30 S pre-ribosomal RNA of Escherichia coli:primary and secondary processing

The metabolism of newly-formed labeled 30 S pre-ribosomal RNA was studied in the Escherichia coli mutant strain AB301-105. Detailed kinetic analysis in rifampicin-treated cells showed that precursors of 23 S and 16 S rRNA are formed from the 30 S RNA species, even in phosphate-limited cultures. To establish the order fo segments along 30 S pre-rRNA, it was allowed to accumulate in replicate chloramphenicol-treated cultures, and pulse-labeled after the addition of rifampicin in segments that progressively contained more 3'-distal label with time. The purified RNAs from the various cultures were then cleaved to a limited extent by RNAase III; the specific activity of the fragments yielded an order from the 5'-end of 17.5S-X-25S, where X refers to some of the sequences released as additional small fragments. More extensive treatment the 25-S and 17.5-S pre-rRNA chains with RNAase III yielded additional fragments and produced RNA species with the mobility of the 23-S and 16-S RNA precursors made in normal cells treated with chloramphenicol. A processing scheme is suggested that distinguishes between early, site-specific cleavage of recognition sites in the RNA itself ('primary processing'), and later, "secondary processing' reactions. The secondary processing reactions are blocked chloramphenicol-treated wild-type cells.