ROLE OF THE SPACER BOXA OF ESCHERICHIA-COLI RIBOSOMAL-RNA OPERONS IN EFFICIENT 23-S RIBOSOMAL-RNA SYNTHESIS IN-VIVO

A boxA sequence, known to be important for transcriptional antitermina tion, is found in both the leader region and in the spacer between the 16S and 23S genes of Escherichia coli ribosomal RNA operons. We have shown that a functional leader boxA is important for efficient complet ion of 16S rRNA transcription. In this study, point mutations were int roduced into the 16S-23S spacer boxA of a plasmid-encoded E. coli rrnB operon in order to study the contribution of this conserved sequence element to ribosomal RNA synthesis in vivo. The rrnB mutant constructs contained an additional point mutation in each of the 16S and 23S gen es, which were used to distinguish rRNA derived from plasmid and chrom osomal rrn operons by primer extension analysis. Mutations in the spac er boxA reduced the proportion of plasmid-derived 23S rRNA without aff ecting synthesis of plasmid-derived 16S rRNA or spacer boxA RNA, indic ating that premature termination of transcription occurred during 23S rRNA synthesis. Reductions in plasmid-derived 23S rRNA were very simil ar for total cellular RNA, 50S subunits and 70S ribosomes, suggesting that plasmid-derived rRNAs from mutant operons were functional in ribo some biogenesis. In the presence of a wild-type leader boxA, single nu cleotide exchanges in the spacer boxA reduced the proportion of plasmi d-derived 23S rRNA from 70% to about 55% under conditions of exponenti al growth in rich medium. This proportion further decreased to 20 to 2 5% with an additional point mutation in the leader boxA. We conclude t hat modification of RNA polymerase into a termination-resistant form h as to be renewed at the spacer boxA in order to ensure the faithful co mpletion of full-length 23S rRNA. (C) 1997 Academic Press Limited.