Search

Satellite DNA (satDNA) is a major component of genomes but relatively little is known about the fine-scale organization of unrelated satDNAs residing at the same chromosome location, and the sequence structure and dynamics of satDNA junctions. We studied the organization and sequence junctions of two nonhomologous satDNAs, pBuM and DBC-150, in three species from the neotropical Drosophila buzzatii cluster (repleta group). In situ hybridization to microchromosomes, interphase nuclei and extended DNA fibers showed frequent interspersion of the two satellites in D. gouveai, D. antonietae and, to a lesser extent, D. seriema. We isolated by PCR six pBuM x DBC-150 junctions: four are exclusive to D. gouveai and two are exclusive to D. antonietae. The six junction breakpoints occur at different positions within monomers, suggesting independent origin. Four junctions showed abrupt transitions between the two satellites, whereas two junctions showed a distinct 10 bp tandem duplication before the junction. Unlike pBuM, DBC-150 junction repeats are more variable than randomly cloned monomers and showed diagnostic features in common to a 3-monomer higher-order repeat seen in the sister species D. serido. The high levels of interspersion between pBuM and DBC-150 repeats suggest extensive rearrangements between the two satellites, maybe favored by specific features of the microchromosomes. Our interpretation is that the junctions evolved by multiples events of illegitimate recombination between nonhomologous satDNA repeats, with subsequent rounds of unequal crossing-over expanding the copy number of some of the junctions.

The crystal structure of the RNA-binding domain of the small nuclear ribonucleoprotein U1A bound to a 21-nucleotide RNA hairpin has been determined at 1.92 A resolution. The ten-nucleotide RNA loop binds to the surface of the beta-sheet as an open structure, and the AUUGCAC sequence of the loop interacts extensively with the conserved RNP1 and RNP2 motifs and the C-terminal extension of the RNP domain. These interactions include stacking of RNA bases with aromatic side chains of proteins and many direct and water-mediated hydrogen bonds. The structure reveals the stereochemical basis for sequence-specific RNA recognition by the RNP domain.

We recently determined the crystal structure of the RNP domain of the U1 small nuclear ribonucleoprotein A and identified Arg and Lys residues involved in U1 RNA binding. These residues are clustered around the two highly conserved segments, RNP1 and RNP2, located in the central two beta strands. We have now studied the U1 RNA binding of mutants where potentially hydrogen bonding residues on the RNA binding surface were replaced by non-hydrogen bonding residues. In the RNP2 segment, the Thr11----Val and Asn15----Val mutations completely abolished, and the Tyr13----Phe and Asn16----Val mutations substantially reduced the U1 RNA binding, suggesting that these residues form hydrogen bonds with the RNA. In the RNP1 segment Arg52----Gln abolished, but Arg52----Lys only slightly affected U1 RNA binding, suggesting that Arg52 may form a salt bridge with phosphates of U1 RNA. Ethylation protection experiments of U1 RNA show that the backbone phosphates of the 3' two-thirds of loop II and the 5' stem are in contact with the U1 A protein. The U1 A protein-U1 RNA binding constant is substantially reduced by A----G and G----A replacements in loop II, but not by C----U or U----C replacements. Based on these biochemical data we propose a structure for the complex between the U1 A ribonucleoprotein and U1 RNA.