RNase III makes the initial cleavages that excise Escherichia coli precursor 16S and 23S rRNA from a single large primary transcript. In mutants deficient in RNase Ill, no species cleaved by RNase Ill are detected and the processing of 23S rRNA precursors to form mature 23S rRNA fails entirely. Instead, 50S ribosomes are formed with rRNAs up to several hundred nucleotides longer than mature 23S rRNA. Unexpectedly, these aberrant subunits function well enough to participate in protein synthesis and permit cell growth. Consistent with the inference that RNase III cleavages are absolutely required for 23S rRNA maturation, when 50S ribosomes from a strain deficient in RNase HI were incubated with a ribosome-free extract from a RNase III+ strain, rRNA species processed by RNase III and species with normal mature 23S rRNA termini were produced. rRNA in Escherichia coli is synthesized as one transcript containing 16S, 23S, and 5S rRNA as well as precursor sequences. This transcript is thought to be processed first by RNase III, which cleaves the precursor at double-stranded stems and separates the three rRNA species from one another before transcription is complete; other processing activities then remove the remaining precursor sequences during ribosome formation, generating the mature termini (1, 2). Because RNase III initiates processing, and processing has been generally considered indispensable to produce functional RNA molecules, it was unexpected that mutants deficient in the enzyme could survive. It was originally suggested that a low level of RNase III might remain in such mutants or that other RNases might form an alternative pathway to yield mature rRNA (3-5). These ideas have persisted in recent discussions, but both are disproven here in the case of 23S rRNA formation. Nuclease S1 mapping is used to show that the lesion in RNase III is complete in these mutants, but no backup pathway replaces the function of RNase III. The 16S rRNA is matured normally without any preliminary RNase III cleavage, but 23S rRNA exists only as unmatured species. Maturation of 23S rRNA is therefore absolutely dependent on RNase III action, but at least some of the unmatured 23S rRNA species detected must function in protein synthesis because these mutant strains are viable. The maturation of the unprocessed 23S rRNA in 50S ribosomes is shown to occur in subcellular extracts of a RNase III+ strain. MATERIALS AND METHODSThe termini of rRNA species in two RNase III-deficient strains, ABL1 and AB301-105 (6, 7), were assessed by their capacity to hybridize to DNA probes composed of complementary sequences of rDNA. ABL1 and AB301-105 cells were grown in Luria broth at 30°C to an optical density of 0.25 at 550 nm and then harvested over ice. Total RNA was prepared by extracting cells directly at 65TC with phenol in the presence of 1% sodium dodecyl sulfate. The RNA was precipitated once with LiCI (8) and twice with ethanol and was then suspended at a standard concentration. Singlestranded DNA hybridization probes for each termi...