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-encoded 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 defects in vitro and in the context of a reporter gene assay. The plasmid-encoded rrnB mutant constructs studied here also contained point mutations in the 16S and 23S rRNA genes, which were used to distinguish rRNAs derived from plasmid and chromosomal rrn operons by primer extension analysis. Point mutations in boxA reduced the fraction of plasmid-derived rRNA in the cell from 75% to about 50%. The reduction was similar for both 30S and 50S subunits as well as 70S ribosomes, suggesting that no transcriptional polarity occurred between the expression of the 16S and 23S rRNA genes in plasmid rrnB operons carrying a mutant boxA. The boxA mutations do not affect the amount of transcription initiation, suggesting that a suboptimal leader boxA causes premature transcription termination at an early stage of transcription. Our results are consistent with a role for antitermination in the completion of full-length rrn transcripts but give no indications of posttranscriptional boxA functions.
Heinrich
T
T
Institut für Biochemie, Freie Universität Berlin, Germany.
Condon
C
C
Pfeiffer
T
T
Hartmann
R K
RK
D001483:Base Sequence; D004275:DNA, Ribosomal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012335:RNA, Ribosomal / Q000096:biosynthesis* / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000096:biosynthesis / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S