POINT MUTATIONS IN THE LEADER BOXA OF A PLASMID-ENCODED ESCHERICHIA-COLI RRNB OPERON CAUSE DEFECTIVE ANTITERMINATION IN-VIVO

We have introduced point mutations into the leader boxA of a plasmid-e ncoded Escherichia coli rrnB operon to study the in vivo role of this regulatory element in the natural context of rRNA synthesis. The same mutations were previously shown to cause severe antitermination defect s in vitro and in the context of a reporter gene assay. The plasmid-en coded rrnB mutant constructs studied here also contained point mutatio ns in the 16S and 23S rRNA genes, which were used to distinguish rRNAs derived from plasmid and chromosomal rm operons by primer extension a nalysis. Point mutations in boxA reduced the fraction of plasmid-deriv ed rRNA in the cell from 75% to about 50%. The reduction was similar f or both 30S and 50S subunits as well as 70S ribosomes, suggesting that no transcriptional polarity occurred between the expression of the 16 S and 23S rRNA genes in plasmid rrnB operons carrying a mutant boxA. T he boxA mutations do not affect the amount of transcription initiation , suggesting that a suboptimal leader boxA causes premature transcript ion termination at an early stage of transcription. Our results are co nsistent with a role for antitermination in the completion of full-len gth rm transcripts but give no indications of posttranscriptional boxA functions.