In vivo RNA sequences and structures required for M1 RNA processing

Abstract

When the rnpB gene encoding M1 RNA, the catalytic RNA component of E. coli RNase P, is transcribed, the primary M1 RNA transcript of 413 nucleotides is produced, and subsequently processed at the 3`` end to generate the mature M1 RNA of 377 nucleotides. However, the mechanism of M1 RNA processing is relatively unexplored. To examine the requirement of the M1 RNA sequence in the primary transcript for the processing reaction, first, we constructed plasmids containing the rnpB gene with a series of internal deletions in the M1 RNA sequence. The deletion plasmids containing at least both 56 nucleotides of the 5`` region and 47 nucleotides of the 3`` region in the M1 RNA sequence generated a truncated M1 RNA transcript processed normally at the 3`` end. The plasmids also produced a larger transcript with the 3`` end at position +402 in the precursor M1 RNA sequence. These results suggest that the intact M1 RNA sequence is not required for M1 RNA processing and the event of the processing involves at least one intermediate that has not been reported yet. In order to investigate the sequence and structural elements required for the processing, mutations were introduced in the cleavage site and the stem formed by the sequences between positions +1 and +10 and between positions +364 and +373 in one of the deletion plasmid. The mutation experiments show that the cleavage site nearly has no effect on the processing but the loss of the stem impairs the processing and/or stability of the truncated transcripts. Additional mutations capable of forming a similar stem by compensatory base change restored the processing, suggesting that a structural motif generated by the stem is important in the M1 RNA processing.