Molecular analysis of a synthetic tetracycline-binding riboswitch

Abstract

Riboswitches are newly discovered regulatory elements that consist solely of RNA, sense their ligand in a preformed binding pocket, and perform a conformational switch in response to ligand binding, resulting in altered gene expression. Regulation by a tetracycline (tc)-binding aptamer when inserted into the 5 untranslated region (UTR) of a reporter gene exhibits all characteristics of a riboswitch. Chemical and enzymatic probing reveals that the aptamer consists of two stems, P1 and P2, which are already present in the absence of tc and form the scaffold of the aptamer. They are separated by a bulge B1-2 and an opposing stem-loop (P3-L3). Tc-dependent changes in the probing pattern only appear in the upper part of the bulge B1-2 (nucleotides 9-13) and the loop L3. Saturating mutagenesis corroborates the involvement of these two regions in regulation. Structural probing of the mutant A55U, which contains a single-nucleotide exchange in loop L3 results in a changed probing pattern of the loop, but also of the opposing bulge B1-2. This denotes that both regions cooperate and form a composite binding pocket. Thus, our model for aptamer-mediated translational regulation is that the ligand-free aptamer has only marginal influence on translational initiation. Tc then leads to an intramolecular connection in a pseudoknot-like manner and turns the aptamer into its inhibitory form. This represents a new mechanism for riboswitch action clearly distinguished from currently known naturally occurring riboswitches, which function by sequestration of the ribosomal binding site, transcriptional attenuation, and ribozyme-mediated degradation.

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

“…This contact might be disturbed in the spin labeled aptamer conformation. However, it has been shown that nucleotide exchanges on position 12 retain the regulatory activity of the aptamer (Hanson et al 2005), and the Tc-binding constants for FIGURE 1. Secondary structure predicted for the Tc aptamer according to Hanson et al (2005).…”

“…However, it has been shown that nucleotide exchanges on position 12 retain the regulatory activity of the aptamer (Hanson et al 2005), and the Tc-binding constants for FIGURE 1. Secondary structure predicted for the Tc aptamer according to Hanson et al (2005). Thiouridines (U) spin labeled with MTS are marked by full gray circles, 29-aminouridine labeled with 4-isocyanato-TEMPO are depicted by open gray circles.…”

“…The RNA aptamer comprises three helices (stems P1, P2, and P3), the single-stranded joining regions J1-2 and J2-3 and the loop L3 ( Fig. 1; Hanson et al 2005;Xiao et al 2008). All three stem regions are not in direct contact with Tc and form the scaffold already in the absence of the ligand.…”

Screening for engineered neomycin riboswitches that control translation initiation

“…The resulting PCR product covers the entire 59 UTR with the aptamer and the first 48 nt of the gfp coding region. RNA transcripts were dephosphorylated and subsequently 59-32 P-end labeled as previously described (Hanson et al 2005). RNA (100,000 cpm) was incubated in 50 mM sodium acetate (pH 4.5), 280 mM NaCl, 4.5 mM ZnSO 4 (S1), or 50 mM Tris-HCl (pH 7.5) (T1) for 2 min at 56°C and for 10 min at 37°C.…”

StreAM- $$T_g$$ : Algorithms for Analyzing Coarse Grained RNA Dynamics Based on Markov Models of Connectivity-Graphs

“…These findings suggest a strong interaction with TC and thus a dramatic, positive change in H. Nucleotides A52 and U54 show a positive entropy difference inside L3. Interestingly, molecular probing experiments show that G51, A52, and U54 of L3 are-in the absence of the antibiotic-the most modified nucleotides [23,34]. Clearly, they change their conformational flexibility upon ligand binding due they direct interaction with the solvent.…”