Ribozyme catalysis from the major groove of group II intron domain 5.

The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

Remarkable morphological variability of a common RNA folding motif: the GNRA tetraloop-receptor interaction.

One of the most common RNA tertiary interactions involves the docking of GNRA hairpin loops into stem-loop structures on other regions of RNA. Domain 5 of the group II intron interacts with Domain 1 through such an interaction, which has been characterized thermodynamically and kinetically for the ai5g intron. Using this system, it was possible to test the morphological tolerances of the GNRA tetraloop involved in tertiary interactions. The data presented herein show that a GNRA tetraloop can still participate in tertiary interaction after being physically cut at any phosphodiester linkage within the loop. The "nicked tetraloop" can be expanded by many nucleotides in either direction and the covalently continuous loop can also be expanded without loss of interaction energy. In the context of the nicked tetraloop, the second nucleotide of the tetraloop sequence can be completely deleted without loss of function. By examining radical alterations in tetraloop structure, this study helps define the minimal sequence and structural requirements of a GNRA motif involved in long-range tertiary interaction. It shows that "tetraloop"-like structures capable of forming tertiary interactions can be imbedded in unexpected contexts, such as internal loops and apparently open structure within RNA. It demonstrates that pentaloops and hexaloops can form the same type of interaction, with almost equal affinity, as a tetraloop. Taken together, these data suggest a more generic term for the GNRA tetraloop-receptor interaction: It is proposed herein that the term "GNRA tetraloop" be replaced by "GNn/RA", where n represents a variable number of nucleotides and / indicates that the loop can be divided and interrupted by other sequences.

Catalytic role of 2'-hydroxyl groups within a group II intron active site.

Domain 5 is an essential active-site component of group II intron ribozymes. The role of backbone substituents in D5 function was explored through synthesis of a series of derivatives containing deoxynucleotides at each position along the D5 strand. Kinetic screens revealed that eight 2'-hydroxyl groups were likely to be critical for activity of D5. Through two separate methods, including competitive inhibition and direct kinetic analysis, effects on binding and chemistry were distinguished. Depending on their function, important 2'-hydroxyl groups lie on opposite faces of the molecule, defining distinct loci for molecular recognition and catalysis by D5.