previously our work on the l. casei preq1-ii riboswitch  identified by breaker and co - workers  resulted in crystals with x - ray diffraction limited to 5.6  resolution . in an effort to improve crystal quality , we conducted blastn refseq rna searches starting with the l. casei riboswitch sequence .
our goal was to identify closely related sequences , especially those with shorter joining regions that might be more amenable to high - resolution structural analysis .
this approach led to the identification of a putative preq1-ii riboswitch sequence in the 5 utr of a cog4708 gene from l. rhamnosus , which is characteristic of other preq1-ii riboswitches .
the cog4708 gene is hypothesized to produce a protein that transports q precursors into the cell . despite genomic synteny and similar probiotic properties , l. casei and l. rhamnosus
, l. rhamnosus has also been associated with endocarditis and is not considered strictly beneficial .
significantly , two differences were apparent in the respective riboswitch sequences between positions 21 and 77  a region documented previously as important for preq1-binding function .
variations included a deletion of l. casei position 34 ( c34 ) in j2/3 of l. rhamnosus , and a c53u change in j2/4 . using itc and in - line probing approaches ( described below )
, we found the l. rhamnosus sequence is responsive to preq1 , and crystallization trials led to well diffracting crystals . at present , the basis for differences in crystal diffraction resulting from the use of the respective sequences is unknown .
1a , b ) and mutants thereof were prepared by in vitro transcription and purified by denaturing page as described .
itc measurements were conducted using a vp - itc calorimeter ( microcal , inc ) at 20 c or 25 c .
lyophilized rna was resuspended in 0.010 m na - hepes ph 7.0 or hepps ph 8.3 and 0.10 m nacl or 0.10 m kcl . for mgcl2-free conditions 0.5 mm edta ph 8.0
the rna was heated to 65 c for 5 min , then mgcl2 was added to a final concentration of 6.0 mm , or 20 mm , or 1.0 mm of co(nh3)6cl3 was added , followed by slow cooling to 24 c ; mgcl2 and co(nh3)6cl3 were omitted completely for folding conditions containing edta .
the rna was dialyzed against 4 l of 0.10 m nacl or 0.10 m kcl , 6.0 mm , 20.0 mm mgcl2 , 1 mm co(nh3)6cl3 or 0.5 mm edta , and 0.050 m na - hepes ph 7.0 or 0.050 m na - hepps ph 8.3 overnight at 4 c , then diluted with dialysis buffer to : 3.3 m for the wild type riboswitch , 13.8 m for the c30u mutant , 23.3  26.2 m for the u41c mutant , 1.5  5.7 m for wild type with 0.5 mm edta , and 3.6 m for wild type with co(nh3)6cl3 .
measurements were carried out by titrating preq1 into the riboswitch located in the sample cell ( cell volume is ~1.7 ml ) using 28 or 29 injections of 10 l each , except for the first injection of 3 l , with 120 or 240 s intervals between injections ; the reference power was 15 cal s. the thermograms were analyzed with origin 7.0 ( microcal ) using a 1:1 binding model .
experiments were performed in triplicate for wild type and mc variant l. rhamnosus sequences with mgcl2 , and in duplicate for mutant riboswitches and for the wild type in all other conditions ( supplementary table 1 ) ; representative titrations and curve fits are shown in supplementary fig .
a solution of 0.25 mm rna ( prepared as described above for itc ) in 0.01 m na - cacodylate ph 7.0 was heated to 65 c for 3 min .
mgcl2 was added to a final concentration of 6 mm and preq1  synthesized as described  was added to a final concentration of 0.5 mm followed by heating to 65 c for 5 min .
a volume of 1.7 l of pre - folded rna was mixed 1:1 with well solution comprising : 14.414.8% ( w / v ) poly(ethylene ) glycol 6000 ( peg6k ) , 0.140.16 m mgoac2 , 0.05 m na - cacodylate ph 6.0 , 1 mm spermine , and 0.15 m cscl .
crystals grew as rectangular plates of size 0.20 mm  0.05 mm  0.02 mm .
cryoprotection was by a 30 s transfer into synthetic mother liquor comprising : 17.217.8% ( w / v ) peg6k , 168192 mm mgoac2 , 0.18 m cscl , 0.06 m na - cacodylate ph 6.0 , and 1.2 mm spermine supplemented with 20% ( v / v ) 2-methyl-2,4-pentanediol and 20% ( v / v ) ethanol .
x - ray diffraction intensities were recorded at stanford synchrotron radiation lightsource ( ssrl , menlo park ca ) beamline 71 at 173 c and reduced with the hkl2000 software package .
a single crystal was used to obtain an 18-fold redundant , 2.6  resolution x - ray data set that was recorded at a wavelength of 1.7  to optimize the cs anomalous signal ( supplementary table 2 ) .
phenix autosol located 14 site - bound cs atoms , which were used for initial phase calculations in combination with density modification ( supplementary fig .
the figure of merit before density modification in resolve ( as implemented in phenix ) was 0.41 .
initial model building was performed by autobuilding in phenix , followed by iterative rounds of building in coot and refinement in phenix .
the trace and solvent content were consistent with a single molecule in the asymmetric unit .
a 2.3  resolution native dataset was collected at ssrl on a second crystal at a wavelength of 1.127  , which was used to extend the resolution of the sad model . at a late stage ,
preq1 was modeled into reduced bias 2fo - fc and fo - fc maps ( e.g. fig .
cs ions were included in the refined model based on their anomalous diffraction signal ( supplementary fig .
the final rwork / rfree values were 19.3/24.4% with reasonable geometry ( supplementary table 2 ) .
nearly the entire 77-nucleotide riboswitch was resolved in electron density maps including the ligand - binding pocket and rbs ( fig .
the area of preq1 that is inaccessible to solvent was calculated using a water probe with a radius of 1.4  using ucsf chimera .
the in - line probing reaction was carried out on an extended preq1-ii sequence ( supplementary fig .
rna was prepared by in vitro transcription ( as described above for itc ) , dephosphorylated with alkaline phosphatase , and radiolabeled with [ p]atp ( perkinelmer ) and t4 polynucleotide kinase ( new england biolabs ) , which was then page purified .
rna was folded by heating in 0.05 m tris - hcl ( ph 8.3 at 25 c ) to 65 c , snap cooling on ice , then adding kcl to 0.10 m , mgcl2 to 0.020 m , and preq1 at one of the concentrations described ( supplementary fig .
probing reactions each contained 1.0  10 cpm and were incubated at 25 c for ~40 h. reaction products were separated by denaturing 7.5% page , the gel was dried , and then exposed to a phosphor storage screen for ~40 h. imaging was carried out using a ge storm 860 , and gel quantification was conducted by use of safa with data normalized against invariant nucleotides selected by safa ; the nucleotides chosen as invariant bases were : 34 , 40 , 50 , 53 and 54 .
the fraction of rna cleaved was determined by setting the maximum amount cleaved to 1 and the minimum to 0 for all concentrations tested at a given nucleotide position , as described .
binding curves were generated in prism 6 by fitting the corrected intensities measured to a dose response curve , with the apparent kd equal to a level of 0.5 cleaved .
protein data bank : coordinates and structure factors have been deposited under accession code 4jf2 .
previously our work on the l. casei preq1-ii riboswitch  identified by breaker and co - workers  resulted in crystals with x - ray diffraction limited to 5.6  resolution . in an effort to improve crystal quality , we conducted blastn refseq rna searches starting with the l. casei riboswitch sequence .
our goal was to identify closely related sequences , especially those with shorter joining regions that might be more amenable to high - resolution structural analysis .
this approach led to the identification of a putative preq1-ii riboswitch sequence in the 5 utr of a cog4708 gene from l. rhamnosus , which is characteristic of other preq1-ii riboswitches .
the cog4708 gene is hypothesized to produce a protein that transports q precursors into the cell . despite genomic synteny and similar probiotic properties , l. casei and l. rhamnosus
, l. rhamnosus has also been associated with endocarditis and is not considered strictly beneficial .
significantly , two differences were apparent in the respective riboswitch sequences between positions 21 and 77  a region documented previously as important for preq1-binding function .
variations included a deletion of l. casei position 34 ( c34 ) in j2/3 of l. rhamnosus , and a c53u change in j2/4 . using itc and in - line probing approaches ( described below )
, we found the l. rhamnosus sequence is responsive to preq1 , and crystallization trials led to well diffracting crystals . at present , the basis for differences in crystal diffraction resulting from the use of the respective sequences is unknown .
1a , b ) and mutants thereof were prepared by in vitro transcription and purified by denaturing page as described .
itc measurements were conducted using a vp - itc calorimeter ( microcal , inc ) at 20 c or 25 c .
lyophilized rna was resuspended in 0.010 m na - hepes ph 7.0 or hepps ph 8.3 and 0.10 m nacl or 0.10 m kcl . for mgcl2-free conditions 0.5
the rna was heated to 65 c for 5 min , then mgcl2 was added to a final concentration of 6.0 mm , or 20 mm , or 1.0 mm of co(nh3)6cl3 was added , followed by slow cooling to 24 c ; mgcl2 and co(nh3)6cl3 were omitted completely for folding conditions containing edta .
the rna was dialyzed against 4 l of 0.10 m nacl or 0.10 m kcl , 6.0 mm , 20.0 mm mgcl2 , 1 mm co(nh3)6cl3 or 0.5 mm edta , and 0.050 m na - hepes ph 7.0 or 0.050 m na - hepps ph 8.3 overnight at 4 c , then diluted with dialysis buffer to : 3.3 m for the wild type riboswitch , 13.8 m for the c30u mutant , 23.3  26.2 m for the u41c mutant , 1.5  5.7 m for wild type with 0.5 mm edta , and 3.6 m for wild type with co(nh3)6cl3 .
measurements were carried out by titrating preq1 into the riboswitch located in the sample cell ( cell volume is ~1.7 ml ) using 28 or 29 injections of 10 l each , except for the first injection of 3 l , with 120 or 240 s intervals between injections ; the reference power was 15 cal s. the thermograms were analyzed with origin 7.0 ( microcal ) using a 1:1 binding model .
experiments were performed in triplicate for wild type and mc variant l. rhamnosus sequences with mgcl2 , and in duplicate for mutant riboswitches and for the wild type in all other conditions ( supplementary table 1 ) ; representative titrations and curve fits are shown in supplementary fig .
a solution of 0.25 mm rna ( prepared as described above for itc ) in 0.01 m na - cacodylate ph 7.0 was heated to 65 c for 3 min .
mgcl2 was added to a final concentration of 6 mm and preq1  synthesized as described  was added to a final concentration of 0.5 mm followed by heating to 65 c for 5 min .
a volume of 1.7 l of pre - folded rna was mixed 1:1 with well solution comprising : 14.414.8% ( w / v ) poly(ethylene ) glycol 6000 ( peg6k ) , 0.140.16 m mgoac2 , 0.05 m na - cacodylate ph 6.0 , 1 mm spermine , and 0.15 m cscl .
crystals grew as rectangular plates of size 0.20 mm  0.05 mm  0.02 mm .
cryoprotection was by a 30 s transfer into synthetic mother liquor comprising : 17.217.8% ( w / v ) peg6k , 168192 mm mgoac2 , 0.18 m cscl , 0.06 m na - cacodylate ph 6.0 , and 1.2 mm spermine supplemented with 20% ( v / v ) 2-methyl-2,4-pentanediol and 20% ( v / v ) ethanol .
x - ray diffraction intensities were recorded at stanford synchrotron radiation lightsource ( ssrl , menlo park ca ) beamline 71 at 173 c and reduced with the hkl2000 software package .
a single crystal was used to obtain an 18-fold redundant , 2.6  resolution x - ray data set that was recorded at a wavelength of 1.7  to optimize the cs anomalous signal ( supplementary table 2 ) .
phenix autosol located 14 site - bound cs atoms , which were used for initial phase calculations in combination with density modification ( supplementary fig .
3 ) . the figure of merit before density modification in resolve ( as implemented in phenix ) was 0.41 .
initial model building was performed by autobuilding in phenix , followed by iterative rounds of building in coot and refinement in phenix .
the trace and solvent content were consistent with a single molecule in the asymmetric unit .
a 2.3  resolution native dataset was collected at ssrl on a second crystal at a wavelength of 1.127  , which was used to extend the resolution of the sad model . at a late stage ,
preq1 was modeled into reduced bias 2fo - fc and fo - fc maps ( e.g. fig .
cs ions were included in the refined model based on their anomalous diffraction signal ( supplementary fig .
the final rwork / rfree values were 19.3/24.4% with reasonable geometry ( supplementary table 2 ) .
nearly the entire 77-nucleotide riboswitch was resolved in electron density maps including the ligand - binding pocket and rbs ( fig .
the area of preq1 that is inaccessible to solvent was calculated using a water probe with a radius of 1.4  using ucsf chimera .
the in - line probing reaction was carried out on an extended preq1-ii sequence ( supplementary fig .
1b ) essentially as described . rna was prepared by in vitro transcription ( as described above for itc ) , dephosphorylated with alkaline phosphatase , and radiolabeled with [ p]atp ( perkinelmer ) and t4 polynucleotide kinase ( new england biolabs ) , which was then page purified .
rna was folded by heating in 0.05 m tris - hcl ( ph 8.3 at 25 c ) to 65 c , snap cooling on ice , then adding kcl to 0.10 m , mgcl2 to 0.020 m , and preq1 at one of the concentrations described ( supplementary fig
probing reactions each contained 1.0  10 cpm and were incubated at 25 c for ~40 h. reaction products were separated by denaturing 7.5% page , the gel was dried , and then exposed to a phosphor storage screen for ~40 h. imaging was carried out using a ge storm 860 , and gel quantification was conducted by use of safa with data normalized against invariant nucleotides selected by safa ; the nucleotides chosen as invariant bases were : 34 , 40 , 50 , 53 and 54 .
the fraction of rna cleaved was determined by setting the maximum amount cleaved to 1 and the minimum to 0 for all concentrations tested at a given nucleotide position , as described .
binding curves were generated in prism 6 by fitting the corrected intensities measured to a dose response curve , with the apparent kd equal to a level of 0.5 cleaved .
protein data bank : coordinates and structure factors have been deposited under accession code 4jf2 .
 preq1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preq1 during biosynthesis of the essential trna base queuosine . 
 we report the first preq1-ii riboswitch structure at 2.3  resolution , which uses a novel fold to achieve effector recognition at the confluence of a three - way - helical junction flanking a pseudoknotted ribosome - binding site ( rbs ) . 
 the results account for preq1-ii - riboswitch - mediated translational control , and expand the known repertoire of ligand binding modes utilized by regulatory rnas . 
Online Methods
Identification of the 
RNA production and isothermal titration calorimetry (ITC)
Riboswitch crystallization and X-ray data collection
Phase determination, structural refinement, and analysis
In-line probing of the 
Accession codes
Supplementary Material