BACKGROUND
over the last two decades numerous rna regulators have been discovered that range in function from directing development in eukaryotes  <cit> , to controlling bacterial virulence  <cit>  and metabolism  <cit> .
these rna regulators vary considerably in their sizes , required processing proteins , and mechanisms of action  .
this extensive variability suggests that different regulatory rnas have very diverse evolutionary origins.
the independent evolution of rna regulators is especially apparent in bacteria due to the large number of sequenced genomes, and the breadth of biological diversity these genomes represent.
several metabolic processes are controlled by completely distinct rna regulatory mechanisms in different bacterial species.
these include methionine biosynthesis, which is regulated by at least three completely distinct s-adenosyl methionine -binding riboswitch architectures , and glucosamine metabolism where both a glucosamine-6-phosphate responsive ribozyme  <cit>  and a series of small rnas  <cit>  regulate the same pathway in different bacterial species.
the independent derivation of rna regulators is not restricted to the control of metabolic pathways.
in e. coli, the over fifty genes encoding ribosomal proteins are localized to approximately twenty transcriptional units, and the stoichiometry of ribosomal proteins is partially maintained through negative autogenous regulation.
in this paradigm, excess levels of a given ribosomal protein that are not rapidly assembled into the ribosome, bind a regulatory rna structure  to prevent further transcription or translation of the ribosomal protein effector and other proteins encoded by the operon  <cit> .
discovered over 40 years ago  <cit> , autogenous regulation of ribosomal protein biosynthesis has been well-characterized in e. coli
<cit> .
while several of these rna regulators appear to be conserved throughout many bacterial species, in most cases these protein-binding sites are narrowly distributed to a few orders of gammaproteobacteria  <cit> .
in addition there are also many cases where alternative rna structures that interact with homologous ribosomal proteins are present in different bacterial phyla  <cit> .
examples include rnas that interact with ribosomal proteins s <dig>  l <dig>  and s <dig> .
the widespread nature of rna-based autogenous regulation as a mechanism for the control of ribosomal protein synthesis coupled with the narrow distribution of most rna regulators, and the existence of alternative rna structures in a few cases, strongly suggests that many similar such mechanisms remain to be discovered in other bacterial phyla.
the rnas that interact with ribosomal protein s <dig> are representative of what is likely a common phenomenon.
to date, three different rna structures that interact with ribosomal protein s <dig> have been identified in escherichia coli, thermus thermophilus, and geobacillus stearothermophilus .
each rna structure appears to have distinct binding determinants, and they bear little resemblance to the rrna binding-site for s <dig>  <cit> .
yet, each allows negative regulation of rpso, the gene encoding s <dig>  despite their shared function, the rna structures show no obvious sequence or structural similarity .
while this collection of rna regulators already highlights rna structural diversity, examination of their phylogenetic distributions indicates that most bacterial phyla have no previously described s <dig> regulation  <cit> .
this suggests that there are many more rnas remaining to be described.figure  <dig>
previously identified ribosomal protein s15-interacting rna leader sequences and structures originating from different phyla of bacteria.
three regulatory rna structures have been previously reported for ribosomal protein s <dig>  a: escherichia coli <cit> , b: thermus thermophilus <cit> , and c: geobacillus stearothermophilus <cit> .
in each structure the rpso start codon is boxed, and a red bar is placed over the ribosome-binding site.
in this work, we implement a framework for computational identification of structured rna in bacterial genomes and apply it to genomic regions proximal to the s <dig> coding region  to assess the diversity of s15-interacting rnas in bacteria.
our search resulted in many putative structured rnas across different phyla of bacteria.
sequence alignments corresponding to several of these putative rna structures were further examined to determine phylogenetic distributions and identify transcription start sites from available rna-seq data.
additionally, to establish the biological relevance of our results, we experimentally demonstrate that one of these rnas, originating from the alphaproteobacterium rhizobium radiobacter , has the expected biological function.
we validate specific interactions between the predicted rna structure  and the s <dig> protein from the same species  using in vitro binding assays, and pinpoint regions of the rna important for protein-interaction using mutagenesis and truncation.
the rna’s secondary structure is further confirmed using structural probing assays.
finally, we also demonstrate that the rra-rna regulates gene expression in response to rra-s <dig> using an e. coli surrogate reporter system.
RESULTS
comparative genomics identifies several putative rna structures associated with rpso
to identify putative rna structures associated with the coding region for ribosomal protein s <dig> , we implemented a computational pipeline, gaisr  for de novo ncrna discovery and candidate refinement.
gaisr is based on existing rna discovery pipelines  <cit>  that have been very successful at identification of ncrna candidates  <cit> .
gaisr utilizes several pre-existing tools, including cmfinder, a de novo ncrna discovery tool  <cit> , and infernal  <dig> , an rna homology search tool  <cit>  to streamline sequence selection, identify potential ncrnas, and efficiently detect additional homologues for putative rna structures.
we used gaisr to examine the genomic region corresponding to the 5′-untranslated region of the gene encoding s <dig>  rpso, in fully sequenced bacterial genomes.
from the initial search we identified  <dig> potential ncrna sequences, originating from  <dig> initial phylogenetic sequence clusters.figure  <dig>
overview of comparative genomic pipeline: genomic analysis for illuminating structured rna .
putative 5′-untranslated regions  of our gene of interest  are identified within completed microbial genomes and clustered by their taxonomic group.
cmfinder is used to identify potential conserved rna structures within these sequence clusters  <cit> .
following rna structure identification, the candidate rna structures are manually inspected and additional homologs are identified using infernal  <dig>   <cit> .
the genomic context of putative homologs is assessed, and they are incorporated into the alignment using cmalign.
the alignment is then typically manually inspected to identify potential pseudoknots or other regulatory features and the curation process may be repeated several times.
finally, transcriptomic data is sought to identify the transcription start site of the putative ncrna.
from these initial sequences, we identified five promising rna structures.
among these structures were the two known rnas that allow regulation of rpso in firmicutes and gammaproteobacteria  <cit> .
of note, the rna structure reported for thermus thermophilus was not identified by our search, suggesting that more rnas may be present that were not uncovered here.
there are several potential reasons for this result including biases in sequence coverage , and our use of a single rna discovery tool for identification of rna structures may limit our ability to identify putative rna structures.
no tool for rna de novo discovery is designed to identify potential pseudoknotted structures, yet these are very common in biologically functional rnas  <cit> .
the pseudoknotted structures we have identified  are typically identified as individual helices by cmfinder and manually merged during the curation process.
alignments corresponding to the three promising novel structures were curated and additional examples identified using infernal homology searches.
in addition, the phylogenetic distribution of each putative ncrna was examined, and each alignment was compared with existing rna-seq data to identify regions likely to be within the rpso transcript.
consensus diagrams of the three candidate ncrnas are shown in figure  <dig>  and the alignments that correspond to these structures may be found as additional files  <dig>   <dig> and  <dig>  rna-secondary structures determined from analysis of large phylogenies are often well defined by co-varying nucleotide positions.
however, individual sequences corresponding to the rna structures we identified contain extensive variability including many non-canonical base-pairs and variable-length regions outside of the very well-conserved regions that are likely directly involved in protein-binding.
thus the secondary structure predictions in figure  <dig> should be considered tentative.
however, the degree of conservation observed here is consistent with that observed for other ribosomal protein-interacting regulatory rnas that have been experimentally validated in the past  <cit> .
therefore despite the sequence and structure variability, we believe that the rnas we identified are likely to have a regulatory function.figure  <dig>
consensus diagrams of novel putative rna structures and individual examples used for transcriptomic analysis.
novel regulatory rnas we have identified upstream of the rpso operon.
h <dig> helices were originally predicted by comparative genomics, but not supported by transcriptomic analysis and therefore are unlikely to be biologically relevant.
a: rna originating from alphaproteobacteria, b: rna originating from actinobacteria, and c: rna originating from chlamydia.
d: alphaproteobacterial rna example originating from rhodobacter spaeroides  showing putative transcription start site determined from analysis of rna-seq reads   <cit> .
e: actinobacterial rna example originating from mycobacterium tuberculosis  showing putative transcription start site determined from analysis of rna-seq reads .
f: chlamydia rna example originating from chlamydia trachomatis with previously determined transcription start site   <cit> .
rnas identified are diverse in sequence and secondary structure
our first rna  was identified in greater than 90% of species within the alphaproteobacteria orders of rhizobiales, rhodobacterales, rhodospirillales, caulobacterales, and sphingomonadales.
however, only a single example of the rna was found in a rickettsiales species , potentially reflective of genome reduction in most rickettsiales species  <cit> .
our original putative rna structure included three predicted pairing elements .
in ~50% of examples there is also a long-linker region between h <dig> and h <dig>  that is typically base-paired, although the precise position of this base-pairing within the sequence does not appear to be well-conserved .
the most highly conserved portion of the putative rna is the h <dig> helix.
this helix shows extensive evidence of co-variation and the loop region is highly conserved suggesting that it is important for protein binding.
the h <dig> helix is less conserved, but typically encompasses a putative ribosome-binding site in the 3′ portion.
while h <dig> shows some co-variation, the loop region is not well conserved in sequence or length.
in addition, transcriptomic analysis of rna-seq data derived from rhodobacter spaeroides
<cit>   suggests that the 5′ portion of this pairing element is not transcribed , thus we believe that the originally predicted h <dig> pairing element is likely not part of the biologically relevant rna.
our second rna  was identified mainly in the actinomycetales order of actinobacteria.
the putative rna structure contains a kissing-loop pseudoknotted structure that bears faint resemblance to the rna structure originating from e. coli , and there are weakly scoring homologs that appear in various gammaproteobacteria  lacking the known e. coli s <dig> regulator  <cit> .
however, the closing pseudoknot occurs prior to any potential regulatory sequences suggesting that the “entrapment” mechanism proposed for the e. coli rna is not likely to play a role here  <cit> .
like the rna described above, a ribosome-binding site is apparent in the 3′ portion of the h <dig> helix, suggesting a potential translational regulatory mechanism .
analysis of rna-seq data from mycobacterium tuberculosis , suggests that the transcription start site for this rna is approximately  <dig> nucleotides upstream from the start of the first predicted pairing element .
our third rna originates from chlamydia, and is the one in which we have the least confidence, mainly due to the limited sequence diversity available for analysis .
however, there is a very strongly conserved hairpin overlapping start codon of rpso in approximately  <dig> sequenced strains of chlamydia and a second potential short pairing element displaying some covariation and compatible mutations.
in our original prediction, this hairpin was significantly extended .
however, pre-existing analysis of transcript start sites in chlamydia trachomatis indicates that the transcript start site is just upstream of h <dig>   <cit> .
therefore we believe that h <dig> is likely not part of the biologically relevant rna.
notably, very few regulatory rnas have been identified in chlamydia.
only examples of the tpp and cobalamin riboswitches have been identified in this class of bacteria  <cit> , and in these cases there appear to be only isolated sequences rather than elements that are conserved in many genomes.
the process of curating our original alignments, and in particular the incorporation of rna-seq data, was critical for narrowing our focus to the portions of the predicted rnas that are most likely to be biologically relevant.
in two cases, transcriptomic data allowed us to determine that putative hairpins predicted through comparative genomics are unlikely to be part of the transcript.
our analysis exemplifies that in assessing the biological relevance of a given ncrna candidate it is important to determine whether a putative rna is actually transcribed as well as identify the transcription start site of the rna candidate  <cit> .
thus, archives that consolidate rna-seq data, and provide easily accessible read-depth information for many bacterial species are of great importance moving forward in rna comparative genomics.
rna from alphaproteobacterium rhizobium radiobacterspecifically interacts with s <dig> protein
to experimentally validate the biological relevance of our results, we further examined an example of the alphaproteobacterial rna originating from rhizobium radiobacter .
the sequence from r. radiobacter conforms well to our consensus structure, containing the highly conserved h <dig>  and the predicted h <dig> pairing element.
in addition, this sequence is one where the region directly preceding our transcription start site has the potential to base-pair with the 5′-most portion of the rna.
we designated this helix h <dig> due to its position 5’ of the predicted transcription start site.
we first tested the full-length version of the rna  and called it rra-rna <dig> because it was the first rna tested from this organism .
to examine whether this rna interacts specifically with s <dig> protein from the same organism  we utilized filter-binding assays  <cit> .
these assays confirmed that rra-rna <dig> binds rra-s <dig> with nanomolar affinity .
this value is similar to those reported for the interactions between s <dig> and the rna structures originating from e. coli   <cit> , g. stearothermophilus   <cit> , and the t. thermophilus   <cit> .figure  <dig>
in vitro
nitrocellulose filter-binding assays confirm the transcription start site as well as indicate h <dig> as the region essential for rra-s <dig> binding.
a: truncation sites and specific mutations to the rra-rna.
the start codon aug is boxed, and a red bar is over the ribosome-binding site.
b: 5′ truncations; c: 3′ truncations; d: putative binding-site mutations.
each curve represents at least three independent replicates.
for the purposes of comparison, the data from rra-rna <dig> was repeated on graphs b &d. reported kd measurements represent the protein concentration at which half of the maximum percentage of rra-rna is protein bound.
max% refers to the maximum percentage of rra-rna that interacts with rra-s <dig> in this in vitro assay .
truncation analysis suggests that rra-s <dig> minimal binding site includes both h <dig> and h2
to experimentally investigate the validity of the putative transcription start site we constructed several 5′ truncations to the rra-rna <dig> sequence and tested their ability to bind rra-s <dig>  based on the putative transcription start site derived from analysis of rna-seq data from rhizobium spaeroides , the potential h <dig> helix predicted from comparative genomics in the absence of rna-seq analysis  is unlikely to be necessary for rra-s <dig> binding.
we performed 5′-race for this rna to further identify the transcription start site.
although c- <dig> was one of the 5′-ends identified , this experiment provided multiple 5′-ends and was ultimately inconclusive.
truncations rra-rna <dig>  and rra-rna <dig>  appear to have negligible effects on rra-s <dig> binding  .
these results indicate that all bases upstream of nucleotide - <dig> are not required for binding rra-s <dig>  consistent with the putative transcription start site prior to this nucleotide at c- <dig>  binding was not significantly affected until the rna was truncated to g- <dig>  rra-rna <dig>  .
collectively, these results suggest the entire h <dig> stem and loop are dispensable and the c- <dig> identified during analysis of rna-seq data from r. sphaeroides likely represents the transcription start site.
to identify the minimal protein binding-site, we examined 3′-truncations to the rra-rna .
in the gammaproteobacterial rna, the initial amino acid encoding nucleotides of rpso form an integral part of the rna structure and function  <cit> .
however, removing the coding region of the alphaproteobacterial rna  has minimal effect on the binding affinity .
rra-rna <dig>  was designed to remove all bases downstream of the predicted h2; again, this rna binds rra-s <dig> with an affinity better than that of rra-rna <dig> .
the observed increase in binding affinity is likely due to removal of potential alternative competing structures, thus allowing a tighter interaction between the protein and the rna.
rra-rna <dig>  was designed to remove all of predicted hairpin h <dig> including the five uracils  through the putative ribosome binding site , start codon and subsequent protein coding nucleotides.
this truncation completely abolishes rra-s <dig> binding .
to assess whether slippage along the predicted h <dig> might allow the five uracils  to base-pair with the putative ribosome binding site , we mutated the polyuridine to a purine-rich sequence to destabilize this alternative pairing .
this mutant was able to bind rra-s <dig> with a similar affinity to the full length rra-rna <dig>  suggesting that the pairing we have drawn is one that allows for protein binding.
based on this data we predict the minimal rna regulatory region includes nucleotides g- <dig> through u- <dig>  which is fully encompassed by our predicted transcript, and includes both of the predicted pairing elements h <dig> and h <dig>
mutation analysis suggests a potential binding-site
the most highly conserved portion of this rna is the h <dig> stem loop .
due to its high sequence conservation, we hypothesize that this region is essential for rra-s <dig> binding.
mutations made in this stem, rra-rna <dig> and rra-rna <dig>  both significantly inhibit binding .
the compensatory mutation, rra-rna <dig>  was able to partially recover rra-s <dig> binding .
in this compensatory mutant, it is likely an alternative base-pair forms with usually unpaired a- <dig> and the dynamic equilibrium of the two rna structures allows, but does not completely restore, rra-s <dig> binding.
in combination with the truncation experiments above, these results suggest that rra-s <dig> binds its rna regulator in the highly conserved stem-loop structure of h <dig> but that h <dig> is still required for binding.
the r. radiobacterrna allows regulation in response to s <dig> in vivo
to determine whether the rra-rna has regulatory activity in addition to s15-binding activity, we conducted in vivo reporter assays to assess regulation.
to do this we used a gfp reporter to measure expression of the gene following the rra-rna in response to different levels of rra-s <dig>  the rna sequence was cloned in-frame as a translational fusion with the gfp reporter under the control of the iptg-inducible trc promoter.
this construct included the rpso start codon, shine-dalgarno sequence, and the first nine codons of the rpso gene to form the rna-gfp fusion.
on a second plasmid, the r. radiobacter rpso coding sequence was placed under the control of an l-arabinose inducible promoter.
the pair of plasmids were co-transformed into an e. coli k12:Δrpso strain.
we chose to use a surrogate organism, e. coli, due to its ease of use and manipulation, and the ability to obtain a knockout organism lacking endogenous s <dig>  using this gfp in vivo reporter system, we assessed the ability of rra-s <dig> to regulate gene expression by measuring the gfp levels in the cells in the presence and absence of induced rra-s <dig>  if the rna interacts with rra-s <dig> to regulate gene expression, we expect to see a decrease in gfp expression in cells expressing rra-s <dig> compared to cells not expressing rra-s <dig>
cells co-transformed with plasmids containing full length rra-rna1-gfp, and rra-s <dig> were grown in the presence and absence of l-arabinose.
the cells grown in the presence of the sugar  displayed a ~4-fold decrease in gfp-reporter expression .
because l-arabinose induces rra-s <dig> production, the decrease in gfp reporter expression is likely due to an interaction between the rna and rra-s <dig>  next, to corroborate that our predicted transcription start site at the c- <dig> allows regulation, the sequence for rra-rna <dig>  was also tested in this system and behaved in a similar manner.
these results indicate an rna sequence starting at the transcription start site derived from r. spaeroides is sufficient to allow regulation in vivo.figure  <dig>
gfp reporter assays validate rra-rna’s regulatory role.
gfp reporter assays validate the regulatory capacity of rra-rna in response to rra-s <dig>  rra-rna structure and sequence are the same as described in figure  <dig>  all relative fluorescence values were calculated by normalizing gfp/od <dig>  all bars are 3+ independent experiments.
*indicates p <  <dig> ; **indicates p <  <dig> .
we also examined whether mutations to h <dig> that abolish rra-s <dig> binding would affect regulation.
cells containing either rra-rna9-gfp and rra-rna10-gfp did not display a significant difference in gfp reporter expression when grown in the presence and absence of l-arabinose.
this is likely because both rra-rna <dig> and rra-rna <dig> do not interact specifically with s <dig> and are unable to regulate the expression of the reporter.
however, it should be noted that gfp expression levels in the absence of arabinose were also significantly lower than those observed with the rra-rna <dig> and rra-rna <dig> constructs.
to assess whether the partial compensation of binding observed for rrarna <dig> in vitro represented a biologically relevant, functional compensation, rra-rna <dig> was also examined in this system.
in this case, cells grown without l-arabinose displayed an increased gfp expression level compared to rra-rna <dig>  and cells grown in the presence of l-arabinose had a ~10-fold decrease in relative gfp fluorescence .
however, the increase in fold-change is solely due to increased rna11-gfp expression levels and the repressed level of gene expression is comparable between the two rna elements.
thus, the rra-rna <dig> compensatory mutation that partially restored the in vitro rna-protein interaction also restored the regulatory interaction between the rra-rna and rra-s <dig>  the partial restoration of in vitro binding by the compensatory mutant rra-rna <dig> is likely due to the presence of several competing structures formed by the rna under these conditions.
however, the in vivo conditions enable the rna to adopt a secondary structure that increases overall reporter expression and enables regulation in response to s <dig>  together, these assays indicate that not only the does this rna interact with rra-s <dig> in vitro, but it is a biologically relevant regulatory element responding to s <dig>
structural probing confirms predicted secondary structure
to further examine the secondary structure of rra-rna in the absence of protein we used several structural probing methods in combination with a minimal rna construct  including nuclease cleavage assays with , and in-line probing.
rnase vi cleaves double stranded rna non-specifically, rnase a cleaves single stranded c’s and u’s, and in-line probing the rna structure reveals the flexible regions of the rna structure  that are more prone to spontaneous self-cleavage.
although the putative stem h <dig> is predicted in our alignment , there are many sequences that contain short polypyrimidine sequences that are unpaired in our sequence alignment.
these sequences may form alternative pairings with the ribosome-binding site .
based on sequence data alone it is difficult to distinguish which bases are interacting with the ribosome-binding site.
however, several lines of evidence indicate that we have identified the correct in vitro base-pairing conformation for our putative h <dig> in the r. radiobacter example of the rna .
first, our mutagenesis and truncation analyses indicate that mutating the polyuridine  does not alter protein-binding activity.
this suggests that this region is unlikely to interact with the putative ribosome-binding site .
however, deleting this region and the following hairpin  abolishes protein binding indicating that h <dig> is important for protein binding.
second, rnase v <dig> cleavage occurs symmetrically in regions that are base-paired in our figure , and rnasea cleavage occurs at c- <dig> as would be expected for a loop region.
in addition, in-line probing shows that the entire 3′ portion of the molecule is somewhat flexible, from bases - <dig> through - <dig> .
in conjunction with our mutagenesis results, this strongly suggests the correct pairing-element has been identified.figure  <dig>
structural probing confirms predicted secondary structure.
a: rnase v <dig> , rnase a , no reaction , hydroxyl cleavage , denaturing rnase t <dig> , and two independent replicates of in-line probing reactions  where the cleavage products have been separated by denaturing 10% page.
cleaved cytosine and uridine residues in the rnase a reaction and cleaved guanosines in the denaturing t <dig> reaction were used to map cleavage to the rna structure, and regions of strong in-line cleavage are labeled.
b: mapping of prominent cleavage sites to the structure of rra-rna6; bases in black are resolved on the gel.
tss indicates predicted transcription start site.
cleavage sites largely confirm structure anticipated from comparative genomics.
consistent with our mutagenesis results, the highly conserved stem h <dig>  is almost certainly double-stranded with a loop from c- <dig> to a- <dig>  bases u- <dig> through c- <dig> are shielded from in-line attack and there are strong cleavage bands in rnase vi probing for bases c- <dig>  bases - <dig> to - <dig>  and g- <dig>  also, there are no rnase a cleavage products for any of these uracils or cytosines, suggesting that these nucleotides are not single-stranded.
there is also evidence for the predicted loop region in h <dig>  probing with rnase a results in cleavage products for c- <dig> and c- <dig> and in-line probing reveals that c- <dig> through a- <dig>  are flexible.
at the base of the h <dig> stem, we predict a bulged adenosine  and the highlighted region c of our in-line probing gel corresponds to this bulged base.
this data corroborates our other evidence that the region essential for rra-s <dig> binding in h <dig> forms a double-helix.
the nature of the junction between the two predicted helices is still unresolved.
this region is not well-conserved so there is little phylogenetic evidence of structure, and in several cases the different assays give conflicting results, which may be the result of multiple folding conformations.
the string of uridines from u– <dig> to u- <dig> does not appear to be flexible based on in-line probing, is cleaved by rnaase v <dig>  and is not cleaved by rnase a, indicating that the region is not single-stranded.
however, there are also no clear binding partners for these nucleotides suggesting that they may be forming a constrained tertiary structure.
the string of cytosines that follows this region, c- <dig> to c- <dig>  do show strong rnase a cleavage suggesting they are single-stranded, and this is corroborated by the in-line cleavage at these positions .
however, these bases also display rnase v <dig> cleavage indicating that they may sometimes adopt a double-stranded conformation.
nucleotides from - <dig> to - <dig> are not cleaved by either rnase v <dig> or rnase a, and appear to be structurally constrained.
this suggests that they are not necessarily double-stranded, but may be participating in some tertiary structure.
nucleotides - <dig> to - <dig>  which potentially could interact with these bases, also show conflicting results, cleaving with both rnase v <dig> and rnase a. we have included the possible base pairing of the nucleotides at the base of h <dig> in our structure figures , but these interactions are likely weak.
the accumulation of these data strongly suggests that both the predicted pairing elements do form, but the nature of the junction between these two elements remains unclear.
CONCLUSIONS
this work demonstrates the premise that nature may invent many unique ways to solve a single biological problem.
in the context of other forms of rna-based regulation the diversity of distinct rna structures allowing cis-regulation of the rpso operon is nearly unmatched.
the only similar example of such diversity in rna regulators for a specific function are the sam-binding riboswitches, where more than three completely distinct classes , and several additional sub-classes with re-arranged or modified secondary structure elements have been characterized  <cit> .
the s <dig> auto-regulatory rna structures we identified are quite diverse from one another, and from the existing known characterized s <dig> regulatory rnas that originate from e. coli, g. stearopthermophilus, and t. thermophilus
.
all of the previously characterized rna structures encompass a predicted shine-dalgarno sequence, but beyond such regulatory features the rnas appear to share very few common sequence features or patterns in secondary structure.
while the s15-interacting rnas potentially share some tertiary structure similarities that are not captured in the secondary structure diagrams, previous studies indicate that the e. coli s <dig> does not interact with the regulatory rna originating from g. stearothermophilus
<cit> .
this finding suggests that there may be no single conserved tertiary structure shared by the s15-binding mrna structures.
in the absence of structural data, it remains to be seen whether the structural diversity apparent in natural s15-interacting mrna structures is a result of rna’s inherent ability to generate a similar tertiary structure from diverse arrangements of primary and secondary structure  <cit> , or from differences between the s <dig> protein homologs that lead to distinct pools of potential rna ligands.
from the structures we describe here, it is clear that there are many ways to solve this particular biological problem.
an exciting question that stems from this work is precisely how prevalent are rna solutions within sequence space that allow autogenous regulation in response to s <dig>  based on the natural diversity of s15-interacting rnas, we expect that this number is large, and that as more genomes are sequenced and the sensitivity of computational searches increases, additional structures with this function will be identified.

 over the last two decades numerous rna regulators have been discovered that range in function from directing development in eukaryotes, to controlling bacterial virulence and metabolism. these rna regulators vary considerably in their sizes, required processing proteins, and mechanisms of action. this extensive variability suggests that different regulatory rnas have very diverse evolutionary origins. the independent evolution of rna regulators is especially apparent in bacteria due to the large number of sequenced genomes, and the breadth of biological diversity these genomes represent. several metabolic processes are controlled by completely distinct rna regulatory mechanisms in different bacterial species. these include methionine biosynthesis, which is regulated by at least three completely distinct s-adenosyl methionine -binding riboswitch architectures, and glucosamine metabolism where both a glucosamine-6-phosphate responsive ribozyme and a series of small rnas regulate the same pathway in different bacterial species. the independent derivation of rna regulators is not restricted to the control of metabolic pathways. in e. coli, the over fifty genes encoding ribosomal proteins are localized to approximately twenty transcriptional units, and the stoichiometry of ribosomal proteins is partially maintained through negative autogenous regulation. in this paradigm, excess levels of a given ribosomal protein that are not rapidly assembled into the ribosome, bind a regulatory rna structure to prevent further transcription or translation of the ribosomal protein effector and other proteins encoded by the operon. discovered over 40 years ago, autogenous regulation of ribosomal protein biosynthesis has been well-characterized in e. coli. while several of these rna regulators appear to be conserved throughout many bacterial species, in most cases these protein-binding sites are narrowly distributed to a few orders of gammaproteobacteria. in addition there are also many cases where alternative rna structures that interact with homologous ribosomal proteins are present in different bacterial phyla. examples include rnas that interact with ribosomal proteins s l and s. the widespread nature of rna-based autogenous regulation as a mechanism for the control of ribosomal protein synthesis coupled with the narrow distribution of most rna regulators, and the existence of alternative rna structures in a few cases, strongly suggests that many similar such mechanisms remain to be discovered in other bacterial phyla. the rnas that interact with ribosomal protein s are representative of what is likely a common phenomenon. to date, three different rna structures that interact with ribosomal protein s have been identified in escherichia coli, thermus thermophilus, and geobacillus stearothermophilus. each rna structure appears to have distinct binding determinants, and they bear little resemblance to the rrna binding-site for s. yet, each allows negative regulation of rpso, the gene encoding s despite their shared function, the rna structures show no obvious sequence or structural similarity. while this collection of rna regulators already highlights rna structural diversity, examination of their phylogenetic distributions indicates that most bacterial phyla have no previously described s regulation. this suggests that there are many more rnas remaining to be described.figure previously identified ribosomal protein s15-interacting rna leader sequences and structures originating from different phyla of bacteria. three regulatory rna structures have been previously reported for ribosomal protein s a: escherichia coli, b: thermus thermophilus, and c: geobacillus stearothermophilus. in each structure the rpso start codon is boxed, and a red bar is placed over the ribosome-binding site. in this work, we implement a framework for computational identification of structured rna in bacterial genomes and apply it to genomic regions proximal to the s coding region to assess the diversity of s15-interacting rnas in bacteria. our search resulted in many putative structured rnas across different phyla of bacteria. sequence alignments corresponding to several of these putative rna structures were further examined to determine phylogenetic distributions and identify transcription start sites from available rna-seq data. additionally, to establish the biological relevance of our results, we experimentally demonstrate that one of these rnas, originating from the alphaproteobacterium rhizobium radiobacter, has the expected biological function. we validate specific interactions between the predicted rna structure and the s protein from the same species using in vitro binding assays, and pinpoint regions of the rna important for protein-interaction using mutagenesis and truncation. the rna’s secondary structure is further confirmed using structural probing assays. finally, we also demonstrate that the rra-rna regulates gene expression in response to rra-s using an e. coli surrogate reporter system.  comparative genomics identifies several putative rna structures associated with rpso to identify putative rna structures associated with the coding region for ribosomal protein s, we implemented a computational pipeline, gaisr for de novo ncrna discovery and candidate refinement. gaisr is based on existing rna discovery pipelines that have been very successful at identification of ncrna candidates. gaisr utilizes several pre-existing tools, including cmfinder, a de novo ncrna discovery tool, and infernal, an rna homology search tool to streamline sequence selection, identify potential ncrnas, and efficiently detect additional homologues for putative rna structures. we used gaisr to examine the genomic region corresponding to the 5′-untranslated region of the gene encoding s rpso, in fully sequenced bacterial genomes. from the initial search we identified potential ncrna sequences, originating from initial phylogenetic sequence clusters.figure overview of comparative genomic pipeline: genomic analysis for illuminating structured rna. putative 5′-untranslated regions of our gene of interest are identified within completed microbial genomes and clustered by their taxonomic group. cmfinder is used to identify potential conserved rna structures within these sequence clusters. following rna structure identification, the candidate rna structures are manually inspected and additional homologs are identified using infernal. the genomic context of putative homologs is assessed, and they are incorporated into the alignment using cmalign. the alignment is then typically manually inspected to identify potential pseudoknots or other regulatory features and the curation process may be repeated several times. finally, transcriptomic data is sought to identify the transcription start site of the putative ncrna. from these initial sequences, we identified five promising rna structures. among these structures were the two known rnas that allow regulation of rpso in firmicutes and gammaproteobacteria. of note, the rna structure reported for thermus thermophilus was not identified by our search, suggesting that more rnas may be present that were not uncovered here. there are several potential reasons for this result including biases in sequence coverage, and our use of a single rna discovery tool for identification of rna structures may limit our ability to identify putative rna structures. no tool for rna de novo discovery is designed to identify potential pseudoknotted structures, yet these are very common in biologically functional rnas. the pseudoknotted structures we have identified are typically identified as individual helices by cmfinder and manually merged during the curation process. alignments corresponding to the three promising novel structures were curated and additional examples identified using infernal homology searches. in addition, the phylogenetic distribution of each putative ncrna was examined, and each alignment was compared with existing rna-seq data to identify regions likely to be within the rpso transcript. consensus diagrams of the three candidate ncrnas are shown in figure  and the alignments that correspond to these structures may be found as additional files and rna-secondary structures determined from analysis of large phylogenies are often well defined by co-varying nucleotide positions. however, individual sequences corresponding to the rna structures we identified contain extensive variability including many non-canonical base-pairs and variable-length regions outside of the very well-conserved regions that are likely directly involved in protein-binding. thus the secondary structure predictions in figure  should be considered tentative. however, the degree of conservation observed here is consistent with that observed for other ribosomal protein-interacting regulatory rnas that have been experimentally validated in the past. therefore despite the sequence and structure variability, we believe that the rnas we identified are likely to have a regulatory function.figure consensus diagrams of novel putative rna structures and individual examples used for transcriptomic analysis. novel regulatory rnas we have identified upstream of the rpso operon. h helices were originally predicted by comparative genomics, but not supported by transcriptomic analysis and therefore are unlikely to be biologically relevant. a: rna originating from alphaproteobacteria, b: rna originating from actinobacteria, and c: rna originating from chlamydia. d: alphaproteobacterial rna example originating from rhodobacter spaeroides showing putative transcription start site determined from analysis of rna-seq reads. e: actinobacterial rna example originating from mycobacterium tuberculosis showing putative transcription start site determined from analysis of rna-seq reads. f: chlamydia rna example originating from chlamydia trachomatis with previously determined transcription start site. rnas identified are diverse in sequence and secondary structure our first rna was identified in greater than 90% of species within the alphaproteobacteria orders of rhizobiales, rhodobacterales, rhodospirillales, caulobacterales, and sphingomonadales. however, only a single example of the rna was found in a rickettsiales species, potentially reflective of genome reduction in most rickettsiales species. our original putative rna structure included three predicted pairing elements. in ~50% of examples there is also a long-linker region between h and h that is typically base-paired, although the precise position of this base-pairing within the sequence does not appear to be well-conserved. the most highly conserved portion of the putative rna is the h helix. this helix shows extensive evidence of co-variation and the loop region is highly conserved suggesting that it is important for protein binding. the h helix is less conserved, but typically encompasses a putative ribosome-binding site in the 3′ portion. while h shows some co-variation, the loop region is not well conserved in sequence or length. in addition, transcriptomic analysis of rna-seq data derived from rhodobacter spaeroides suggests that the 5′ portion of this pairing element is not transcribed, thus we believe that the originally predicted h pairing element is likely not part of the biologically relevant rna. our second rna was identified mainly in the actinomycetales order of actinobacteria. the putative rna structure contains a kissing-loop pseudoknotted structure that bears faint resemblance to the rna structure originating from e. coli, and there are weakly scoring homologs that appear in various gammaproteobacteria lacking the known e. coli s regulator. however, the closing pseudoknot occurs prior to any potential regulatory sequences suggesting that the “entrapment” mechanism proposed for the e. coli rna is not likely to play a role here. like the rna described above, a ribosome-binding site is apparent in the 3′ portion of the h helix, suggesting a potential translational regulatory mechanism. analysis of rna-seq data from mycobacterium tuberculosis, suggests that the transcription start site for this rna is approximately nucleotides upstream from the start of the first predicted pairing element. our third rna originates from chlamydia, and is the one in which we have the least confidence, mainly due to the limited sequence diversity available for analysis. however, there is a very strongly conserved hairpin overlapping start codon of rpso in approximately sequenced strains of chlamydia and a second potential short pairing element displaying some covariation and compatible mutations. in our original prediction, this hairpin was significantly extended. however, pre-existing analysis of transcript start sites in chlamydia trachomatis indicates that the transcript start site is just upstream of h. therefore we believe that h is likely not part of the biologically relevant rna. notably, very few regulatory rnas have been identified in chlamydia. only examples of the tpp and cobalamin riboswitches have been identified in this class of bacteria, and in these cases there appear to be only isolated sequences rather than elements that are conserved in many genomes. the process of curating our original alignments, and in particular the incorporation of rna-seq data, was critical for narrowing our focus to the portions of the predicted rnas that are most likely to be biologically relevant. in two cases, transcriptomic data allowed us to determine that putative hairpins predicted through comparative genomics are unlikely to be part of the transcript. our analysis exemplifies that in assessing the biological relevance of a given ncrna candidate it is important to determine whether a putative rna is actually transcribed as well as identify the transcription start site of the rna candidate. thus, archives that consolidate rna-seq data, and provide easily accessible read-depth information for many bacterial species are of great importance moving forward in rna comparative genomics. rna from alphaproteobacterium rhizobium radiobacterspecifically interacts with s protein to experimentally validate the biological relevance of our results, we further examined an example of the alphaproteobacterial rna originating from rhizobium radiobacter. the sequence from r. radiobacter conforms well to our consensus structure, containing the highly conserved h and the predicted h pairing element. in addition, this sequence is one where the region directly preceding our transcription start site has the potential to base-pair with the 5′-most portion of the rna. we designated this helix h due to its position 5’ of the predicted transcription start site. we first tested the full-length version of the rna and called it rra-rna because it was the first rna tested from this organism. to examine whether this rna interacts specifically with s protein from the same organism we utilized filter-binding assays. these assays confirmed that rra-rna binds rra-s with nanomolar affinity. this value is similar to those reported for the interactions between s and the rna structures originating from e. coli, g. stearothermophilus, and the t. thermophilus.figure in vitro nitrocellulose filter-binding assays confirm the transcription start site as well as indicate h as the region essential for rra-s binding. a: truncation sites and specific mutations to the rra-rna. the start codon aug is boxed, and a red bar is over the ribosome-binding site. b: 5′ truncations; c: 3′ truncations; d: putative binding-site mutations. each curve represents at least three independent replicates. for the purposes of comparison, the data from rra-rna was repeated on graphs b &d. reported kd measurements represent the protein concentration at which half of the maximum percentage of rra-rna is protein bound. max% refers to the maximum percentage of rra-rna that interacts with rra-s in this in vitro assay. truncation analysis suggests that rra-s minimal binding site includes both h and h2 to experimentally investigate the validity of the putative transcription start site we constructed several 5′ truncations to the rra-rna sequence and tested their ability to bind rra-s based on the putative transcription start site derived from analysis of rna-seq data from rhizobium spaeroides, the potential h helix predicted from comparative genomics in the absence of rna-seq analysis is unlikely to be necessary for rra-s binding. we performed 5′-race for this rna to further identify the transcription start site. although c- was one of the 5′-ends identified, this experiment provided multiple 5′-ends and was ultimately inconclusive. truncations rra-rna and rra-rna appear to have negligible effects on rra-s binding. these results indicate that all bases upstream of nucleotide - are not required for binding rra-s consistent with the putative transcription start site prior to this nucleotide at c- binding was not significantly affected until the rna was truncated to g- rra-rna. collectively, these results suggest the entire h stem and loop are dispensable and the c- identified during analysis of rna-seq data from r. sphaeroides likely represents the transcription start site. to identify the minimal protein binding-site, we examined 3′-truncations to the rra-rna. in the gammaproteobacterial rna, the initial amino acid encoding nucleotides of rpso form an integral part of the rna structure and function. however, removing the coding region of the alphaproteobacterial rna has minimal effect on the binding affinity. rra-rna was designed to remove all bases downstream of the predicted h2; again, this rna binds rra-s with an affinity better than that of rra-rna. the observed increase in binding affinity is likely due to removal of potential alternative competing structures, thus allowing a tighter interaction between the protein and the rna. rra-rna was designed to remove all of predicted hairpin h including the five uracils through the putative ribosome binding site, start codon and subsequent protein coding nucleotides. this truncation completely abolishes rra-s binding. to assess whether slippage along the predicted h might allow the five uracils to base-pair with the putative ribosome binding site, we mutated the polyuridine to a purine-rich sequence to destabilize this alternative pairing. this mutant was able to bind rra-s with a similar affinity to the full length rra-rna suggesting that the pairing we have drawn is one that allows for protein binding. based on this data we predict the minimal rna regulatory region includes nucleotides g- through u- which is fully encompassed by our predicted transcript, and includes both of the predicted pairing elements h and h mutation analysis suggests a potential binding-site the most highly conserved portion of this rna is the h stem loop. due to its high sequence conservation, we hypothesize that this region is essential for rra-s binding. mutations made in this stem, rra-rna and rra-rna both significantly inhibit binding. the compensatory mutation, rra-rna was able to partially recover rra-s binding. in this compensatory mutant, it is likely an alternative base-pair forms with usually unpaired a- and the dynamic equilibrium of the two rna structures allows, but does not completely restore, rra-s binding. in combination with the truncation experiments above, these results suggest that rra-s binds its rna regulator in the highly conserved stem-loop structure of h but that h is still required for binding. the r. radiobacterrna allows regulation in response to s in vivo to determine whether the rra-rna has regulatory activity in addition to s15-binding activity, we conducted in vivo reporter assays to assess regulation. to do this we used a gfp reporter to measure expression of the gene following the rra-rna in response to different levels of rra-s the rna sequence was cloned in-frame as a translational fusion with the gfp reporter under the control of the iptg-inducible trc promoter. this construct included the rpso start codon, shine-dalgarno sequence, and the first nine codons of the rpso gene to form the rna-gfp fusion. on a second plasmid, the r. radiobacter rpso coding sequence was placed under the control of an l-arabinose inducible promoter. the pair of plasmids were co-transformed into an e. coli k12:Δrpso strain. we chose to use a surrogate organism, e. coli, due to its ease of use and manipulation, and the ability to obtain a knockout organism lacking endogenous s using this gfp in vivo reporter system, we assessed the ability of rra-s to regulate gene expression by measuring the gfp levels in the cells in the presence and absence of induced rra-s if the rna interacts with rra-s to regulate gene expression, we expect to see a decrease in gfp expression in cells expressing rra-s compared to cells not expressing rra-s cells co-transformed with plasmids containing full length rra-rna1-gfp, and rra-s were grown in the presence and absence of l-arabinose. the cells grown in the presence of the sugar displayed a ~4-fold decrease in gfp-reporter expression. because l-arabinose induces rra-s production, the decrease in gfp reporter expression is likely due to an interaction between the rna and rra-s next, to corroborate that our predicted transcription start site at the c- allows regulation, the sequence for rra-rna was also tested in this system and behaved in a similar manner. these results indicate an rna sequence starting at the transcription start site derived from r. spaeroides is sufficient to allow regulation in vivo.figure gfp reporter assays validate rra-rna’s regulatory role. gfp reporter assays validate the regulatory capacity of rra-rna in response to rra-s rra-rna structure and sequence are the same as described in figure  all relative fluorescence values were calculated by normalizing gfp/od all bars are 3+ independent experiments. *indicates p < ; **indicates p < . we also examined whether mutations to h that abolish rra-s binding would affect regulation. cells containing either rra-rna9-gfp and rra-rna10-gfp did not display a significant difference in gfp reporter expression when grown in the presence and absence of l-arabinose. this is likely because both rra-rna and rra-rna do not interact specifically with s and are unable to regulate the expression of the reporter. however, it should be noted that gfp expression levels in the absence of arabinose were also significantly lower than those observed with the rra-rna and rra-rna constructs. to assess whether the partial compensation of binding observed for rrarna in vitro represented a biologically relevant, functional compensation, rra-rna was also examined in this system. in this case, cells grown without l-arabinose displayed an increased gfp expression level compared to rra-rna and cells grown in the presence of l-arabinose had a ~10-fold decrease in relative gfp fluorescence. however, the increase in fold-change is solely due to increased rna11-gfp expression levels and the repressed level of gene expression is comparable between the two rna elements. thus, the rra-rna compensatory mutation that partially restored the in vitro rna-protein interaction also restored the regulatory interaction between the rra-rna and rra-s the partial restoration of in vitro binding by the compensatory mutant rra-rna is likely due to the presence of several competing structures formed by the rna under these conditions. however, the in vivo conditions enable the rna to adopt a secondary structure that increases overall reporter expression and enables regulation in response to s together, these assays indicate that not only the does this rna interact with rra-s in vitro, but it is a biologically relevant regulatory element responding to s structural probing confirms predicted secondary structure to further examine the secondary structure of rra-rna in the absence of protein we used several structural probing methods in combination with a minimal rna construct including nuclease cleavage assays with, and in-line probing. rnase vi cleaves double stranded rna non-specifically, rnase a cleaves single stranded c’s and u’s, and in-line probing the rna structure reveals the flexible regions of the rna structure that are more prone to spontaneous self-cleavage. although the putative stem h is predicted in our alignment, there are many sequences that contain short polypyrimidine sequences that are unpaired in our sequence alignment. these sequences may form alternative pairings with the ribosome-binding site. based on sequence data alone it is difficult to distinguish which bases are interacting with the ribosome-binding site. however, several lines of evidence indicate that we have identified the correct in vitro base-pairing conformation for our putative h in the r. radiobacter example of the rna. first, our mutagenesis and truncation analyses indicate that mutating the polyuridine does not alter protein-binding activity. this suggests that this region is unlikely to interact with the putative ribosome-binding site. however, deleting this region and the following hairpin abolishes protein binding indicating that h is important for protein binding. second, rnase v cleavage occurs symmetrically in regions that are base-paired in our figure, and rnasea cleavage occurs at c- as would be expected for a loop region. in addition, in-line probing shows that the entire 3′ portion of the molecule is somewhat flexible, from bases - through -. in conjunction with our mutagenesis results, this strongly suggests the correct pairing-element has been identified.figure structural probing confirms predicted secondary structure. a: rnase v, rnase a, no reaction, hydroxyl cleavage, denaturing rnase t, and two independent replicates of in-line probing reactions where the cleavage products have been separated by denaturing 10% page. cleaved cytosine and uridine residues in the rnase a reaction and cleaved guanosines in the denaturing t reaction were used to map cleavage to the rna structure, and regions of strong in-line cleavage are labeled. b: mapping of prominent cleavage sites to the structure of rra-rna6; bases in black are resolved on the gel. tss indicates predicted transcription start site. cleavage sites largely confirm structure anticipated from comparative genomics. consistent with our mutagenesis results, the highly conserved stem h is almost certainly double-stranded with a loop from c- to a- bases u- through c- are shielded from in-line attack and there are strong cleavage bands in rnase vi probing for bases c- bases - to - and g- also, there are no rnase a cleavage products for any of these uracils or cytosines, suggesting that these nucleotides are not single-stranded. there is also evidence for the predicted loop region in h probing with rnase a results in cleavage products for c- and c- and in-line probing reveals that c- through a- are flexible. at the base of the h stem, we predict a bulged adenosine and the highlighted region c of our in-line probing gel corresponds to this bulged base. this data corroborates our other evidence that the region essential for rra-s binding in h forms a double-helix. the nature of the junction between the two predicted helices is still unresolved. this region is not well-conserved so there is little phylogenetic evidence of structure, and in several cases the different assays give conflicting results, which may be the result of multiple folding conformations. the string of uridines from u– to u- does not appear to be flexible based on in-line probing, is cleaved by rnaase v and is not cleaved by rnase a, indicating that the region is not single-stranded. however, there are also no clear binding partners for these nucleotides suggesting that they may be forming a constrained tertiary structure. the string of cytosines that follows this region, c- to c- do show strong rnase a cleavage suggesting they are single-stranded, and this is corroborated by the in-line cleavage at these positions. however, these bases also display rnase v cleavage indicating that they may sometimes adopt a double-stranded conformation. nucleotides from - to - are not cleaved by either rnase v or rnase a, and appear to be structurally constrained. this suggests that they are not necessarily double-stranded, but may be participating in some tertiary structure. nucleotides - to - which potentially could interact with these bases, also show conflicting results, cleaving with both rnase v and rnase a. we have included the possible base pairing of the nucleotides at the base of h in our structure figures, but these interactions are likely weak. the accumulation of these data strongly suggests that both the predicted pairing elements do form, but the nature of the junction between these two elements remains unclear.  this work demonstrates the premise that nature may invent many unique ways to solve a single biological problem. in the context of other forms of rna-based regulation the diversity of distinct rna structures allowing cis-regulation of the rpso operon is nearly unmatched. the only similar example of such diversity in rna regulators for a specific function are the sam-binding riboswitches, where more than three completely distinct classes, and several additional sub-classes with re-arranged or modified secondary structure elements have been characterized. the s auto-regulatory rna structures we identified are quite diverse from one another, and from the existing known characterized s regulatory rnas that originate from e. coli, g. stearopthermophilus, and t. thermophilus. all of the previously characterized rna structures encompass a predicted shine-dalgarno sequence, but beyond such regulatory features the rnas appear to share very few common sequence features or patterns in secondary structure. while the s15-interacting rnas potentially share some tertiary structure similarities that are not captured in the secondary structure diagrams, previous studies indicate that the e. coli s does not interact with the regulatory rna originating from g. stearothermophilus. this finding suggests that there may be no single conserved tertiary structure shared by the s15-binding mrna structures. in the absence of structural data, it remains to be seen whether the structural diversity apparent in natural s15-interacting mrna structures is a result of rna’s inherent ability to generate a similar tertiary structure from diverse arrangements of primary and secondary structure, or from differences between the s protein homologs that lead to distinct pools of potential rna ligands. from the structures we describe here, it is clear that there are many ways to solve this particular biological problem. an exciting question that stems from this work is precisely how prevalent are rna solutions within sequence space that allow autogenous regulation in response to s based on the natural diversity of s15-interacting rnas, we expect that this number is large, and that as more genomes are sequenced and the sensitivity of computational searches increases, additional structures with this function will be identified. 
this work illustrates the importance of integrating comparative genomic and transcriptomic approaches during de novo ncrna identification and reveals a diversity of distinct natural rna regulators that support analogous biological functions. furthermore, this work indicates that many additional uncharacterized rna regulators likely exist within bacterial genomes and that the plasticity of rna structure allows unique, and likely independently derived, solutions to the same biological problem. to validate the biological relevance of our findings, we demonstrate that an example of the alphaproteobacterial rna from rhizobium radiobacter specifically interacts with s in vitro, and allows in vivo regulation of gene expression in an e. coli reporter system. three distinct rna structures from different bacterial phyla have demonstrated interactions with s to regulate gene expression; however, these rnas are distributed across a small fraction of bacterial diversity. we used comparative genomics in combination with analysis of existing transcriptomic data to identify three novel putative rna structures associated with the s coding region in microbial genomes. in addition, structural probing and nuclease protection assays confirm the predicted secondary structure and indicate nucleotides required for protein interaction. autogenous cis-regulators of ribosomal protein synthesis play a critical role in maintaining the stoichiometry of ribosome components. structured portions within an mrna transcript typically interact with specific ribosomal proteins to prevent expression of the entire operon, thus balancing levels of ribosomal proteins across transcriptional units.
 autogenous cis-regulators of ribosomal protein synthesis play a critical role in maintaining the stoichiometry of ribosome components. structured portions within an mrna transcript typically interact with specific ribosomal proteins to prevent expression of the entire operon, thus balancing levels of ribosomal proteins across transcriptional units. three distinct rna structures from different bacterial phyla have demonstrated interactions with s to regulate gene expression; however, these rnas are distributed across a small fraction of bacterial diversity.  we used comparative genomics in combination with analysis of existing transcriptomic data to identify three novel putative rna structures associated with the s coding region in microbial genomes. these structures are completely distinct from those previously published and encompass potential regulatory regions including ribosome-binding sites. to validate the biological relevance of our findings, we demonstrate that an example of the alphaproteobacterial rna from rhizobium radiobacter specifically interacts with s in vitro, and allows in vivo regulation of gene expression in an e. coli reporter system. in addition, structural probing and nuclease protection assays confirm the predicted secondary structure and indicate nucleotides required for protein interaction.  this work illustrates the importance of integrating comparative genomic and transcriptomic approaches during de novo ncrna identification and reveals a diversity of distinct natural rna regulators that support analogous biological functions. furthermore, this work indicates that many additional uncharacterized rna regulators likely exist within bacterial genomes and that the plasticity of rna structure allows unique, and likely independently derived, solutions to the same biological problem. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 