The post-transcriptional regulation of gene expression underlies several critical developmental phenomena. In metazoa, gene products that are expressed, silenced and packaged during oogenesis govern early developmental processes prior to nascent transcription activation. Furthermore, tissue-specific alternative splicing of several transcription factors controls pattern formation and organ development. A highly conserved family of proteins containing a STAR/GSG RNA-binding domain is essential to both processes. Here, we identify the consensus STAR-binding element (SBE) required for specific mRNA recognition by GLD-1, a key regulator of Caenorhabditis elegans germline development. We have identified and verified new GLD-1 repression targets containing this sequence. The results suggest additional functions of GLD-1 in X-chromosome silencing and early embryogenesis. The SBE is present in Quaking and How mRNA targets, suggesting that STAR protein specificity is highly conserved. Similarities between the SBE and the branch-site signal indicate a possible competition mechanism for STAR/GSG regulation of splicing variants.

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme 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.

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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.

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