ANALYSIS OF THE IN-VIVO DECAY OF THE ESCHERICHIA-COLI DICISTRONIC PYRF-ORFF TRANSCRIPT - EVIDENCE FOR MULTIPLE DEGRADATION PATHWAYS

Messenger RNA decay III Escherichia coli is slowed in pnp-7 (PNPase) r nb-500 (RNase II) rne-1(RNase E) multiple mutants. We have used Northe rn blots, S-1 nuclease protection and primer extension analysis to map 18 endonucleolytic cleavage sites within the pyrF-orfF dicistronic tr anscript. Although examination of a total of 27 cleavage sites includi ng those determined for the monocistronic trxA transcript revealed a c omplex pattern, the central four nucleotides within a cluster of 12 re sidues encompassing the cleavage sites showed a definite A/U preferenc e. Also of interest was the processing of the dicistronic transcript t o remove the downstream orfF sequence as a stable but untranslated RNA fragment. The data provide further support for the hypothesis that mu ltiple decay pathways are involved in the decay of a single transcript . In particular, the pyrF-orfF transcript apparently can be degraded e ither in the 5' to 3' or the 3' to 5' direction. Our results are discu ssed in light of current models of mRNA decay involving polyadenylatio n and multiprotein decay complexes. (C) 1997 Academic Press Limited.