Cooperative binding of effectors by an allosteric ribozyme

Abstract

An allosteric ribozyme that requires two different effectors to induce catalysis was created using modular rational design. This ribozyme construct comprises five conjoined RNA modules that operate in concert as an obligate FMN- and theophylline-dependent molecular switch. When both effectors are present, this 'binary' RNA switch self-cleaves with a rate enhancement of approximately 300-fold over the rate observed in the absence of effectors. Kinetic and structural studies implicate a switching mechanism wherein FMN binding induces formation of the active ribozyme conformation. However, the binding site for FMN is rendered inactive unless theophylline first binds to its corresponding site and reorganizes the RNA structure. This example of cooperative binding between allosteric effectors reveals a level of structural and functional complexity for RNA that is similar to that observed with allosteric proteins.

Figure 1

Design and function of a binary RNA switch. (A) Modular rational design of TF1 RNA using five distinct RNA modules (numbered 1–5). Individual modules are depicted as an integrated sequence whose boundaries are schematically represented underneath. Communication modules cm⁺theo3 (15) and cm⁺FMN1 (13) are labeled theo3 and FMN1, respectively. Four putative ligand-dependent base pairs are labeled a–d. An arrowhead denotes the site of ribozyme cleavage. (B) Allosteric function of the TF1 RNA. Precursor RNA (Pre, internally ³²P-labeled) was incubated in the absence (–) or presence (+) of 1 mM theophylline (T) and/or 1 mM FMN (F) for 0 or 1 h. The region of gel containing the precursor and the 5′-cleavage fragment (Clv) is shown.

Figure 2

Activation of ribozyme function by two effectors. Plots of the natural logarithm of the fraction of TF1 that remains uncleaved versus time. (A) Both FMN (encircled F, 1 mM final concentration) and theophylline (encircled T, 1 mM final concentration) were added simultaneously to a reaction mixture at t = 120 min. Similarly, effectors were added independently in (B) and (C) at t = 60 min and t = 120 min, as indicated. The fraction of precursor RNAs that self-cleaved were established as described in Materials and Methods.

Figure 3

Kinetic modulation of a binary RNA switch. (A) Diagrammatic representation of the kinetic framework for binary allosteric ribozyme function. Encircled T and F represent bound theophylline and FMN, respectively. (B) Effector-dependent activation of TF1 self-cleavage. The maximum k_obs for ribozyme function in 1 mM each of theophylline and FMN is ∼1.2 × 10^–2 min^–1. (C) Double reciprocal plots reflecting the dependence of the observed rate constant for TF1 self-cleavage with various concentrations of effectors. Open, shaded and filled symbols represent data collected at 0.01, 0.1 and 1 mM theophylline, respectively.

Figure 4

RNA structure probing of various effector–RNA complexes. (A) Autoradiogram depicting the distribution of cleavage products resulting from spontaneous transesterification of TF1-v1 RNA. The top-most band represents 5′-³²P-labeled TF1-v1 RNA, while lower bands correspond to various 5′-cleavage fragments. Each band reflects spontaneous cleavage at a different site along the polynucleotide chain, which were identified by comparison to TF1-v1 cleavage products generated by partial digestion with alkali (^–OH) or with ribonuclease T1 (T1). Regions exhibiting the greatest frequency of spontaneous cleavage are denoted by nucleotide sequence and number. RNA strand scission occurs 3′ to each nucleotide listed. The arrowhead identifies the site of ribozyme cleavage. Cleavage products <18 nt in length are not depicted. For reactions grouped in lanes 5–7, 8–10 and 11–13, the three lanes in each contain 0.01, 0.1 and 1 mM, respectively, of the effector indicated. Lanes 11–13 additionally contain 1 mM theophylline. (B) Sequence and secondary structure model of TF1-v1 in the absence of effectors. An asterisk denotes the single A→G mutation at position 12 relative to TF1 that was incorporated to reduce ribozyme-mediated RNA cleavage during the probing reaction. Encircled nucleotides identify those positions that exhibit the highest frequencies of spontaneous cleavage under the respective probing conditions. (C) Model of TF1-v1 bound to theophylline (encircled T). Labeled rectangles identify RNA modules that are properly folded for binary switch function as depicted in Figure 1A. Other details are as described in (B). (D) Model of TF1-v1 bound to theophylline and FMN (encircled F). Other details are as described in (B) and (C).