Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch

Abstract

Riboswitches are cis-acting elements that regulate gene expression by affecting transcriptional termination or translational initiation in response to binding of a metabolite. A typical riboswitch is made of an upstream aptamer domain and a downstream expression platform. Both domains participate in the folding and structural rearrangement in the absence or presence of its cognate metabolite. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls the ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the aptamer domain and the expression platform. Pausing at strategic locations may be a general mechanism for coordinated folding and conformational rearrangements of riboswitch structures that underlie their response to environmental cues.

Fig. 1.

Secondary structures of the E. coli btuB riboswitch. (A) The secondary structure of the aptamer domain in the off structure encompassing residues 1–202 is the same as that derived by Breaker and coworkers (6, 7). The structurally important regions are shown in orange (anti-anti-RBS, residues 148–161), purple (pseudoknot 3′ region, residues 187–191), green (antiaptamer, residues 162–209), and brown (anti-RBS = 212–216 and RBS = 229–233, residues 210–235). In the coenzyme B12 bound off structure, the anti-anti-RBS region is part of the aptamer, allowing the pairing of the anti-RBS with the RBS regions. In the apo, or on structure, the anti-anti-RBS region pairs with the anti-RBS region to form an antiaptamer structure, leaving the RBS available for ribosome binding. (B) Phylogenetic support for the anti-RBS and RBS hairpin in the off structure, and for the anti-anti-RBS and anti-RBS interaction (the antiaptamer structure) in the on structure. (C) Structural mapping using oligonucleotide hybridization/RNase H cleavage assay in the absence and presence of 100 μM coenzyme B12. Sequences of all oligos and their complementary regions in the btuB riboswitch are listed in Fig. S2. In the first round, a total of 26 oligos were used spanning residues 12–243. Examples of the first round mapping are shown in the upper panel where the transcripts are 5′ ³²P labeled. Oligo length is determined by having similar melting temperatures and varies from 6 to 26 nucleotides. In the second round, a total of 31 10-mer oligos were used spanning residues 142–242. Examples of the second round mapping are shown in the lower panel where the transcripts are internally labeled. The full-length RNA transcript also contains the first 20 codons of the btuB gene (residues 243–302); this region is not probed in our experiment. FL, full-length transcript; P, RNase H cleavage product. Star corresponds to RNAs that are either paused transcripts or randomly cleaved fragments. (D) Structural probing data are plotted as fraction protected ± coenzyme B12 versus nucleotide position. The y axis is plotted in log ₂ scale. Aptamer, gray; antiaptamer, green; pseudoknot, purple; RBS hairpin, brown.

Fig. 2.

Pausing of btuB riboswitch during transcription. (A) A large number of pauses are present. All transcripts are 5′ ³²P labeled using initiating dinucleotide [³²P]pAU. The aptamer domain is indicated by a vertical bar. Most prominent pauses occur near the end (P_A) and downstream (P_B and P_C) of the aptamer domain. Mapping of the sites A–C is shown in Fig. S8. (B) Location of the pause sites A, B, and C. Site A is between the anti-anti-RBS region and the pseudoknot region. Site B is in the RBS hairpin loop between the RBS and its pairing partners—i.e., the anti-RBS region. When RNAP pauses at sites A and B, the anti-anti-RBS region is outside, whereas the anti-RBS region is in the RNAP exit channel. In the paused complex C, the anti-RBS region is available, but the RBS region is still in the exit channel. Both the aptamer and the antiaptamer structures can form in paused complex C.

Fig. 3.

Pausing-deficient, mutant RNAP transcription affects folding fraction of the btuB riboswitch. (A) Both mutant RNAPs significantly affect pausing at sites A–C. Lane 240+ corresponds to high [NTP] chase after 4 min. (B) Schematic representation of the two RNA constructs. The full-length construct has 302 residues and contains the entire aptamer domain, the expression platform, and the first 20 codons of the BtuB gene. The aptamer-only construct has 202 residues and contains just the aptamer domain as defined in the literature (6). The location of the pause sites A–C is also indicated. (C) Transcription by the fast RNAPs reduces the ratio of protection in the absence and presence of coenzyme B12 for both the aptamer domain and the expression platform. The cleavage reaction with the RBS probe was carried out with 25% the amount of RNase H, as compared to the cleavage reaction with the aptamer probe. (D) Transcription by fast RNAPs has little effect on differential fraction folded of the aptamer-only construct.

Fig. 4.

Pausing-deficient RNAP affects folding kinetics. The cleavage reaction with the RBS probe was carried out with 25% the amount of RNase H, as compared to the cleavage reaction with the aptamer probe. (A) Folding kinetics of the aptamer and expression platform during transcription by the wild-type RNAP. (B) The folding rate constants of the aptamer domain and the expression platform are within 1.5-fold to each other when transcribed by the wild-type enzyme, both in the absence and presence of coenzyme B12 ligand. In contrast, transcription by the pausing-deficient, fast RNAPs leads to increased divergence of the folding rates, especially in the presence of coenzyme B12. (C) Folding rates of the aptamer-only construct do not depend on the RNAP used in the transcription.