Sensing complex regulatory networks by conformationally controlled hairpin ribozymes

Abstract

The hairpin ribozyme catalyses RNA cleavage by a mechanism utilizing its conformational flexibility during the docking of two independently folded internal loop domains A and B. Based on this mechanism, we designed hairpin ribozyme variants that can be induced or repressed by external effector oligonucleotides influencing the docking process. We incorporated a third domain C to assimilate alternate stable RNA motifs such as a pseudo-half-knot or an internal stem-loop structure. Small sequence changes in domain C allowed targeted switching of ribozyme activity: the same effector oligonucleotide can either serve as an inducer or repressor. The ribozymes were applied to trp leader mRNA, the RNA sequence tightly bound by l-tryptophan-activated trp-RNA-binding attenuation protein (TRAP). When domain C is complementary to this mRNA, ribozyme activity can be altered by annealing trp leader mRNA, then specifically reverted by its TRAP/tryptophan-mediated sequestration. This approach allows to precisely sense the activity status of a protein controlled by its metabolite molecule.

Figure 1

TRAP-responsive and mRNA-regulated hairpin ribozymes. (a) Hairpin ribozyme and fluorescent substrate (orange). Ribozyme and substrate sequences are in uppercase and lowercase letters, respectively. Substrate-binding arm A and the B domain are indicated in blue and green letters, respectively. The arrow indicates the substrate-cleavage site. Cleavage of substrate results in product dissociation and FRET decay (green light at 520 nm). F, fluorescent label (FAM); Q, fluorescence quencher (TAMRA). The flanking helical stems are abbreviated as H1 to H4. (b) Comparison of a classical three-way junction (3WJ) and the so-called pseudo-three-way junction (pseudo-3WJ). Insertion of a pseudo-3WJ motif at the hinge region allows the cleavage reaction to proceed in the trans-mode. (c) Design of the hairpin ribozyme rHP–TRAP, repressed by trp-mRNA. The colour scheme is the same as in (a). The sequence shown in black letters is complementary to trp-mRNA. Domain C is inserted at the hinge region between H2 and H3 [between positions A14 and A15 in (a)] and comprises the terminal connecting domain, TCD and the unpaired loop. The grey shadowed TCD sequence plus the unpaired loop of domain C hybridize to trp-mRNA, resulting in repression of rHP–TRAP catalysis. (d) Insertion of an additional seven nucleotides (boxed) into the trp-mRNA binding region of domain C produces an inducible trp-mRNA-responsive ribozyme (iHP–TRAP). The boxed region in domain C can hybridize to the ribozyme-part of domain A (the sequence shown in blue), preventing substrate (orange) binding to the ribozyme to create an inactive ribozyme construct.

Figure 2

Trp-mRNA-dependent repression and induction of hairpin ribozymes. (a) Hybridization of trp-mRNA to rHP–TRAP inhibits catalysis by forcing A and B domains into an extended conformation. (b) The same trp-mRNA induces a conformational switch in iHP–TRAP which creates a pseudo-half-knot structure stabilized by the terminal helical sequence shown in red, thereby inducing cleavage activity. (c) Inactivation of rHP–TRAP and (d) induction of iHP–TRAP in the presence of increasing concentrations of trp-mRNA as indicated. Maximal rate enhancement occurrs at a ratio of 1 : 1 of trp-mRNA and iHP–TRAP, or rHP–TRAP, respectively. Reactions were carried out under MTO conditions with 50 nM iHP–TRAP/rHP–TRAP; 1 μM substrate. (e) Cleavage reactions were monitored using fluorescence signal measurement as previously described (38). Shown here is a time course of substrate-RNA cleavage catalysed by rHP–TRAP (circles) and iHP–TRAP (squares) in the presence of equimolar trp-mRNA (filled squares and circles) or the absence of any mRNA (open squares and circles). Reactions were carried out under multiple turnover conditions (MTO) to show maximum substrate turnover.

Figure 3

(a) Mg²⁺ dependence of the inducible construct iHP–TRAP in the presence of 50 nM trp-mRNA. (b) Spermidine dependence, with increasing Mg²⁺ concentrations, of the inducible construct iHP–TRAP in the presence of 50 nM trp-mRNA, in FBB and 1.0 mM l-tryptophan at 37°C. Notably, at a concentration of 8.0 mM spermidine iHP–TRAP shows activity in the presence of 50 nM mRNA despite the low concentration of Mg²⁺ (4.0 mM).

Figure 4

Sensing TRAP regulatory networks dependent on l-tryptophan. (a) In the presence of l-tryptophan (open circles) TRAP protein (grey shaded circle) is able to bind trp leader mRNA (grey). The presence or absence of l-tryptophan and TRAP causes the sequestration or release of trp-mRNA and thus shifts the equilibrium either in favour of inactive iHP–TRAP or active rHP–TRAP ribozyme conformations. (b) iHP–TRAP ribozyme activity regulated by increasing TRAP protein concentrations. Column 1, ribozyme alone; column 2, ribozyme/trp-mRNA (1 : 1); columns 3–7, ribozyme/trp-mRNA (1 : 1) with increasing concentrations of l-tryptophan-activated TRAP (10-1000 nM) (black bars). l-tryptophan concentration was 1.0 mM. The grey bars represent similar negative control experiments with 1.0 mM d-tryptophan. (c) Analogous to (b), but with the rHP–TRAP construct. Both ribozyme formats specifically respond to the interaction of l-tryptophan with the TRAP protein and the resulting formation of the TRAP/trp-mRNA complex. (d) l-tryptophan dependence of TRAP protein activation investigated using the iHP–TRAP ribozyme and a constant concentration of TRAP (1 μM). Assays were performed in the presence of l-tryptophan (black bars) or d-tryptophan (grey bars) at the indicated concentrations.