The self-splicing Group I introns have a highly specific binding site for the substrate guanosine. Mutant versions of the Tetrahymena ribozyme have been used in combination with guanosine analogues to identify the nucleotide in the ribozyme that is primarily responsible for recognition of the guanine base.

The biochemical and genetic analyses of P-glycoprotein (P-gp) have indicated that the membrane-associated regions of P-gp play an important role in drug recognition and drug transport. Predicted transmembrane domain 11 (TM11) maps near a major drug binding site revealed by photoaffinity labeling, and mutations in this domain alter the substrate specificity of P-gp. To investigate further the role of TM11 in P-gp function in general, and substrate specificity in particular, each of the 21 residues of TM11 of the P-gp isoform encoded by the mouse mdr3 gene was independently mutated to alanine, or to glycine in the case of endogenous alanines. After transfection and overexpression in Chinese hamster ovary cells, pools of stable transfectants were analyzed for qualitative or quantitative deviations from the profile of resistance to vinblastine, adriamycin, colchicine, and actinomycin D displayed by the wild-type protein. While mutations at eight of the positions had no effect on P-gp function, 13 mutants showed a 2-10-fold reduction of activity against one of the four drugs tested. Although the phenotype of individual mutants was varied, replacements at most mutation-sensitive positions seemed to affect the drug resistance profiles rather than the overall activity of the mutant P-gp. When TM11 was projected in a alpha-helical configuration, the distribution of deleterious and neutral mutations was not random but segregated with a more hydrophobic (mutation-insensitive) face and a more hydrophilic (mutation-sensitive) face of a putative amphipathic helix. The alternate clustering pattern of deleterious vs neutral mutations in TM11 together with the altered drug resistance profile of deleterious mutants suggest that the more hydrophilic face of the TM11 helix may play an important structural or functional role in drug recognition and transport by P-gp. Finally, the conservation of the two residues most sensitive to mutations (Y949 and Y953) in TM11, and in the homologous TM5, of all mammalian P-gps and also in other ABC transporters, suggests that these residues and domains may play an important role in structural as well as mechanistic aspects common to this family of proteins.

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The group I self-splicing introns are RNA enzymes that catalyze phosphodiester-exchange reactions. These ribozymes have a highly specific binding site for guanosine, a substrate for the first self-splicing reaction (Bass & Cech, 1984). The binding site for guanosine has been localized to a specific region of the ribozyme (Michel et al., 1989), but the conformation of the bound guanosine substrate remains unknown. Most analogs of guanosine with substituents at C8 have a preference for the syn conformation; however, some C8-substituted analogs have the potential to form a hydrogen bond between the C8 substituent and the 5'-hydroxyl that would stabilize the anti conformation; we have found that analogs with the potential to form such a hydrogen bond are more active substrates than those that cannot form such a hydrogen bond. These observations led us to test 8-5'-O-cycloguanosine, which is locked in the anti conformation, and 8-(alpha-hydroxyisopropyl)guanosine, which is locked in the syn conformation; the former is active as a substrate, while the latter is inactive. These results strongly suggest that guanosine is bound to the ribozyme in the anti conformation and provide an additional constraint on structural models of this RNA enzyme. We have also examined a series of N7-substituted guanosine analogs; this position had previously been assumed to be unimportant for substrate binding since 7-methylguanosine is an excellent substrate. However, we have found that 7-deazaguanosine and 7-methyl-7-deazaguanosine are less active substrates than guanosine. We discuss several models for the role of N7 in guanosine binding.