Structural analysis of an RNA molecule involved in replication control of plasmid Rl

Abstract

The replication control circuit of the FII plasmids R1, R100 and R6-5 involves a small untranslated RNA molecule of about 93 nucleotides. This RNA, designated CopA RNA in plasmid R1, acts in vivo as a post-transcriptional inhibitor of the expression of the RepA protein whose synthesis is required for each round of plasmid replication. We have studied the structure of CopA RNA in solution by probing with single-strand- as well as double-strand-specific nucleases. Our data show that CopA RNA has two stem-loop structures connected by a long spacer region and a single-stranded 3'-tail. Enzyme reactions were performed under buffer conditions where we find strong and specific binding of CopA RNA to its target RNA (to be published elsewhere), and the patterns were compared to digests in two other buffer systems. All partial cleavages supported the proposed secondary structure. We also analyzed the stem-loop regions of CopA RNAs from two R1 copy number mutants. The data show that both RNAs have altered loop structures. The implications of these findings are discussed.