Phylogenetic analysis of coenzyme B12 riboswitches reveals a complex RNA motif
The 5′-UTRs of the btuB
genes from E.coli
each contain a large RNA domain that functions as a metabolite-dependent genetic control element (10
). Both RNAs bind coenzyme B12
(5′-deoxy-5′-adenosylcobalamin or AdoCbl) with high affinity and specificity, and thus function as natural aptamers for this important metabolite. These RNAs, along with the 5′-UTR of the cob
gene from S.typhimurium
, carry a short conserved sequence domain termed the B12
), and therefore they likely share structural and functional homology.
Although we proposed a partial secondary structural model for the riboswitch domains present in the btuB
mRNAs from E.coli
), the model was not in agreement with that proposed recently for the 5′-UTR of the cob
). To improve upon the structural model, additional examples of the coenzyme B12
class of riboswitches were identified in other organisms. This was achieved through an iterative approach to secondary-structure modeling and comparative sequence analysis using a computer algorithm. The algorithm (SequenceSniffer; J. E. Barrick and R. R. Breaker, unpublished) searches genomic databases for domains that correspond closely to specific sequence and structural constraints, and has been used previously to identify homologs for the TPP (9
), guanine (5
), SAM (7
) and lysine riboswitches (15
). More specifically, database searches such as these are effective in revealing similar aptamer domains, as the metabolite-binding portions of riboswitches are the most highly conserved amongst various organisms and amongst different mRNAs within a single organism.
A total of 92 different representatives of the coenzyme B12
class of riboswitches were identified and compared by sequence alignment (Fig. ; see also the Rfam database for additional representatives) (16
). Upon examination of the sequence alignments, a pattern of conserved sequence and secondary-structure elements that are present in all representatives becomes evident (Fig. A). The most striking observation is that the B12
box encompasses only a small portion of the conserved sequence and structural domain. The phylogenetic analysis indicates that this complex RNA motif has at least 10 base-paired elements and ~57 nucleotide positions whose identity is conserved in >90% of the representative sequences.
Figure 1 Sequence alignment of putative coenzyme B12 riboswitches. Conserved sequence and base pairing elements are highlighted for 43 of 92 representative motifs identified by searching GenBank (see Materials and methods). The domain’s strand orientation (more ...)
Figure 2 Consensus sequence and secondary-structure model for the metabolite-binding domain of the B12 class of riboswitches. (A) Red nucleotides depict bases whose identities are conserved in at least 90% of the representatives of the phylogeny assembled (more ...)
The secondary-structure model proposed previously for the riboswitch from E.coli btuB
) corresponds only partially with the new model that is based on phylogenetic data. Specifically, pairing elements P4 (part) and P5 to P8 of the original structural model are retained in the revised model with the modified designations P5 and P8 to P12, respectively (Fig. B). In addition, the P9 stem from the original model is retained without designation in the revised model, as it resides outside the domain of highest sequence and structural conservation. In contrast, the revised model includes numerous short base-paired elements that explain the role of nucleotides that were left unfolded in the earlier model, and also that preclude formation of certain other pairing possibilities. Specifically, stems P1 to P3, as predicted in the original model, are replaced and supplemented in the revised model by stems P1 to P4, P6, P7 and P10.
The revised secondary-structure model is similar to a model proposed recently for this same RNA motif (17
). Most notably, these two models differ with respect to how the B12
box is folded, and on the structural details of the bridge between the left and right halves of the E.coli btuB
aptamer. Several lines of evidence support our revised structural model for the B12
aptamer. First, the new secondary-structure model is largely consistent with structural probing data that was presented previously (10
). Likewise, mutational analysis supported the formation of a bridging stem element that is retained in the revised model (P8). Thirdly, representatives within the sequence alignment (Fig. ) exhibit considerable sequence covariation within the stem elements that are common to most B12
riboswitches (Fig. B), indicating that these pairing elements are important for riboswitch function. Specifically, the covariation in sequences of proposed stems P3 and P6 (Fig. ) suggest that these structures are more likely to be formed than the alternative structure proposed recently (17
The revised secondary-structure model is more complex than those of other riboswitches. Interestingly, biochemical evidence (see below) suggests that, at least in some cases, RNA constructs that encompass only the minimal aptamer domain are not sufficient to exhibit maximal binding affinity to coenzyme B12. These latter results indicate that the more highly conserved portion of the riboswitch might not comprise the complete and fully functional aptamer motif. Thus, the likely involvement of more distal RNA elements (such as a possible pseudoknot interaction between the loop of P5 and downstream sequences) adds even greater complexity to the RNA’s structure.
5′-UTRs from the btuB and cob mRNAs of S.typhimurium bind coenzyme B12
, genetic expression of the cob
mRNA from S.typhimurium
is known to be regulated by B12
-related compounds (14
). We speculated that the presence of the conserved B12
box also could be an indication that the mRNA that encodes for the majority of the genes required for de novo
synthesis of coenzyme B12
might be negatively regulated by the metabolic end product. Consistent with this hypothesis is the observation that our database search, along with another reported recently (17
), for sequence elements with similarity to the B12
aptamer identified the 5′-UTR of the cob
operon from S.typhimurium
As expected, the 5′-UTR from S.typhimurium btuB
mRNA has considerable sequence and predicted structural similarity to that of the homologous E.coli
leader (Fig. A). In contrast, the sequence of the corresponding cob
5′-UTR varies significantly from that of btuB
. Furthermore, the secondary-structure model proposed recently (14
) for the cob
RNA differs substantially from our model for the coenzyme B12
aptamer presented in Figure . However, we find that the cob
5′-UTR does carry the consensus sequence elements that are indicative of the B12
class of riboswitches, despite the considerable sequence differences between the two RNAs. Furthermore, the cob
5′-UTR can be folded into a secondary structure (Fig. B) that carries the major features of the B12
aptamer model (Fig. A). The differences in sequence and predicted base-pairing potential reside in portions of the RNA that are not conserved through evolution. Thus, the two leader domains from the btuB
mRNAs from S.typhimurium
each carry a putative ligand binding aptamer domain for a B12
Figure 3 Structure and function of two B12 riboswitch domains from S.typhimurium. Sequence and secondary structure models for the B12 aptamer domains residing in the 5′-UTRs of the btuB mRNA (A) and the cob operon mRNA (B) from S.typhimurium. Constructs (more ...)
In-line probing analyses were conducted with fragments of both the btuB
mRNA leader sequences from S.typhimurium
that correspond to the putative B12
aptamer domains. As indicated previously (10
), the btuB
mRNA undergoes B12
-dependent structure modulation with a pattern that is similar to that observed for the homologous mRNA from E.coli
(Fig. C). The data also reveal that substantial ligand-dependent structure modulation occurs with the cob
mRNA fragment (Fig. D). Upon closer inspection, the spontaneous cleavage patterns for both RNAs correspond well with the revised secondary-structure model for the B12
aptamer, wherein the sites of modulation largely correlate with conserved bulges and sites of constant high cleavage correlate with the non-conserved loops of stems. Therefore, we conclude that the 5′-UTR of the cob
mRNA directly binds coenzyme B12
without the participation of protein factors, and that the biosynthetic pathway for this metabolite is regulated, at least in part, by a coenzyme B12
Ligand-binding affinity varies with the length of the 5′-UTR fragment
The ligand-dependent changes in spontaneous RNA cleavage can be quantitated and used to derive an apparent KD
value for ligand–aptamer complex formation (21
). This approach was used previously to obtain an apparent KD
value for the interaction of coenzyme B12
with the 202 btuB
construct from E.coli
of ~300 nM (10
). Similarly, the corresponding 206 btuB
construct from S.typhimurium
exhibits an apparent KD
value of ~400 nM (Fig. A).
Figure 4 Measurements of binding affinities of various coenzyme B12 aptamers. (A) Representative plot of the ligand concentration-dependent modulation of spontaneous RNA cleavage using the 206 btuB fragment from S.typhimurium. The fraction of RNA cleaved at sites (more ...)
The binding affinities for certain riboswitch constructs are known to vary depending upon the length of the construct used. For example, the thiamine pyrophosphate (TPP) aptamers from the thiM
mRNAs of E.coli
exhibit the highest affinity for TPP, while larger constructs that carry portions of the expression platform each bind with significantly lower affinity (4
). This is likely due to the presence of competing RNA structures that detract from tight ligand binding by reducing preorganization of the aptamer domain. In other words, the allosteric nature of the full riboswitch element (aptamer plus expression platform) might be expected to result in a poorer equilibrium constant for complex formation with its target ligand due to these dynamic but mutually exclusive folds.
We examined several RNA constructs that encompass the minimal consensus sequence and structure for the B12 aptamer to determine whether such molecules might exhibit higher affinity for the coenzyme. Interestingly, the shortened constructs for the two btuB RNAs and for the cob RNA experience a significant (>100-fold) loss of binding affinity when the riboswitch is trimmed to represent only the conserved aptamer domain (Fig. B). This suggests that sequence or structural element(s) within the presumed non-conserved domain of the riboswitch aid in proper formation of the more highly conserved aptamer.
In contrast to other instances where extra-consensus sequences detract from binding affinity, the consensus B12
aptamer domain (at least in some cases) makes productive use of flanking sequences to form an aptamer domain with improved affinity. Previously, we had suggested that a pseudoknot might play an important role in the formation of the B12
riboswitch structure of E.coli btuB
). This speculative proposal was based on the fact that structural probing data were consistent with such an interaction, although mutational analysis was inconclusive. This proposed pseudoknot, which is depicted as a base pairing interaction between the loop of P5 and the loop of a non-conserved hairpin (Fig. B), might play a role in supporting the consensus domain in its function as a B12
aptamer. If true, this putative interaction is not obviously present in all other representatives of the phylogeny examined in this study.
Confirmation of coenzyme B12–cob RNA interaction by equilibrium dialysis
Equilibrium dialysis was used to confirm that the cob
mRNA sequence directly binds coenzyme B12
in the absence of proteins (data not shown), as had been determined previously (10
) for the corresponding btuB
mRNA fragment from E.coli
. A Scatchard analysis was also carried out for the 386 cob
RNA by using equilibrium dialysis with various concentrations of tritiated coenzyme B12
(data not shown). Unfortunately, coenzyme B12
is highly photo-labile, and the data were not of sufficient quality to establish stoichiometry with confidence.
Specifically, we found that 27% of the tritium in a control equilibrium dialysis assay failed to bind the RNA, which is most likely due to breakdown of the compound during assembly of the assay mixtures, despite taking precautions to minimize exposure to light. The uncertainty with respect to the extent of degradation in each independent equilibrium dialysis run likely contributes scatter in the placement of data points on the Scatchard plot. Although the plot should be interpreted with caution, a best-fit line to the data indicates a dissociation constant for the cob aptamer of ~200 nM, which is similar to that determined by in-line probing (Fig. B). The x intercept of this line resides between 0.75 and 1, which is most consistent with a 1:1 relationship between ligand and RNA when in complex.
A variant coenzyme B12 riboswitch occurs in the B.subtilis yvrC mRNA
The phylogenetic survey of domains that resemble B12 riboswitches revealed a single example from B.subtilis (Figs and A). This representative resides immediately upstream of an apparent four-gene operon that carries the open reading frames (ORFs) yvrC, yvrB, yvrA and yvqK. Although the functions of these ORFs remain unproven, the yvr genes appear to encode proteins involved in metal import and processing, and thus modulation of their levels of genetic expression in response to coenzyme B12 concentrations seems reasonable since this coenzyme carries a metal ligand.
Figure 5 A variant riboswitch aptamer retains selective binding of coenzyme B12. (A) The B12 riboswitch-like domain residing upstream of the yvrC gene of B.subtilis. The 149 yvrC and 120 yvrC constructs carry an additional two nucleotides preceding position 1 (more ...)
Interestingly, the B.subtilis variant is lacking the P8, P10 and P11 helices, which otherwise would present sequence and structural elements that are conserved in most other representatives (Fig. A). The absence of this highly conserved portion of the B12 aptamer makes the yvrC RNA one of the most extreme variants identified in our phylogenetic analysis. Therefore, we conducted several experiments to determine whether the yvrC RNA binds coenzyme B12 and whether the riboswitch-like domain responds genetically to this effector when appended to a reporter gene.
Three RNA constructs were created based on the UTR sequence of B.subtilis yvrC. The two largest constructs, 259 yvrC and 149 yvrC, both exhibit structural modulation upon addition of coenzyme B12, which indicate that they both retain ligand-binding function (Fig. B and C) despite the absence of the P8, P10 and P11 helices and the associated conserved nucleotides. However, the 120 yvrC construct, which carries only those nucleotides that correspond closely to the left-most portion of the consensus aptamer motif, fails to bind coenzyme B12 as indicated by the absence of ligand-induced structural modulation (Fig. D).
This finding suggests that a structural element that is critical for high-affinity binding resides within nucleotides 121 and 149 of the larger yvrC
constructs. In addition, this result suggests that the B12
riboswitch might make use of a two-lobed aptamer, where the left and right halves of the RNA form distinct folds that independently recognize different parts of the ligand. In this model, the novel 29-nucleotide domain that forms the 3′ terminus of 149 yvrC
might serve as a functional replacement for the absence of stems P8 to P12 that are found in the E.coli
and S.typhimurium btuB
RNAs. In a preliminary assessment of this hypothesis, we have examined whether the molecular recognition characteristics of the yvrC
variants might be different. Specifically, the yvrC
leader exhibits poorer discrimination against purinyl cobalamin (PurCbl) (10
) than does the btuB
riboswitch from E.coli
(data not shown). Although other explanations for this observation can be envisioned, it is possible that the right half of the aptamer binds the adenosyl moiety of coenzyme B12
and that the 29-nucleotide alternative domain from yvrC
recognizes this moiety somewhat differently.
The 5′-UTR of the B.subtilis yvrC mRNA also permits coenzyme B12-dependent modulation of a reporter gene when expressed in vivo. To assess riboswitch function in vivo, a DNA construct carrying the first 275 nucleotides of the yvrC sequence (Fig. A) was appended to a β-galactosidase gene in a transcriptional reporter vector. When integrated into B.subtilis, the construct exhibits an ~3-fold reduction of β-galactosidase activity when coenzyme B12 is added to the culture medium. Specifically, Miller units of 142 and 47 were obtained in the absence and presence of B12 supplementation to the medium, respectively. These findings indicate that the yvrC variant is a functional coenzyme B12-specific riboswitch, despite its weaker correspondence to the sequence and structure of most other representatives in the phylogeny.