We plan to continue our ongoing studies on ribosomal RNA (rRNA). In the first section, we will conclude experiments dealing with evolution of ribosomal DNA. One of these defines the minimum length of sequence identity necessary for homologous recombination in yeast rDNA. Another study proposed in this section deals with selective forces of evolution acting upon rDNA mutations that have been inserted by gene replacement into yeast rDNA. The second section will fill the majority of the five year grant period; it addresses RNA-RNA interactions involving rRNA, and is divided into 3 parts: (1) U3-rRNA (2) peptidyl transferase domain (3) SRP-rRNA. In the first part, U3 snRNA will be sequenced in Xenopus laevis and Xenopus borealis to determine regions that may be complementary to the ITS 2 (internal transcribed spacer), suggesting a role of U3 snRNA in 5.8S RNA processing during 32S to 28S rRNA maturation. Genomic clones of U3 Xenopus will be prepared and their sequence organization studied. Xenopus oocyte infections will be done to study U3 transport to the nucleolus, and antisense U3 snRNA will provide a functional test for the role of U3 snRNA. In another experiment, an inducible form of yeast U3 will be constructed to assay possible U3 function in rRNA processing. The second part looks at rRNA-rRNA interactions within the peptidyl transferase domain, using reverse transcriptase primer extension structure probing of Xenopus 28S rRNA in various functional states. The results may provide experimental support for RNA """"""""switches"""""""" as a driving force for mRNA movement through the ribosome via a tRNA change of state. In vitro binding of complementary synthetic oligonucleotides provide a functional test for this. In the third part, experiments are described to study interactions between 7SL RNA of the SRP (signal recognition particle) and rRNA, resulting in inhibition of translation.
