Maturation of the escherjchia cou tyrt transcript: involvement of multiple endo- and exoribonucleases

The TyrT transcript of E. coll contains a tandem repeat of tRNA-Tyrl followed by rtT mRNA. In vivo rtT mRNA is present in small amounts compared to the tRNA implying either that it is degraded rapidly or that shortening by 3'->5' exoribonucleases during maturation of tRNA-Tyrl destroys the mRNA in the process. In earlier studies we showed that endonucleolytic cleavage(s) at two sites separate the tRNA and the mRNA and that the exoribonuclease, RNase II. efficiently degrades the mRNA. In order to identify the enzyme(s) responsible for these cleavages, mutant strains lacking RNase P, RNase III, or RNase E, in addition to most of the exoribonucleases, were examined by Northern analysis tor the production of tRNA-Tyrl and rtT mRNA using probes complementary to different regions of the transcript. This analysis suggested an involvement of RNase E, but not RNase P or III in the cleavages. To test this point, processing of the TyrT transcript in vitro was examined. The TyrT transcript is cleaved efficiently by a cell extract derived from an exonuclease-deficient strain, generating tRNA and mRNA. The same products are observed when RNase III is missing. In an extract lacking RNase P, tRNA production is affected, but mRNA synthesis continues. In contrast, in a RNase E" extract the formation of both tRNA and mRNA is seriously affected and long precursors remain uncut. Based on these observations, we propose the following detailed maturation pathway for the TyrT transcript: RNase E makes at least one of the cleavages separating tRNA-Tyrl from rtT mRNA; RNase P cleavage at the 5' end of the tRNA precursor proceeds once the mRNA sequence is removed; exoribonucleases then trim extra residues to generate the mature 3' end of tRNA. (Supported by NIH Grant GM16317).