5S RNA, a small RNA of the large ribosomal subunit required for protein synthesis, has a conserved five domain stem-loop secondary structure. Like most eukaryotic RNAs, D. melanogaster 5S RNA is not utilized directly after transcription. The 135 nt primary transcript has 15 nt removed from its 3' end by processing. This proposal concerns the structural basis for 5S RNA processing. 5S RNA processing is relatively unaffected by changes in the nucleotides downstream from the processing site at +120, and by deletion of 30 nt from stem IV-V. On the other hand, deletions in stem III, removing all of stem IV-V, and disruption of the +1-118 base pair, the first paired nucleotides in stem I, all interfere with processing. Herein I propose to thoroughly investigate the domains required for 5S RNA processing. Site-directed mutagenesis of 5S RNA genes linked to a T7 RNA polymerase promoter will be performed. Mutant 5S RNAs will be transcribed with T7 RNA polymerase and tested using a Drosophila melanogaster processing extract for their ability to be processed. Sets of three mutations will be introduced into the top or bottom of helical stems to disrupt base pairing, or into both top and bottom to restore it. Paired deletions and insertions will be made to alter the length of helixes. The effect of length and sequence will also be analyzed in each of the single stranded loops. By means of these experiments, we shall evaluate in detail the relative contribution of nucleotide sequence and base paired structure to the processing ability of Drosophila melanogaster 5S RNA.
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