translation initiation region (TIR) including the Shine-Dalgarno (SD) sequence is highlighted in green, while the αTIR and ααTIR sequences are highlighted in red and blue, respectively.uMetE2 and uMetE4 are described in the text.( C ) Predicted str uct ure of AdoB 12 -bound metE switch.Paired regions in the aptamer are labelled sequentially (P0-P11).The conserved 'B 12 box' (G1 99-C21 5) matc hes the consensus f or this element ( 68 ).B ases of L5 that could potentially form a kissing loop (KL) with L13 are highlighted in purple and the base pairs between the loops are shown in the inset.The region of hypercleavage at G231-C233 is highlighted in grey.Other coloured features are the same as in panel B. SL -stem-loop.( D ) Beta-galactosidase assays of reporters expressed in M. smegmatis wildtype strains expressing translational LacZ reporters fused to the native or mutant riboswitch bearing nucleotide substitutions in L5 (92-94AAA → CCC), complementary substitutions in L13 (258-259GG → UU), or both.Data represents mean ± standard deviation of at least three biological replicates.P -values one-way ANO V A: *** P < 0.001; **** P < 0.0001.

Figure 4 .
Figure 4. Translation of uMetE modifies MetE expression.( A ) Outline of the metE leader including control elements (TIR, αTIR and ααTIR) identified in Figure 3 , the potentially translated leader-encoded peptides, uMetE2 and uMetE4 and location of the U124C mutation.( B ) Left, outline of reporter constructs for the validation of the translational expression platform using gradual 5 extensions; numbers indicate 5 ends of each construct; right, β-gal activities of the reporter fusions shown in Miller Units (MU) / mg protein; note the log-scale.ctrl: no expression control.Data represents mean ± standard deviation of at least three biological replicates.p -values One-way ANO V A: **** P < 0.0 0 01.( C ) Assessing the potential for translation of uMetE2 and uMetE4 and the effect of uMetE2 translation.The expression of MetE'-LacZ was measured in the context of wildtype umetE2 , non-translated umetE2 (U124C) or umetE2 ( 115-188), all expressed in either wildtype M. smegmatis (B 12 producing) or cobK (B 12 deficient).Data represents mean ± standard deviation of at least three biological replicates, P -values t -test: * P < 0.05; **** P < 0.0 0 01.

Figure 5 .
Figure 5. Str uct ure of ppe2 riboswitch.( A ) T he clea v age pat tern of the ppe2 switc h o v er a concentration gradient of A doB 12 .Strongly modulated positions are indicated by labels on the right side of the gel.G positions are shown on the left.NR -no-reaction control; T1 -RNase T1 ladder; OH −alkaline digest ladder; NL -no-ligand control; lanes, 1: 4 mM; 2: 2 mM; 3: 1 mM; 4: 0.5 mM; 5: 0.1 mM; 6: 10 μM; 7: 1 μM; 8: 100 nM; 9: 10 nM; 10: 1 nM.( B ) Predicted secondary str uct ure of the Apo-form of the ppe2 switch.The translation initiation region (TIR) including the SD motif is highlighted in green, while the αTIR and ααTIR sequences are highlighted in red and blue, respectively.( C ) AdoB 12 -bound ppe2 switch.Paired regions in the aptamer are labelled sequentially from P1 to P11.The 'B 12 box' located G1 21-G1 35 matc hes the consensus ( 68 ).T he region of h yperclea v age at A1 31-C1 34 is highlighted in grey.Bases of L5 that could potentially form a kissing loop (KL) with L13 are highlighted in purple and the base pairs between them are shown in the inset.