Mature 5S RNA has five stems connected and terminated by loops; the primary transcript has a 3' single stranded tail which is removed by processing. The internal domain consisting of loop D, stem IV and loop E is dispensable for processing. We have made numerous substitutions in Drosophila 5S RNA to map the influence of sequence and structure on processing, and interpreted the distribution of processing elements dispersed through stems I, II, III and loops B and C in two ways. First, sequence changes which stimulate processing cooperate when combined; we suggest that a processing protein makes multiple contacts with 5S RNA (long range cooperation). Second, poorly processed sequence variants cluster in the center of stems I and II, flanked by positions where changes which weaken base pairing improve processing. We suggest that a polypeptide arm reaches in from loop A yields stem I and from loop A yields stem II to make required central contracts (the breathing model). We propose to investigate long range cooperation and helix breathing at stem/loop junctions by separating the proteins involved in 5S RNA processing. Once the processing proteins are isolated, we will assess their sites of interaction with wild type and variant 5S RNA by footprinting. Protein contacts in stems I, II, III and loops B and C would be consistent with long range cooperation; such contacts should be weakened or absent from sequence variants with reduced processing. We will probe wild type and variant 5S RNA structure to evaluate athe breathing model. Substitutions which improve processing should increase probe access to central stem contracts in the event of breathing at stem/loop junctions. Finally, a randomization/processing size selection experiment will enable us to analyze many combinations of sequence variants for processing which would be impractical by site-specific mutagenesis. These investigations will contribute to our understanding of nucleic acid- protein interactions by using a small stable RNA as a processing substrate.
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