S1 nuclease mapping analysis of ribosomal RNA processing in wild type and processing deficient Escherichia coli

Abstract

S1 nuclease mapping was used to assess rRNA processing in Escherichia coli. Single-stranded DNA probes complementary to the sequences bordering each terminus of 16 S and 23 S rRNA were end-labeled, hybridized to total E. coli RNA, and treated with S1 nuclease. The resultant DNA fragments were then displayed on denaturing polyacrylamide gels. Measurements of steady state levels of precursor rRNA species and measurements of the rates of decay of precursors after transcription arrest by rifampicin gave consistent results. 1) The rRNA precursor species identified in wild type cells corresponded to those previously identified by other means. 2) In RNase III-deficient strains, mature 16 S rRNA termini form at the same rate as in wild type cells; but the normal mature termini of 23 S rRNA are never generated. 3) RNase III cleavage at the 5' end of 23 S rRNA can occur before the 3' end of the same molecule is synthesized. 4) The cleavages that generate the mature termini of 16 S rRNA are interdependent; in the BUMMER strain, slow processing at the 5' end is accompanied by slow processing at the 3' end. Thus, the kinetically observed order of processing reactions is obligate for some cleavages but not for others, and the assumption that complete rRNA processing is required for function fails for 23 S rRNA.