Ordered processing of Escherichia coli 23S rRNA in vitro.

In an RNase III-deficient strain of E. coli 23S pre-rRNA accumulates unprocessed in 50S ribosomes and in polysomes. These ribosomes provide a substrate for the analysis of rRNA maturation in vitro. S1 nuclease protection analysis of the products obtained in in vitro processing reactions demonstrates that 23S rRNA processing is ordered. The double stranded stem of 23S rRNA is cleaved by RNase III in vitro to two intermediate RNAs at the 5' end and one at the 3' end. Mature termini are then produced by other enzyme(s) in a soluble protein fraction from wild-type cells. The nature of the reaction at the 5' end is not clear, but the reaction at the 3' end is exonucleolytic, producing three heterogeneous mature termini. The two reactions are coordinated; 3' end maturation progresses concurrently with cleavages at the 5' end. Two results suggest a possible link between final maturation and translation: in vitro, mature termini are formed efficiently in the presence of additives required for protein synthesis; and all the processing intermediates detected from in vitro reactions are also found in polysomes from wild-type cells.