A 3' splice site-binding sequence in the catalytic core of a group I intron.

Ribozymes use specific RNA-RNA interactions for substrate binding and active-site formation. Self-splicing group I introns have approximately 70 nucleotides constituting the core, a region containing sequences and structures indispensable for catalytic function. The catalytic core must interact with the substrates used for the two steps of the self-splicing reaction, that is, guanosine, the 5'-splice-site helix (P1) and the 3' splice site. Mutational evidence suggests that core sequences near segment J6/7 that joins the base-paired stems P6 and P7, and the bulged base of P7(5'), participate in binding guanosine substrate, but nothing is known about the interactions between the core, the 5'-splice-site helix and the 3' splice site. On the basis of comparative sequence data, it has been suggested that two specific bases in the catalytic core of group I introns might form a binding sequence for the 3' splice site. Here we present genetic evidence that such a binding site exists in the core of the Tetrahymena large subunit ribosomal RNA intron. We demonstrate that this pairing, termed P9.0, is functionally important in the exon ligation step of self-splicing, but is not itself responsible for 3'-splice-site selection.

Synthesis and characterization of an affinity label for brain receptors to psychotomimetic benzomorphans: differentiation of sigma-type and phencyclidine receptors.

Brain sigma-type receptors and phencyclidine receptors are thought to mediate the psychotomimetic effects of benzomorphans and phencyclidine in humans. Recently, we reported the characterization of a selective sigma receptor ligand, 1,3-di-o-tolyl-guanidine (DTG), that shows negligible crossreactivity with phencyclidine receptors. Here we describe the synthesis and characterization of an isothiocyanate derivative of DTG, di-o-tolyl-guanidine-isothiocyanate (DIGIT). Guinea pig brain membranes treated with nanomolar doses of DIGIT followed by extensive washing exhibit a dose dependent reduction of [3H]DTG and (+)[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [+)[3H]3-PPP) binding to sigma receptors. Binding of radiolabelled ligands for phencyclidine, mu-opioid, benzodiazepine and dopamine-D2 receptors is not affected by membrane treatment with DIGIT, indicating specificity of the affinity label for sigma-type receptors. Treatment of DIGIT-derivatized membranes with 2 M NaCl does not result in recovery of sigma binding activity, suggesting that DIGIT's interaction with sigma receptors is not of an ionic nature. Equilibrium saturation binding experiments show that the inhibition of [3H]DTG binding to sigma receptors by DIGIT pretreatment of membranes is attributable to an irreversible reduction in the affinity (increase in Kd) of sigma receptors for DTG. The finding that sigma receptors are irreversibly modified by DIGIT whereas phencyclidine receptors are not affected suggests that sigma receptors are physically separate from phencyclidine receptors. The availability of a selective affinity label for the sigma receptor should facilitate the purification of the receptors and the characterization of sigma-type pharmacological effects in vivo and in vitro.