A second RNA-binding protein is essential for ethanol PNAS PLUS tolerance provided by the bacterial OLE ribonucleoprotein complex

Kimberly A. Harrisa,b,1, Zhiyuan Zhoub,1, Michelle L. Petersb, Sarah G. Wilkinsb, and Ronald R. Breakera,b,c,2

aHoward Hughes Medical Institute, Yale University, New Haven, CT 06520-8103; bDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103; and cDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8103

Contributed by Ronald R. Breaker, May 24, 2018 (sent for review February 22, 2018; reviewed by Venkat Gopalan and Karen Wassarman) OLE (ornate, large, extremophilic) RNAs comprise a class of structured isms that harbor ole (2, 6, 7), suggesting that the function of OapA noncoding RNAs (ncRNAs) found in many extremophilic bacteria is intimately tied to the function of OLE RNA. OapA is a 21-kDa species. OLE RNAs constitute one of the longest and most wide- protein predicted to have four transmembrane helices containing spread bacterial ncRNA classes whose major biochemical function several conserved amino acid motifs (Fig. 2A). We have previously remains unknown. In the Gram-positive alkaliphile Bacillus halo- shown that OapA from the bacterium Bacillus halodurans binds durans, OLE RNA is abundant, and localizes to the cell membrane OLE RNA in vitro, and that this RNA localizes to cell membranes by association with the transmembrane OLE-associated protein in an OapA-dependent fashion (6). Specifically, the protein ap- called OapA (formerly OAP). These characteristics, along with the pears to exhibit at least a 2:1 (OapA:OLE RNA) binding in- well-conserved sequence and structural features of OLE RNAs, sug- teraction with a KD of ∼50 nM. The protein requires portions of gest that the OLE ribonucleoprotein (RNP) complex performs impor- the P3 and P3.1 stems (Fig. 1A) for maximal binding affinity (6). tant biological functions. B. halodurans strains lacking OLE RNA Because OLE RNAs are found in many species of extrem- (Δole)orOapA(ΔoapA) are less tolerant of cold (20 °C) and short- ophilic bacteria and localize to the cell membrane, it was pre- chain alcohols (e.g., ethanol). Here, we describe the effects of a viously speculated that the OLE ribonucleoprotein (RNP) mutant OapA (called PM1) that more strongly inhibits growth under complex might play a role in a pathway protecting these species cold or ethanol stress compared with strains lacking the oapA gene, from their extreme environments. Notably, Δole, ΔoapA, and even when wild-type OapA is present. This dominant-negative effect Δole-oapA strains of B. halodurans have phenotypes consistent of PM1 is reversed by mutations that render OLE RNA nonfunctional. with membrane stress. These strains exhibit growth defects when This finding demonstrates that the deleterious PM1 phenotype re- exposed to media containing short-chain alcohols, such as eth- quires an intact RNP complex, and suggests that the complex has anol, or when grown in cold temperatures (7). one or more additional undiscovered components. A genetic screen Analysis of the transcriptome of B. halodurans revealed that uncovered PM1 phenotype suppressor mutations in the ybzG gene, OLE RNA represents one of the most abundant transcripts which codes for a putative RNA-binding protein of unknown biolog- under normal growth conditions (6, 7). These RNAs are rela- ical function. We observe that YbzG protein (also called OapB) selec- tively stable for a bacterial transcript, with a half-life approaching tively binds OLE RNA in vitro, whereas a mutant version of the 3 h (7). Interestingly, OLE RNA abundance increases fivefold protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an B. important component of the functional OLE RNP complex in Significance halodurans. Although large bacterial noncoding RNAs (ncRNAs) are rare, alcohol toxicity | Bacillus halodurans | noncoding RNA | OLE RNA | YbzG those whose functions have been experimentally established perform fundamental roles in genetic information transfer, oncoding RNAs (ncRNAs) are involved in numerous, di- RNA processing, and protein production and localization. OLE Nverse cellular processes in all domains of life (1). In bacteria, (ornate, large, extremophilic) RNAs represent one of the most bioinformatic search strategies that use sequence and structure widespread and well-conserved classes of bacterial ncRNAs conservation have revealed several possible classes of novel large whose activities remain unknown. We have identified muta- ncRNAs whose functions remain unknown (2–5). Each of these tions in an OLE-associated protein (OapA), an essential partner classes is remarkable for its size, complex structural features, and for OLE RNA, which cause more severe inhibition of host bac- sequence conservation. Moreover, these classes are distinct from terial growth under cold or ethanol stress conditions compared all other known RNA classes, suggesting that their biological and with knockout strains lacking OLE or OapA. A genetic screen biochemical roles are likely to be novel. using a bacterial strain carrying the mutant OapA protein Of these, OLE (ornate, large, extremophilic) RNAs (Fig. 1A), revealed another protein partner that also forms a complex named for their ornate structure, large size, and prevalence in with OLE RNA, and is essential for the biological function of extremophilic bacteria, represent one of the longest ncRNA this mysterious bacterial ncRNA. classes to be discovered in bacteria (2). OLE RNAs are typically ∼600 nucleotides long, have highly conserved and complex Author contributions: K.A.H., Z.Z., and R.R.B. designed research; K.A.H., Z.Z., M.L.P., and structures, and are widespread in anaerobic, Gram-positive bac- S.G.W. performed research; K.A.H., Z.Z., M.L.P., S.G.W., and R.R.B. analyzed data; and MICROBIOLOGY teria. To date, we have identified 795 nonidentical representatives K.A.H. and R.R.B. wrote the paper. from various species in the Firmicutes phylum. Intriguingly, OLE Reviewers: V.G., The Ohio State University; and K.W., University of Wisconsin–Madison. RNAs represent the most widespread and most conserved bac- The authors declare no conflict of interest. terial ncRNA class whose function remains unknown. Published under the PNAS license. The ole gene commonly resides in tandem with a gene encoding 1K.A.H. and Z.Z. contributed equally to this work. a transmembrane protein of unknown function (Fig. 1B). This 2To whom correspondence should be addressed. Email: [email protected]. protein was originally named OAP (for “OLE-associated protein”) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. but hereafter we call this protein OapA for reasons described 1073/pnas.1803191115/-/DCSupplemental. below. The presence of the oapA gene is exclusive to the organ- Published online June 18, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1803191115 PNAS | vol. 115 | no. 27 | E6319–E6328 Downloaded by guest on October 1, 2021 GC A G C G A P9.3 A A A G U G A 0-27 0-23 C A nt nt R A U C Y C Y P14a P9.2 G R G G P14 R U C RY Y G Y R Y U Y A G P13 R R G A Y Y P14.1 Y G A R GG A P15 C Y R Y Y G A R Y G G R G R C R C C A A C U A A G Y A U R Y G G P12.2 P9.1 U A R Y R Y G C P8 R G U A C U G R U G C A U A A A C A Key Y R G A R R C G P7 R Y U G A G C base pair annotations Y U A U A R R A AC P12.1 Y R R C G C G P9 G Y covarying mutations Y G C G Y Y A G U A A compatible mutations C GUG C C U G U A R A no mutations observed R RRR R R R G Y CA G GG U A A Y Y YY Y Y U U connectors G U P4a A U G P3.1 A U G C R A G G C A zero length Y G Y GR Y R R Y variable length G A R Y C U R Y variable-length hairpin Y G G P4 C G R A A Y A A C G P12 Y U G U G U G nucleotide present A U A A C A 97% 90% 75% 50% C G Y P5 A A P11 A C G P6 G C A U G Y R G C AA G C nucleotide identity G G U P10 R Y G U R Y R N 97% N 90% N 75% C C G R Y Y R Y P2 Y R R U YUG CUAGR C U G U R: A/G Y: C/U R R R R Y Y U R C R R C CUAG Y G A C G Y C R Y R Y G R Y U U U A Y R P3 A G Y P6.1 U Y R Y Y G R U A R A A G C P5.1 C Y G U G G G Y P1 Y Y R G C A G 5′ C C C DNA repair isoprenoid B biosynthesis nusB folD xseA xseB ispA ole oapA dxs yqxC nadF argR recN 0

50 proportion (%)

100 -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 gene position relative to ole

Fig. 1. OLE RNA sequence, structure, and genome context. (A) Updated consensus sequence and secondary structure model for OLE RNAs based on 795 unique representatives from bacterial genomic and metagenomic DNA sequences. (B) Conservation of the genomic region carrying the gene for OLE RNA. The most common gene arrangement adjacent to ole is depicted at the top of the bar graph. The colored section of each bar represents the proportion of all representatives that carry the identified gene in this position relative to ole. Gray represents all other genes found at that position.

when cells are exposed to ethanol (7). Thus, OLE RNAs are up- uncovered a mutant OapA (called protein mutant 1 or PM1) that regulated under particular cellular stresses, and might possess a causes a more severe version of the same phenotype resulting function that helps cells respond to these extreme conditions. from OLE RNA or OapA knockouts. Moreover, the strain To further investigate the biological and biochemical roles of producing PM1 OapA exhibits a dominant-negative phenotype, the OLE RNP complex, herein we examined the growth of wherein cells are exceptionally sensitive to cold and ethanol B. halodurans strains carrying mutations in either OLE RNA or stresses even when WT OapA is also present. A series of OLE OapA under ethanol or cold stress conditions. These studies RNA mutations were examined in cells carrying PM1 OapA,

E6320 | www.pnas.org/cgi/doi/10.1073/pnas.1803191115 Harris et al. Downloaded by guest on October 1, 2021 PNAS PLUS L G Y G vitro. These findings demonstrate that the OLE RNP complex A B. halodurans OapA Q E L N 130 requires YbzG to form its active complex. Therefore, we rec- L S M G A S ommend naming the OLE-associated proteins “OapA” (for- F V L G “ ” T 30 S 120 Q Q extracellular merly OAP) and OapB (YbzG), and will use these names in S W I V 40 V A V V I 110 A F Q this report. S G Y L I F I G M A T V S L A A L L V G T A T F L 140 V 20 I S S Results and Discussion I T L V L V G I V A V V membrane I A V L I G D I S T L A Updated Sequence and Consensus Model for OLE RNAs. Only 15 ex- A G V 150 T L 50 D F D V I K G G 10 W T GxxxGxxxG amples of OLE RNA from fully sequenced bacterial genomes

I DxxxD S I N 60 V G F C N G I A K A N K were described in the initial report of the discovery of this ncRNA 100 G 1234A A T intracellular class (2). Subsequently, the consensus sequence and structural L T F L KxxxK I N model was revised based on ∼200 unique OLE RNA represen- S A R A 160 N F K M L M A D I tatives (7). We have now uncovered a total of 795 unique exam- K P D A E 180 E N ples, mostly due to the tremendous expansion of bacterial sequence E 90 N H conservation E 190 G K R S databases. All examples found in genomic DNA sequences from L K P T T X invariant F K phylogenetically assigned bacterial species (as opposed to meta- F I A 70 Y K X high H A I genomic sequences) are predominantly restricted to the Firmicutes F K A I X moderate V V E phylum, and are divided among three classes: Bacilli, Clostridia, M A Y K K X low Q V G and Tissierellia. Indeed, all newly found OLE RNAs are in species A Q 170 N 200 X none K S of bacteria characterized as extremophilic, spore-forming, Gram- S 80 R K E A positive, and anaerobic (either facultative or obligate). R L G K Even with the addition of newly uncovered sequences, the L G B Q Q predicted secondary structure for OLE RNAs reported pre- V V I A F Q viously (7) remains extraordinarily well conserved, and includes T Y S L PM2 A V numerous base-paired substructures and multistem junctions 1234 G T A T F L (G to A) S S G I V A V V (Fig. 1A). Covariation between nucleotides that results in re- D I S T L A tention of Watson–Crick base-pairing between nucleotides is I G G V PM1 D V K G used as strong evidence for the formation of stem regions. OapA (D to A) F C N PM3 G I A N 34(K to A) K Strikingly, about half of the nucleotides are conserved in more A T than 75% of the representatives. This high conservation of nu- F L R A cleotide identities and secondary structure indicate that OLE RNAs form an intricate tertiary structure that is critical for its C 1.0 5% ethanol biochemical function.

Genome Contexts of OLE RNA Genes Between Distal Bacterial Species

600 Are Similar. The ole gene of B. halodurans is typically found in a 0.5 large operon (2), although a separate promoter was also iden- OD tified immediately upstream of the ole gene in this organism (8). normalized This distinctive genetic context is similar even between distantly 0.0 related bacterial species (Fig. 1B). In over 96% of the organisms plasmid empty WT PM1 PM2 PM3 PM1 that carry OLE RNA, its gene is located directly upstream of oapA, strain WT ∆ole-oapA WT the gene coding for the OLE-associated protein OapA. Typically, these two genes are immediately flanked by the isoprenoid bio- Fig. 2. Analysis of the OLE-associated protein OapA. (A) Amino acid se- synthesis genes ispA (upstream) and dxs (downstream). In many quence and predicted topology of B. halodurans OapA. The sequence con- archaeal species, isoprenoid compounds are produced to modulate servation reflects both the conservation of the identity and physicochemical the fluidity and integrity of cell membranes (9), which is likely also properties of the amino acid at each position (see Materials and Methods for a major challenge for bacteria that live under extreme environmental details). Black circles represent 100% conservation of amino acid identity conditions. among all 735 unique OapA sequences examined. (B) Location of OapA Additional genes, predicted to be involved in DNA repair and mutations examined in this study. (C) Growth of each OapA mutant strain after 48 h incubation in media containing 5% ethanol. Each bar represents other activities, are arranged in a common order on both sides of the average of at least three experiments normalized to WT, and error bars this core gene arrangement (Fig. 1B). In roughly half of the rep- are the SEM. resentatives noted above, ole and oapA are located in a genomic region with the following gene order: nusB, folD, xseA, xseB, ispA, dxs, yqxC, nadF, argR,andrecN. However, in the analogous region and were found to rescue the dominant-negative phenotype. present in B. halodurans (the organism examined in the present These findings suggest that PM1 OapA requires a functional study), nadF is absent. Otherwise, the remaining genes are present OLE RNA for its negative effect, and that the OLE RNP in the order listed. Previous reverse-transcription and PCR ex- complex requires one or more additional components to form periments in B. halodurans revealed that the 11 genes in this cluster

its active state. indeed can yield a single transcriptional unit (2), despite the ac- MICROBIOLOGY We conducted a genetic screen with a B. halodurans strain tivity of the promoter immediately upstream of the ole gene (7, 8). carrying the oapA-pm1 gene to search for additional components This common assembly of genes flanking ole and oapA among of the RNP complex. It was speculated that extragenic sup- diverse bacterial species presumably is relevant to the biological pressors for the extreme cold- and ethanol-sensitivity phenotypes function of the RNA and protein products. These flanking genes caused by PM1 could accrue in genes that were essential for are involved in several major but distinct cellular processes. As formation of the complete OLE RNP complex structure. From noted above, IspA (geranyltranstransferase) and Dxs (1-deoxy-D- this genetic screen, a gene for a protein of unknown function, xylulose-5-phosphate synthase) are involved in the biosynthesis of YbzG (AYT26_RS00915), was identified. B. halodurans YbzG isoprenoids (10). XseA (exoDNase VII large subunit), XseB was then shown to specifically bind B. halodurans OLE RNA in (exoDNase VII small subunit), and RecN (recombination protein N)

Harris et al. PNAS | vol. 115 | no. 27 | E6321 Downloaded by guest on October 1, 2021 function in DNA repair (11, 12). FolD (bifunctional methylenete- K157) are consistent with a KxxxK motif, which has been shown to trahydrofolate dehydrogenase and methenyltetrahydrofolate cyclo- bind RNA in other proteins (24). ) and NadF (ATP-dependent NAD kinase) are related to To investigate the functions of these conserved residues in coenzyme metabolism (13, 14). Both NusB (transcription OapA, alanine substitution mutations were made to each key antitermination protein), required for transcription of rRNA residue (SI Appendix, Table S1). Specifically, three mutant B. genes (15), and YqxC (rRNA methylase), predicted to meth- halodurans oapA constructs were generated that encode trans- ylate rRNA due to the presence of S4 and FtsJ domains (16), lation products named PM1, PM2, and PM3. PM1 represents an are related to the production of ribosomes. Finally, ArgR (ar- OapA protein that carries D100A/D104A mutations in the ginine repressor) down-regulates arginine biosynthesis (17). DxxxD motif. Similarly, the OapA construct PM2 carries G103A/ Unfortunately, at this time we cannot draw a connection be- G107A/G111A mutations in the GxxxGxxxG motif, and the tween all of these genes to infer a specific biological process OapA construct PM3 carries K153A/K157A mutations in the that might involve the function of OLE RNA. KxxxK motif (Fig. 2B and SI Appendix, Table S1). Plasmid Moreover, no hypotheses have been derived from the remain- constructs carrying an unaltered ole gene plus one of the mutant ing ∼50% of neighboring genes because they vary widely and do versions of the oapA gene were transformed into a B. halodurans not exhibit a pattern indicative of a common biological process. It strain whose genome lacks ole and oapA (Δole-oapA). All OapA is possible that this genomic region carries genes related to diverse mutant strains exhibit reduced growth in the presence of 5% processes that all broadly respond to certain stress conditions. ethanol compared with WT B. halodurans cells (Fig. 2C). This However, the levels of mRNAs for genes in the B. halodurans ethanol-dependent reduction in cell growth caused by OapA operon do not change substantially during exposure to ethanol mutations is mostly analogous to that observed for B. halodurans stress compared with the increase in OLE RNA abundance, which strains wherein one or two components of the OLE RNP com- Δ Δ Δ is detected at five times the level compared with cultures without plex have been deleted ( ole, oapA, and ole-oapA) (7). For ethanol (7). Thus, the OLE RNP complex could function in- example, strains lacking genomic ole and oapA genes, but car- dependently from the other gene products in this operon, but rying WT ole and either PM2 or PM3 oapA genes on a plasmid contribute to the overall stress response through its involvement in exhibit reductions in cell growth in 5% ethanol (Fig. 2C). a biological process that is different from those influenced by the However, growth of the PM2 and PM3 strains is not readily classified as representative of the strain carrying only the geno- neighboring genes. Δ Interestingly, genes rarely associated with ole might still pro- mic ole-oapA deletion or the same strain rescued by delivery of vide valuable clues regarding the function of OLE RNA. For the WT ole and oapA genes on a plasmid. Thus, we cannot yet de- ∼4% of the ole representatives that reside outside the genomic termine if the strictly conserved amino acids in the GxxxGxxxG region described above, oapA still remains between ispA and dxs. and KxxxK motifs are critically relevant for ethanol resistance. Most importantly, the PM1 strain grew far worse than the In these instances, the displaced ole gene is often located directly knockout or other OapA mutant strains upon exposure to eth- upstream of a c-di-AMP riboswitch. Riboswitches for c-di-AMP anol (Fig. 2C). This same trend revealing the special sensitivity of frequently regulate genes related to cell wall metabolism and the the PM1 strain to ethanol exposure is also observed when the transport of ions and other osmoprotectants (18). Indeed, each number of viable cells is quantitated by colony forming units c-di-AMP riboswitch adjacent to a displaced ole gene is predicted − (CFU). Plots of the CFU μL 1 values (SI Appendix, Fig. S1A) for to regulate a downstream gene encoding an NlpC/P60 family WT cells and most other Δole-oapA strains remain relatively protein. Such proteins are known to function as cell wall hy- constant, despite the fact that their cultures increase in OD600 drolase (19, 20). This genomic arrangement of dis- (Fig. 2C). This suggests that cell division for these strains is placed ole genes again suggests that the OLE RNP complex counterbalanced by rapid cell death in 5% ethanol. However, might be related to the integrity of the barriers between the cell more than two orders-of-magnitude difference in the CFU contents and the environment, namely the functional status of density curve between the PM1 strain and all other strains is the cell wall or cell membrane. evident after 48-h incubation. These observations indicate that, for the PM1 strain, the production of new viable cells cannot OapA Mutations Cause Reduced Cell Growth Under Ethanol-Mediated offset ethanol-triggered cell death, which results in a rapid de- − Stresses. Our updated bioinformatics data also yielded a large cline in CFU μL 1 values. However, we have not yet determined increase in the number of nonidentical OapA sequences. We if ethanol stress decreases the rate of cell replication, increases identified 735 unique OapA representatives. The amino acid the rate of cell death, or both. sequence alignment revealed that approximately half of the po- The strong deleterious effect caused by PM1 indicates that sitions exhibit at least modest conservation (Fig. 2A). All of the D100 and D104 of the DxxxD motif are essential for the most strongly conserved amino acids reside in the putative trans- mechanism of the OLE RNP complex in a manner that is distinct membrane and intracellular regions, suggesting that the function from the PM2 and PM3 mutations, and thus also distinct even of OapA likely does not involve interactions with the peptidogly- from deletion of the entire oapA gene. This unusual effect is also can matrix of the cell wall or with extracellular molecules. observed when cells are cultured in the presence of other short- Six amino acids whose identities are strictly conserved reside chain alcohols (SI Appendix, Fig. S1B). However, the PM1 strain in or near transmembrane helices 3 and 4. Each of these invariant described above grows normally when the culture medium lacks residues, which presumably are essential for OapA function, added alcohols (SI Appendix, Fig. S1C). corresponds to known consensus protein motifs, as previously reported (6). Specifically, there are two aspartic acid residues The PM1 Strain Exhibits a Dominant-Negative Sensitivity to Ethanol (D100, D104) in a DxxxD motif (21) located in transmembrane and Cold. Strikingly, the PM1 construct yields a dominant- helix 3 (Fig. 2A). This sequence (or a similar peptide motif) is negative trait, whereby the mutant protein causes a deleterious present in some enzymes involved in the biosynthesis of certain effect on cells in the presence of ethanol even when they carry a isoprenoid compounds (21, 22). Also in helix 3, two strictly con- normal copy of the oapA gene. Specifically, WT B. halodurans served glycine residues (G103, G111) and another glycine that is cells that carry natural ole and oapA genes plus a plasmid car- not universally conserved (G107) appear to form a GxxxGxxxG rying both the unaltered ole gene and a mutated oapA-pm1 gene motif. Peptide sequences with this periodic “glycine zipper” ar- are adversely affected by 5% ethanol nearly as strongly as B. rangement are known to promote docking between helices (23). halodurans PM1 cells that lack the WT oapA gene (Fig. 2C). In Finally, at the base of helix 4, two invariant lysine residues (K153, these cells, both the WT and the PM1 versions of OapA should

E6322 | www.pnas.org/cgi/doi/10.1073/pnas.1803191115 Harris et al. Downloaded by guest on October 1, 2021 PNAS PLUS be present, but the WT protein is not able to overcome the A RM2 A A U G A deleterious effects of PM1. Similar results are observed when U A A A G A RM1 U G A 530 G A A A these cells are cultured at reduced temperature (20 °C) (SI Ap- U A U C C A C G A 520 C G P14a pendix, Fig. S2), which is another stress that was previously G G A C U A G U C 500 U C 510 C U G P14.1 Δ Δ G U G A known to more strongly affect the growth of ole, oap, and U G C U G G 540 C C U P14 U C Δ P13 G A G A C G ole-oapA cells (7). However, the strength of the deleterious G A A A C U U 560 C U 550 G GG PM1-mediated phenotype at 20 °C is somewhat diminished in C G P15 C 490 U G C G C G A G the presence of WT OapA compared with the results with P12.2 A UA C A C C U A RM3 U 480 C U A ethanol toxicity. G U A A G A G U G 570 G C These surprising findings suggest that the amino acid changes U A G C 580 in PM1 might cause OapA to have a deleterious interaction with another member of the pathway in which the OLE RNP complex functions. Although the severe PM1 phenotype could be due to a problem that is unrelated to the function of the RNP complex, this is unlikely to be the case based on our additional findings. First, neither WT cells carrying the PM1 construct nor Δole-oapA cells carrying the PM1 construct exhibit growth defects when grown under normal culture conditions (SI Appendix, Fig. S1B). This finding suggests that PM1 does not simply interfere with cell growth. Second, the same stress conditions that are necessary to RM4 UG A A A inhibit growth of mutants expressing defective OLE RNA or U U A G A C G 410 290 G C RM4 OapA protein are required for the strong lethal phenotype to A C G AG G 420 A U P11 U G A 400 A G C G C 430 GUGCCU U A A C A C become apparent. Indeed, PM1 appears to cause the same ethanol- AGAGAUGG UUG C UAGA CU Δ GGGCAC and cold-sensitivity phenotypes observed with ole-oapA cells to P5 80 A CCAUUCUU CAA UCUAG AG A G 50 U C U 30 UU become much worse. A A P3.1 C U P3 A GC A P2 These observations suggest that the strong distress caused by GC 40 PM1 might be due to the disruption of a factor associated with 5% ethanol the OapA component of the RNP complex. Moreover, this hy- B 1.0 pothetical OapA partner also most likely functions in the same biological pathway to yield stronger versions of the same phe- notypes observed for the Δole-oapA strain. Perhaps the normal status of this factor yields partial resistance to stresses, such as 600 ethanol and cold, whereas its disruption by the defective PM1 0.5

OapA causes a more severe response to these same stresses. OD normalized Mutations to OLE RNA also Can Reduce Cell Growth Under Ethanol Stress. To further evaluate the strong sensitivity phenotype caused by the PM1 OapA construct, we sought to establish the 0.0 importance of the OLE RNA to this effect. A series of OLE RM1 RM2 RM3 RM4 ole plasmid empty RNA mutants were constructed (SI Appendix, Table S1) and WT oapA oapA evaluated in bacterial strains carrying either the WT or PM1 OapA constructs. strain WT ∆ole ∆ole-oapA One RNA region we examined is the base-paired substruc- C tures P12 through P15 (Fig. 3A, Upper). The OLE RNA mutant 1 5% ethanol (RM1) construct includes two mutations that disrupt base pairs 0.5 within the P14 stem, whereas the RM2 construct carries the same mutations as RM1, but also includes two additional mutations 600

that completely restore P14 stem base-pairing. B. halodurans OD

Δ normalized ole-oapA cells transformed with a plasmid either carrying the 0.0 RM1 or RM2 ole gene variant plus a WT oapA gene were tested WT ∆ole RM1 RM2 RM3 RM4 ole for growth in the presence of 5% ethanol (Fig. 3B). The results plasmid PM1 oapA oapA reveal that destabilization of the P14 stem in the RM1 construct yields an OLE RNA sequence that fails to improve growth of strain ∆ole-oapA cells lacking a WT OLE RNA. In comparison, the RM2 con- struct that carries compensatory mutations to restore P14 base- Fig. 3. Disruptive OLE RNA mutations preclude the function of the OLE RNP pairing improves growth to a level similar to the WT rescue strain complex and overcome the strongly deleterious phenotype caused by PM1. (A) Sequence and secondary structure of B. halodurans OLE RNA regions that (Fig. 2C). These results demonstrate that even the subtle disrup- carry mutations examined in this study. (B) Growth of B. halodurans strains tion of an OLE RNA substructure in the RM1 construct likely carrying WT OapA with OLE RNA mutations as annotated, in media con-

causes a loss of the biological function of the RNP complex. taining 5% ethanol and incubated for 48 h. (C) Growth of strains with PM1 MICROBIOLOGY Similar results are observed with additional OLE RNA mutant OapA and OLE RNA mutations as annotated, in media containing 5% eth- constructs. The RM3 construct carries mutations converting a anol and incubated for 48 h. Growth data are normalized to the analogous GNRA tetraloop to a UNCG tetraloop (Fig. 3A). These are data for WT cells depicted in B. Additional details for B and C are as de- types of RNA loop families that are commonly found in natural scribed for Fig. 2C. structured ncRNAs (25, 26). However, OLE RNA function ap- pears to require the presence of this GNRA tetraloop because the RM3 strain has a phenotype that is indistinguishable from an that reside in highly conserved regions encompassed by the OLE RNA deletion strain (Fig. 3B). Another RNA construct, P2 through P5 substructures (Fig. 3A, Lower), which are known RM4, was prepared to examine the effects of two mutations to be important for OapA binding (6). The nucleotide changes

Harris et al. PNAS | vol. 115 | no. 27 | E6323 Downloaded by guest on October 1, 2021 carried by RM4 should diminish binding between OLE RNA and or BH0157), was mutated in multiple strains (Fig. 4A). The ybzG OapA, thereby preventing RNP complex formation. Again, B. gene encodes a protein of unknown function that contains a KOW halodurans Δole-oapA cells carrying the RM4 construct in place RNA-binding motif (conserved domain cd06088) commonly found of WT OAP RNA do not show improved growth in the presence in ribosomal RNA-binding proteins and transcription factors (27). of 5% ethanol (Fig. 3B), suggesting that these mutations indeed Specifically, 6 of the 10 strains carried four distinct mutations to disrupt proper formation and function of the RNP complex. ybzG, all of which occur in highly conserved regions of the gene. However, we have not measured the effects of the mutations on Two of these mutations (G42V and H57Y) reside at the only two the levels of these OLE RNA mutants in cells. Thus, the loss of invariant amino acids in the natural protein (Fig. 4A). One of the OLE RNA, rather than a disruption of its function, cannot yet be two remaining mutations (M1I) occurs in the start codon, which ruled out. presumably disrupts protein translation. The ybzG gene appears to be nonessential in Bacillus subtilis (28), a species related to B. The PM1 Phenotype Requires a Structurally Intact OLE RNA. To de- halodurans. Therefore, it is possible that this mutation at the termine whether the strongly deleterious effect of PM1 is de- start codon acts as a knockout. The final mutation results in pendent on functional versions of its OLE RNA partner, we substitution of a highly conserved amino acid (G19S) located examined the growth of the B. halodurans Δole-oapA strains immediately adjacent to amino acids of the KOW motif. This carrying PM1 and the OLE RNA mutants RM1 through RM4 mutation, like the others, probably causes substantial disruption (SI Appendix, Table S1) in the presence of ethanol. When OLE of the protein’s natural function. RNA is not present, cells carrying the PM1 construct exhibit It is unclear how the remaining four strains confer resistance growth that is substantially better than when OLE RNA is pre- against cold and ethanol through the mutations in their genomes. sent (Fig. 3C). Similar results are observed when OLE RNA is Other than ybzG, there were no other genes that were identified supplied in the form of the mutants RM1, RM3, and RM4. Only as mutated in more than one strain. Additional screening efforts, RM2, which carries mutations that retain the original OLE RNA currently underway, should reveal the mutation targets in these structure, permits strong ethanol toxicity of PM1 to persist (Fig. other strains that allow cells to overcome the PM1 phenotype. 3C). These results are consistent with our hypothesis that PM1 OapA retains its interaction with OLE RNA, and that this in- YbzG Selectively Binds OLE RNA. Because YbzG is a protein of teraction is required for the dominant-negative phenotype. unproven function, the mechanism by which it could participate in the biological pathway related OLE RNP complex was not A Genetic Screen Reveals Another Protein Necessary for OLE RNP certain. However, we hypothesized that the protein might di- Complex Function. We recognized that two characteristics of the rectly interact with OLE RNA due to the presence of the pu- OLE RNP complex provide a strong basis for conducting a ge- tative RNA-binding motif (27). To evaluate the RNA-binding netic screen. First, B. halodurans strains carrying PM1 OapA can ability of YbzG, EMSAs were performed with the full-length be exploited to identify genetic mutants that relieve the strong OLE RNA construct (OLE1–637) and various truncated OLE inhibitory effects of ethanol. Second, the disruption of OLE RNA constructs (Fig. 4B and SI Appendix, Fig. S4). RNA is one mechanism to overcome the PM1 phenotype, and so Indeed, WT YbzG binds full-length OLE RNA and a trun- we reasoned that a genetic screen might reveal additional part- cated transcript (OLE449–608) spanning regions P12 through P15 ners necessary for the function of the OLE RNP complex. Thus, (Fig. 4 B and C). In contrast, two other OLE RNA segments that suppressor mutations might occur in genes in the biochemical exclude these regions fail to be bound by YbzG. Similarly, YbzG pathway in which the OLE RNP complex participates, or they does not bind unrelated RNAs from B. halodurans, including a might occur in genes that code for additional protein or RNA transfer-messenger RNA or an adenosylcobalamin riboswitch (SI factors that directly interact with the complex. Appendix, Fig. S5). These results demonstrate that YbzG func- Initial attempts using the B. halodurans Δole-oapA strain tions as an RNA-binding protein and that its is likely transformed with a plasmid carrying ole and the oapA-pm1 genes to be selective for its target sequence and structure. (∼108 cells) failed to yield spontaneously occurring suppressor When the fraction of bound RNA versus free RNA was mutants under either ethanol- or cold-stress conditions. Conse- plotted against the protein concentration, we observed an ap- quently, cells were first treated with ethyl methanesulfonate parent dissociation constant (KD) of no poorer than 60 nM (Fig. (EMS) to introduce mutations into the starting population 4D). This KD value is sufficient for YbzG to form a stable in- (Materials and Methods). After EMS treatment, cells were cul- teraction with OLE RNA even if the two molecular species are tured at 20 °C and growth was observed after 1 wk of incubation. not abundant in cells. Moreover, this KD is similar to that ob- Single colonies were isolated, verified to grow at 20 °C, and served for the OapA protein (6). Therefore, we speculate that plasmids from each strain were sequenced to confirm that the YbzG also might be an important component of a functional transcriptional promoter and the original sequences for the ole OLE-OapA-YbzG RNP complex, although we have not exam- and oapA-pm1 genes remained unaltered. ined the system for simultaneous binding by both proteins. The genomic DNA from each of 14 verified strains was iso- Importantly, we observed that a YbzG variant isolated in our lated and sequenced. Ten unique genomes emerged from this genetic screen, wherein the histidine at position 57 has been effort, along with four isolates that were duplicates. To confirm mutated to a tyrosine (H57Y) (Fig. 4A), does not bind OLE that these 10 strains carried suppressor mutations that broadly RNA under the conditions tested (Fig. 4E). The H57Y mutant overcome the PM1 phenotypic defect, each was successively was chosen for examination because it appears in two strains tested for growth at 20 °C and in the presence of 5% ethanol (SI from our genetic screen and it alters an invariant residue in the Appendix, Fig. S3). Each strain exhibits growth under both stress KOW RNA-binding motif. This suggests that it is the failure of conditions, but yields cell densities that more closely approxi- the H57Y YbzG variant to bind OLE RNA that yields the mate those obtained with the Δole, Δoap, and Δole-oapA strains, suppressor phenotype in the B. halodurans PM1 strain. When rather than with WT cells. These phenotypes suggest that the YbzG is unable to bind, it is equivalent to the loss of functional genomic mutations acquired in the genetic screen likely disrupt OLE RNA. These findings are consistent with our hypothesis the RNP complex to overcome the more severe deleterious ef- that YbzG is a necessary partner for the function of the RNP fects caused by the PM1 mutation. complex. Analyses of the DNA sequences of the 10 suppressor strains Given that OLE RNA is the only known natural RNA target revealed several unique mutations per genome (SI Appendix,Table for YbzG, and that this interaction has a biological purpose, we S2). However, only one gene, ybzG (annotated as AYT26_RS00915 propose renaming the ybzG gene “oapB” and its protein

E6324 | www.pnas.org/cgi/doi/10.1073/pnas.1803191115 Harris et al. Downloaded by guest on October 1, 2021 A PNAS PLUS YbzG/OapB (8) )01,6( )4( IS V MSDSGPSPEI GQIVKIVKGR DRDQFSVIIK RVDDRFVYIA DGDKRKVDRA 10 20 30 40 50 (2,5) Y KRKNMNHLKL IDHISPEVRH SFEETGKVTN GKLRFALKKF LEEHADLLKE GE 60 70 80 90 100 102 B C WT YbzG/OapB (nM) 055 10 25 50 75 100 250 00057 G A A A A G U G A 530 G A C A U C U C bound G A 520 C G P14a OLE G G A G C U C 510 P14 A U C 1-637 500 U C U G A G G U U G U C G U G 540 free C C U A C G A G C G P13 G A A P14.1 A C U U 560 C U 550 G GG C G P15 C 490 U G C G C G A G OLE bound P12.2 A UA C A C C U A 449-608 C U A G U A A G free G U G 570 U 480 A G C 580 A C 1.0 A U U A D C A A G C G OLE1-637 G C OLE 470 U A P12.1 449-608 OLE G C 590 449-608 G U A A G C A A G U U U 460 U 0.5 A U G G C A A U G C 600 A U A U K ≤ 60 nM fraction bound D P12 A U U A U A U A 450 A U 0.0 5′ gg C G -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 log c (M) H57Y YbzG/OapB (nM) E 0710 25 50 75 100 250 500 500001

OLE OLE 1-637 1-637 free

Fig. 4. YbzG/OapB binds OLE RNA. (A) The YbzG/OapB protein sequence from B. halodurans (WP_010896340.1) is depicted with annotations denoting sequence and structural features. Amino acid conservation is indicated with colored shading as described for Fig. 2A: black, invariant; magenta, high; cyan, moderate; yellow, low. Underlined residues identify amino acids forming the putative KOW motif (26). Mutations identified in the genetic screen (SI Ap- pendix, Table S2) are colored red and reside directly above the altered amino acid of the WT protein. Numbers in parentheses identify the strains carrying the

mutation indicated. (B) Sequence and secondary structure of OLE449–608 from B. halodurans, which serves as a truncated RNA binding target for YbzG/OapB. 32 (C) EMSA results using 5′ P-labeled full-length (OLE1–637) or truncated (OLE449–608) OLE RNA with purified WT YbzG/OapB. (D) Plot of the fraction of RNA bound by protein versus the concentration (c) of YbzG/OapB. Fraction bound values are derived from the EMSA results in C, and the KD value is an estimate based on visual inspection of the plot. Additional experiments are necessary to precisely determine KD values and the stoichiometry between the RNA and 32 protein components. (E) EMSA results using 5′ P-labeled OLE1–637 with mutant H57Y YbzG/OapB.

OapB. We have detected the ybzG/oapB gene in all species that OapA interferes with the status of this unknown factor, which contain ole. However, it is also present in additional organisms in causes a phenotype worse than that observed when oapA is de- the Firmicutes phylum that do not carry the ole gene. Thus, it leted. This factor could be another protein partner, a second seems certain that OLE RNA is not the only natural binding RNA molecule, or perhaps a small signaling molecule. However, partner for YbzG/OapB. Efforts to further define the RNA other explanations for the current observations are also possible. binding site for this identified protein partner might aid in Notably, the amino acid changes creating PM1 reside in the identifying additional targets that have sequence or structural DxxxD motif that overlaps part of the GxxxGxxxG motif (Fig. homology with its binding site in OLE RNA. 3B). Previously, it was proposed that OapA interacts with OLE RNA as a protein homodimer (6). If G103, G107, and G111 Concluding Remarks comprise a helix-docking motif, then a helix–helix interaction The identification of PM1 OapA as a dominant-negative mu- might occur between two OapA molecules. Alternatively, this tation that produces strong ethanol- and cold-sensitivity phe- GxxxGxxxG motif might be used to interact with another protein. notypes is consistent with the hypothesis that the OLE RNP Regardless, the mutations to the DxxxD motif in PM1 likely do MICROBIOLOGY complex has a role in responding to these stresses. The not disrupt proper formation of the OLE–OapA interaction, or PM1 phenotype is more severe than the level of sensitivity the strong deleterious phenotype for PM1 would not be observed. caused by complete elimination of OLE RNA, OapA, or both. To search for additional molecules that interact with OapA The data indicate that these components of the RNP complex and OLE RNA, we conducted a genetic screen for mutations in likely only partially contribute to alleviating these stresses. One the bacterial genome that could overcome the severe PM1 possible explanation for our findings is that OapA has a hypo- phenotype. This screen yielded another protein partner of the thetical, and yet unknown, partner that also contributes to eth- OLE RNP complex that appears to interact independently with anol- and cold-stress responses (Fig. 5). In this scenario, PM1 OLE RNA. This mutated protein, YbzG/OapB, might alleviate

Harris et al. PNAS | vol. 115 | no. 27 | E6325 Downloaded by guest on October 1, 2021 Cell Membrane able conserved sequences and substructures for additional factors to bind the RNA. Biochemical methods to purify cell-derived OapA OapA OLE RNA or its protein factors by affinity tagging might also be an effective route to identifying additional factors of the RNP complex. If such additional binding partners for OLE RNA have known biochemical or biological functions, their identification G zipper G zipper might aid in assigning a function to this widespread and well-

DxxxD DxxxD conserved ncRNA. Materials and Methods OLE RNA positive positive Bioinformatic Analyses. Recently uncovered OLE RNA representatives were charge charge identified based on homology searches and sequence alignments with In- fernal v1.1 (29) using the previous alignment of ∼200 OLE RNAs (7) as a seed file. Sequences were compiled using the bacterial and archaeal section of RefSEq v76 (30), and environmental DNA sequences as previously described (31). After manual removal of duplicate and truncated sequences, the OapB resulting alignment of 795 unique OLE RNA sequences was used to establish unknown a revised consensus sequence and structural model by exploiting nucleotide factor(s) (YbzG) proportions and covariation data using the R2R computer algorithm (32). A collection of OapA sequences was compiled from RefSeq v76 and en- vironmental DNA sequences. Additional OapA sequences were identified n = ? using BLAST (33). Alignment was completed using Clustal Omega (34) and manually edited to remove duplicate and truncated sequences. Amino acid conservation was determined with JalView (35) using a scoring algorithm wherein residue identity is the highest contributor; consideration is also given when amino acid changes are within the same physicochemical class. Transmembrane topology was predicted using the TMHMM Server (36). YbzG/OapB protein sequence representatives were obtained using BLAST, aligned with Clustal Omega, and analyzed using JalView as described for Fig. 5. Schematic representation of the RNP complex formed by OLE RNAs. OapA. Depicted are the known components of the RNP complex drawn to scale based on molecular mass, except for the hypothetical involvement of an ad- Bacterial Strains, Plasmids, and Cultures. B. halodurans C-125 was purchased ditional factor. An OapA dimer is embedded in a lipid bilayer and in contact from the ATCC (Catalog #BAA-125). The pHCMC05 plasmid was obtained with OLE RNA in a 2:1 complex (6). Notable features of each OapA protein from The Bacillus Genetic Stock Center (The Ohio State University). Specific include the DxxxD motif (site of the PM1 mutations), GxxxGxxxG motif or OLE RNA and OapA mutants were generated by site-directed mutagenesis glycine zipper (G zipper), and a region enriched with positive-charged amino of the WT pHCMC05::ole-oapA plasmid described previously (7) using the acids. Although one YbzG/OapB molecule is depicted, the true stoichiometry QuikChange II XL kit (Agilent) according to the manufacturer’s instructions. remains to be determined. We predict that an additional factor or factors Primers are listed in SI Appendix, Table S3. Transformation of plasmids into interact with OapA and/or OLE RNA, but the nature, size, stoichiometry, and B. halodurans was completed following established protocols (7, 37). physical locations of any additional factors remain unknown. Unless otherwise specified, B. halodurans was grown in LB broth (USB Corp.) that was prepared at 90% volume, autoclaved, and adjusted to full

volume and pH 10 with 10% (wt/vol) filter-sterilized Na2CO3 [1% (wt/vol) the PM1 phenotype only by interfering with OLE RNA folding final concentration]. Media was solidified with 1.5% (wt/vol) agar. Unless and function, rather than serving as the critical factor that causes otherwise indicated, all cells were grown aerobically at 37 °C and shaken the dominant-negative effect. We know this is a possible mech- at 200 rpm. anism for overcoming the PM1 phenotype because the mutation or deletion of OLE RNA eliminates this severe PM1 effect. Bacterial Growth Assays. Experiments assessing the growth of B. halodurans Perhaps YbzG/OapB simply serves as an RNA folding chaper- strains in the presence of ethanol or at 20 °C were conducted in 14-mL one that helps OLE RNA adopt its active conformation as the round-bottom Falcon culture tubes using 3-mL culture volumes. B. halodurans RNP complex assembles. cells in exponential growth phase were used for inoculation, according to the method previously described (7). Cells were first grown overnight in 3 mL The genetic screen generated four additional strains that ex- −1 of LB (pH 10) with 3 μgmL chloramphenicol and 1 mM isopropyl-β-D- hibit reduced sensitivity to both ethanol and cold, but that lack thiogalactopyranoside (IPTG). These cultures were then diluted to 0.01

changes to the ybzG/oapB gene. This finding suggests that there OD600 in fresh media and incubated for 3 h. Then, for ethanol stress, cells are other genetic loci whose mutation can affect the function of were diluted again to 0.01 OD600 in media containing a final concentration the OLE RNP complex, or contribute in some other manner to of 5% ethanol (vol/vol). Tube caps were sealed and the cultures shaken at its biological pathway. However, each strain carries about a 37 °C for the indicated times. For cold stress, cells were diluted to 0.01 OD600 dozen mutations, and without multiple strains that carry mutations in fresh medium, then shaken at 20 °C for the indicated times. At each time in the same gene, we currently do not know which of these indi- point, OD600 values were determined using a Cary 60 UV-Vis Spectropho- tometer (Agilent Technologies). vidual mutations is necessary for overcoming the PM1 phenotype. It seems unlikely that the dominant-negative phenotype is Genetic Screen for Suppressor Mutations. Mutagenesis was conducted as caused by PM1 OapA interfering with the function of YbzG/ described previously (38). In brief, B. halodurans Δole-oapA cells carrying a OapB. Mutant strain #8 from the genetic screen (Fig. 4A) lacks plasmid with ole and oapA-pm1 genes were cultured to exponential phase

a start codon for the ybzG gene, which presumably precludes (OD600 = 0.4–0.8) and EMS was added to a final concentration of 1% (vol/vol). translation. If nonfunctional YbzG/OapB is the cause of the Cultures were incubated at 37 °C with shaking for 2 h. Cells were then severe ethanol toxicity phenotype, then elimination of YbzG/ pelleted and washed with Davis Salt [0.7% K2HPO4,0.3%KH2PO4, 0.1% • OapB should also yield the same severe phenotype. Thus, we are (NH4)2SO4,0.005%MgSO4 7H2O], then resuspended in LB (pH 10) and in- continuing the genetic screening effort to identify additional cubated for 3 h at 37 °C for recovery. For selection, EMS-treated cells were cultured at 20 °C for 7 d. Cells grown factors related to the function of the OLE RNP complex to from the mutagenized parental strain were collected and grown in separate determine the cause of the dominant-negative phenotype. Given cultures either at 20 °C or in the presence of 5% ethanol to confirm the the large size of OLE RNA relative to its protein binding part- stability of the suppressor phenotype. Once confirmed, these cells were ners OapA and YbzG/OapB (Fig. 5), there are plenty of avail- plated and single colonies were isolated. Each single colony was tested for

E6326 | www.pnas.org/cgi/doi/10.1073/pnas.1803191115 Harris et al. Downloaded by guest on October 1, 2021 the ability to grow at 20 °C and in the presence of 5% ethanol as described 300 mM NaCl, 25 mM imidazole, pH 7.8 at 23 °C) until signal of the eluant, PNAS PLUS above. If confirmed, plasmids were extracted from these strains and se- measured with Bradford reagent, was baseline. His-tagged constructs were quenced to confirm that the promoter region and ole and oapA-pm1 gene eluted with buffer containing imidazole (20 mM sodium phosphate, 300 mM inserts were not mutated. Genomic DNA was isolated from each confirmed NaCl, 250 mM imidazole, pH 7.8 at 23 °C). The protein was dialyzed in a strain using the ZR Fungal/Bacterial DNA Miniprep kit (Zymo Research) Slide-A-Lyzer Dialysis Cassette (Thermo Fisher) with a 10-kDa molecular mass according to the manufacturer’s instructions. The DNA was further purified cut-off against the equilibration buffer to remove imidazole. Purified pro- with the gDNA Clean & Concentrator kit (Zymo Research) according to tein was concentrated using an Amicon Ultra-15 Centrifugal Filter Unit (EMD the manufacturer’s instructions. Whole-genome sequencing with single- Millipore) with a 10-kDa molecular mass cut-off. Samples were aliquoted to nucleotide mutation detection was completed with data analysis by prevent more than one freeze–thaw cycle when used. Aliquots were flash- Genewiz, Inc. frozen with liquid nitrogen and stored at −80 °C. The purity of YbzG/OapB was assessed by SDS/PAGE with Coomassie staining, and only one band Overexpression and Purification of YbzG/OapB. Constructs for expression of was detected. Protein concentrations were determined using a standard wild-type YbzG/OapB and mutant YbzG/OapB-H57Y proteins were designed Bradford assay. with N-terminus hexahistidine tags with Factor Xa protease cleavage sites. Inserts of His-Xa-ybzG/oapB (WT) (NC_002570.2/180746–181054) and His-Xa- Binding Assays with YbzG/OapB Proteins and OLE RNA Constructs. The DNA

ybzG/oapB (H57Y) were synthesized and cloned into a pMK vector and template for the full-length OLE RNA (OLE1–637) was amplified from B. transformed into Escherichia coli K12 OmniMAX 2 T1R cells by GeneArt halodurans genomic DNA with incorporation of a T7 promoter using primers (Thermo Fisher). The insert was amplified by PCR using Phusion high-fidelity BhOLE-F and BhOLE-R (SI Appendix, Table S3). The OLE RNA truncation

polymerase (New England BioLabs) and standard M13 primers. The PCR (OLE449–608) template was created similarly using primers BhOLE449-F and product was purified using the Qiagen PCR purification kit, verified by se- BhOLE608-R (SI Appendix, Table S3). RNAs were generated by in vitro quencing, digested with NhaI-HF and BamHI-HF to obtain the ybzG/oapB transcription reactions, purified, and 5′ 32P-labeled as previously described inserts, and subcloned into a pET11a vector. Ligation was conducted using the (6). All other RNA constructs tested were prepared in the same fashion. ’ Quick Ligation Kit (New England BioLabs) according to the manufacturer s Protein samples were initially diluted in dialysis buffer to 10 μM to prepare instructions. a stock solution. A series of diluted protein samples was then prepared, BL21(DE3) Chemically Competent E. coli (New England BioLabs) were where each dilution was fivefold more concentrated than the protein con- ’ transformed according to the manufacturer s instructions with the pET11a centration in the respective final binding reaction. Each 10-μL binding re- plasmids containing the WT or H57Y His-Xa-ybzG/oapB constructs. Trans- action contained 2.5 μL protein solution, 2 μL5ˊ 32P-labeled RNA (∼10 nM), formations were confirmed by colony PCR with primers flanking the multi- 5 μL2× binding buffer [40 mM Hepes (pH 7.5 at 23 °C), 60 mM MgCl2, − − ple cloning sites, and subsequently verified by DNA sequencing. 160 mM KCl, 10% glycerol, 0.6 mg mL 1 E. coli tRNA (Sigma-Aldrich), 2 U μL 1 For protein expression, each strain was plated, and a single colony was SUPERase-In RNase Inhibitor (Thermo Fisher)], and deionized H O. Reac- μ −1 2 used to inoculate a 10-mL culture of LB with 100 gmL ampicillin. Cultures tions were incubated for 5 min at 23 °C, at which point the unbound and were incubated overnight with shaking at 37 °C. The resulting culture was bound products were directly separated by nondenaturing 6% PAGE. Non- μ −1 used to inoculate 1 L of LB with 100 gmL ampicillin. The culture was denaturing PAGE was prepared with 19:1 acrylamide:bisacrylamide (Bio-Rad), incubated with shaking at 37 °C until the OD600 reached 0.4–0.6. Protein 90 mM Tris-base, 90 mM boric acid, 5 mM Mg(OAc)2, pH 8 at 23 °C (6). Elec- expression was then induced by addition of IPTG to a final concentration of trophoresis was conducted at 4 °C using 40 W for various times depending on 1 mM. After growth at 37 °C for an additional 3 h, cells were harvested by the length of the RNA (6.5 h for full-length OLE RNA, 3 h for truncations). The × centrifugation for 15 min at 5,000 g and 4 °C. Cells were resuspended in resulting gels were imaged and quantified using a Typhoon PhosphorImager 50-mL cell wash buffer [20 mM Hepes (pH 7.5 at 23 °C), 150 mM NaCl], and (GE Healthcare). The fraction of bound RNA was derived from the ratios of × harvested by centrifugation for 15 min at 5,000 g and 4 °C. Each cell pellet band intensities using ImageQuant software (GE Healthcare). The values were ∼ was resuspended in 5 mL of cell lysis buffer [20 mM Hepes (pH 7.5 at 23 °C), plotted versus the logarithm of the protein concentration. The apparent 300 mM NaCl, 4 mM β-mercaptoethanol, EDTA-free protease inhibitors dissociation constant (KD) value was determined using a sigmoidal dose- (1 tablet per 50 mL; Thermo Fisher)] per gram of cell pellet. Cells, kept on ice, response equation in GraphPad Prism 7. were lysed by sonication. Nonlysed cells and debris were pelleted by cen- trifugation for 30 min at 15,000 × g and 4 °C. The supernatant was trans- ACKNOWLEDGMENTS. We thank Narasimhan Sudarsan, Adam Roth, Christina ferred to a clean tube chilled on ice. (Lünse) Weinberg, Anna Chase, Shira Stav, and Tamina Kienka for their con- For purification, HisPur Ni-NTA Resin (Thermo Fisher) was first equilibrated tributions to the OLE RNA project; and members of the R.R.B. laboratory for with equilibration buffer (20 mM sodium phosphate, 300 mM NaCl, pH 7.8 at helpful discussions. K.A.H. was supported by NIH Grant F32GM116426 and the 23 °C) at 25 °C. The cell lysate was loaded onto the column and run through project was supported by NIH Grant GM022778 (to R.R.B.). RNA research in the twice. The column was washed with buffer (20 mM sodium phosphate, R.R.B. laboratory is also supported by the Howard Hughes Medical Institute.

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