Probing Drosophila 5S RNA loop C using an in vitro processing model.
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I Arjun
Publications and source records attributed to I Arjun.
Point mutations distal to the processing site affect Drosophila pre-5 S RNA processing. Long range cooperation and a breathing model.
Drosophila pre-5 S RNA, which consists of five conserved stem-loop domains and a 15-nucleotide 3' tail, is 3'-end processed to 120 nucleotide mature 5 S RNA before ribosome assembly. Large deletions in stems II and III, all of stems IV and V, and loop C prohibit Drosophila 5 S RNA processing; deletion of stem IV and half of V does not (Preiser, P. R., and Levinger, L. (1991a) J. Biol. Chem. 266, 7509-7516). Several point mutations in stem I reduce, while certain neighboring sequence changes stimulate, processing (Levinger, L., Vasisht, V., Greene, V., and Arjun, I. (1992) J. Biol. Chem. 267, 23683-23687). Herein we extend this 5 S RNA fine structure analysis to regions farther from the processing site. Most point mutations in loop B, stem III, and loop C severely inhibit processing. One loop C substitution stimulates processing; when combined with stimulatory sequence changes in stem I and loop A, these dispersed mutations improve processing manyfold, perhaps by stabilizing a required conformation or strengthening a protein-binding site. Central stem II sequence changes inhibit processing; several adjacent sequence substitutions which weaken base pairing improve processing. Combining these results with earlier work from stem I and loop A, we hypothesize that slight reduction in base pairing may improve groove access of polypeptide chains to essential contact positions.
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The effects of stem I and loop A on the processing of 5 S rRNA from Drosophila melanogaster.
The 135-nucleotide Drosophila melanogaster 5 S RNA precursor is processed by removal of 15 nucleotides from its 3' end before incorporation into the large ribosomal subunit. Mature 5 S RNA consists of five helical stem-loops; stem IV and part of V are dispensable, whereas stem III and the 1/118 G-C base pair closest to the processing site at nucleotide 120 are required for processing (Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 7509-7516; Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 23602-23605). We have investigated the effects of stem I and loop A transversions, transitions, selected additions and deletions on 5 S RNA processing. Stem I single substitutions generally prevent processing, whereas compensatory double substitutions restore a range of processing rates. Proximal to the processing site, stem I double substitutions inhibit processing. In the distal portion of stem I and loop A, the processing effect of paired sequence changes varies widely in an irregular pattern. The 7/112 GU pair and nucleotide 13A least tolerate sequence changes; several mutations clustered close to the stem I-loop A boundary stimulate processing. We interpret these results in terms of the RNA helix path and possible RNA-protein contacts.
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