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Although the three structural genes, aceB, aceA and aceK, which code for the E.coli glyoxylate cycle enzymes, reside in a single operon, the aceK is known to be expressed very inefficiently compared with the upstream gene, aceA. In this study, Northern blot and S1 nuelease mapping analysis have been employed to investigate the transcriptional role in the differential expression between the two genes. When three different probes, each derived from different regions of an openn clone, were used for Northern hybridizatioiL the amount and size of the message revealed by each probe were quite distinct; two abundant species of operon RNA were revealed by a probe derived from the 3´ region of aceA, but a single species of RNA with relatively low abundance was detected by a 5´ aceK probe. More interestingly, virtually no message was visible under the same experimental conditions when using a 3´ aceK probe. This dramatic reduction in the level of message corresponding to the 3´ coding region of aceK was confirmed in the subsequent S1 nuclease protection analysis, which clealy demonstrated that the majority of the operon messages have their 3´ end just about 300 base pairs downstream from the start codon of aceK. Thus the results from Northern and S1 hybridizations are consistentwith the conclusion that most of the operon messnges do not reach even the middle of the coding sequence of aceK, providing direct evidence for the participation of some important transcriptional regulation in the downregulation of aceK expression. The previous localizationof one of the major determinants for the polarity to the aceA/aceK intergenic region was also confirmed in this study by constructing an aceA::aceK fusion gene.
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