RNA aptamers that specifically bind dopamine have been isolated by in vitro selection from a pool of 3.4 x 10(14) different RNA molecules. One aptamer (dopa2), which dominated the selected pool, has been characterized and binds to the dopamine affinity column with a dissociation constant of 2.8 microM. The specificity of binding has been determined by studying binding properties of a number of dopamine-related molecules, showing that the interaction with the RNA might be mediated by the hydroxyl group at position 3 and the proximal aliphatic chain in the dopamine molecule. The binding domain was initially localized by boundary experiments. Further definition of the dopamine binding site was obtained by secondary selection on a pool of sequences derived from a partial randomization of the dopa2 molecule. Sequence comparison of a large panel of selected variants revealed a structural consensus motif among the active aptamers. The dopamine binding pocket is built up by a tertiary interaction between two stem and loop motifs, creating a stable framework in which five invariant nucleotides are precisely arrayed. Minimal active sequence and key nucleotides have been confirmed by the design of small functional aptamers and mutational analysis. Enzymatic probing suggests that the RNA might undergo a conformational change upon ligand binding that stabilizes the proposed tertiary structure.

We have detected two paralogs of the tRNA endonuclease gene of Methanocaldococcus jannaschii in the genome of the crenarchaeote Sulfolobus solfataricus. This finding has led to the discovery of a previously unrecognized oligomeric form of the enzyme. The two genes code for two different subunits, both of which are required for cleavage of the pre-tRNA substrate. Thus, there are now three forms of tRNA endonuclease in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaea and the Nanoarchaea. The last-named enzyme, arising most likely by gene duplication and subsequent ''subfunctionalization,'' requires the products of both genes to be active. molecular evolution ͉ RNA-protein interactions ͉ tRNA splicing G ene duplication is the primary source of new genes. The ''subfunctionalization'' hypothesis argues that duplicate genes experience degenerate mutations that divide the activity encoded by a single ancestral gene among its descendants (1). Here, we report a striking example of subfunctionalization.In Archaea, the tRNA endonuclease plays a key role in assuring the correct removal of the intron from pre-tRNAs and pre-rRNA (2-6), which constitute the core of the translation machinery. Crystal structures of the tRNA endonucleases from Methanocaldococcus jannaschii (METJA) and Archaeoglobus fulgidus (ARCFU), both belonging to the phylum Euryarchaeota, are available (7,8). These structures differ in a remarkable way. The structure of the homotetrameric endonuclease from METJA reveals two different functional roles for the monomeric units. The METJA endonuclease is organized as a dimer of dimers, with one subunit from each dimer participating in the catalytic cleavage reaction (the catalytic subunit) and the other (structural) subunit acting to place the two catalytic subunits correctly in space.The crystal structure of the ARCFU endonuclease, by contrast, shows it to be a homodimer. Each subunit contains two similar repeating domains that are homologous to the subunit structure of the homotetrameric enzyme from METJA; the C-terminal repeat (CR) is the active domain, and the N-terminal repeat (NR) acts to stabilize the dimer.The overall shape and size of the homodimeric ARCFU endonuclease resembles that of the homotetrameric METJA enzyme.Both METJA and ARCFU belong to the Euryarchaeota. Nothing is known about the tRNA endonuclease of Crenarchaeota, the other main family of Archaea. To determine the properties of a crenarchaeal endonuclease, we searched the genome sequence of Sulfolobus solfataricus (SULSO) for homologs of the METJA endonuclease and found two candidate sequences.Characterization of the two gene products reveals that both are required for tRNA endonuclease activity, each presumably functioning like one-half of the ARCFU enzyme. Detailed analysis of the amino acid sequences of the two proteins supports the idea that they evolved by the process called subfunctionalization (1, 9, 10). Materials and MethodsIdentification of the Homologs. Th...

Members of the three kingdoms of life contain tRNA genes with introns. The introns in pre-tRNAs of Bacteria are self-splicing, whereas introns in archaeal and eukaryal pre-tRNAs are removed by splicing endonucleases. We have studied the structures of the endonucleases of Archaea and the architecture of the sites recognized in their pre-tRNA substrates. Three endonuclease structures are known in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaeota. The homotetramer cleaves only the canonical bulge-helix-bulge structure in its substrates. Variants of the substrate structure, termed bulge-helix-loops, appear in the pretRNAs of the Crenarcheota and Nanoarcheota. These variant structures can be cleaved only by the homodimer or heterotetramer forms of the endonucleases. Thus, the structures of the endonucleases and their substrates appear to have evolved together. molecular evolution ͉ RNA-protein interactions ͉ splicing

Accuracy in transfer RNA (tRNA) splicing is essential for the formation of functional tRNAs, and hence for gene expression, in both Eukaryotes and Archaea. The specificity for recognition of the tRNA precursor (pre-tRNA) resides in the endonuclease, which removes the intron by making two independent endonucleolytic cleavages. Although the eukaryal and archaeal enzymes appear to use different features of pre-tRNAs to determine the sites of cleavage, analysis of hybrid pre-tRNA substrates containing eukaryal and archaeal sequences, described here, reveals that the eukaryal enzyme retains the ability to use the archaeal recognition signals. This result indicates that there may be a common ancestral mechanism for recognition of pre-tRNA by proteins.