In vitro analysis of processing at the 3 -end of precursors of M1 RNA, the catalytic subunit of Escherichia coli RNase P: multiple pathways and steps for the processing

Abstract

M1 RNA of 377 nucleotides, the catalytic subunit of Escherichia coil RNase P, is produced by a 3' processing reaction from precursor M1 RNA, a major transcript from the rnpB gene. We analyzed products and intermediates generated by the in vitro processing reaction using a 40% ammonium sulfate precipitate of the S30 fraction (ASP-40) and determined their involvement in the processing. From the results we proposed a model of two pathways for 3' processing of M1 RNA, In this model, one pathway (pathway I) involves +385/+386 intermediates and the other pathway (pathway II) does not. The position of the 3'-end of the precursor molecule determined the choice of the pathways. The precursor having the 3'-end of +413 was processed by both pathways while that having the +415 end was processed only by pathway II. The ASP-40 fraction generated processing products (termed +3781+379 RNA) containing one or two more nucleotides at the 3'-end than M1 RNA, regardless of which pathway was used. Therefore, both pathways require the final 3' trimming for complete processing. The endonucleolytic generation of +3781+379 RNA by pathway II was blocked by the rne-3071 mutation, suggesting that this step is carried out by RNase E.