In vitro genetic analysis of the hinge region between helical elements P5-P4-P6 and P7-P3-P8 in the sunY group I self-splicing intron

Modeling of the group I intron RNA suggests that its catalytic core is primarily composed of two extended structural elements (stacked helices P5-P4-P6 and P7-P3-P8) whose relative orientation is partially determined by base-triple interactions between paired regions P4 and P6, and single-stranded joining regions J6/7 and J3/4, respectively. In vitro genetic selection was used to isolate functional sequence variants of the proposed triple helical domain of the sunY intron. Comparative sequence analysis of the selected variants provided supporting evidence for the two previously established base-triples between P4 and J6/7 and provided the first experimental evidence for an interaction between P6(1) and J3/4(3). Sequence covariations also indicated that a simple relationship exists between the length of a single-stranded joining region, J3/4, and the identity of a particular base-pair, P4(1). Selected variants based on a core structure with an extra nucleotide inserted in J3/4 revealed two different responses to this structural perturbation: a base-triple interaction and an intrahelical bulged pyrimidine. Chemical modification analysis supported the existence of these alternative structures. The function of this region of the ribozyme can therefore be fulfilled by at least three different structures.