We propose to obtain complete DNA sequences, both within and adjacent to four of the seven ribosomal RNA operons (rrn) of Escherichia coli. We also propose to sequence the terminator region of the rplJL-rpoBC operon which specifies the beta and beta' subunits of RNA polymerase. This DNA sequence information will provide the basic foundation required to evaluate other experiments defining the structure, function and organization of these important operons. We are using both the Maxam & Gilbert and the Sanger dideoxy DNA sequencing methods in conjunction with the cloning system worked out by Messing which uses the single stranded DNA bacteriophage, M13. The primary sequence data will be analysed for significant secondary structure features which may be related to control, structure and function of the gene products. Additional complementary techniques will be used to examine the role that these nucleic acid sequences play in determining the expression and function of the operons. RNA-DNA hybridization combined with nuclease S1 mapping will be used to accurately delineate RNA synthesis and processing patterns in the rrn operons. We propose to obtain transcript maps of genes preceeding and following the rrn operons and of the region following the rpo terminator. The combined DNA sequences and RNA transcript maps will be used to study rRNA processing and to examine the hypothesis that flanking DNA regions are involved in the expression of rrn operons. We will also define the termination process in the rpo and rrn operons. Experiments designed to probe the genetic organization of rRNA genes will attempt to inactivate one or more rrn operons to see whether all are necessary for survival of the cell. Lastly, experiments will be performed to determine whether the observed re-assortment of segments in some of the rrn clones is a consequence of the cloning process or, rather, reflects recombination between rrn cistrons in the E. coli genome itself.
| Harvey, S; Hill, C W; Squires, C et al. (1988) Loss of the spacer loop sequence from the rrnB operon in the Escherichia coli K-12 subline that bears the relA1 mutation. J Bacteriol 170:1235-8 |
