Identification and characterization of transcription termination sites in the Escherichia coli lacZ gene.
The Escherichia coli lacZ gene contains a series of latent transcriptional terminators that are responsible for the polar effects of certain mutations. We demonstrate, using gel electrophoretic size analyses and nuclease S1 mapping procedures, that RNA polymerase terminates RNA synthesis in the vicinity of five positions 180, 220, 379, 421 and 463 base-pairs downstream from the start point during transcription of lacZ DNA in vitro in the presence of rho factor. Termination at all but the 421 position depends on rho factor. In the in vitro assays with 0.05 M-KCl and excess rho (36 nM), the terminators are moderately effective, having efficiencies that range from about 8% at the 180 base-pair site to 56% at the 463 base-pair site. These termination stop points correspond to five of the 11 transcriptional pause sites between 180 and 463 base-pairs. Several stop points also correspond to 3' end points of lacZ mRNA isolated from cells containing the strongly polar lacZ-U118 mutation and from cells starved for serine, thus confirming that these latent terminators are responsible for the polar effect and demonstrating that they also function under a condition of physiological stress that prevents the transcription from being translated properly. Two other potential termination factors, NusA protein and cyclic AMP receptor protein have no effect in vitro on the efficiency of termination at the five lacZ sites.
Ruteshouser
E C
EC
Department of Chemistry, Indiana University, Bloomington 47405.
Richardson
J P
JP
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005809:Genes, Regulator; D007763:Lac Operon; D008969:Molecular Sequence Data; D011189:Potassium Chloride / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics; D015183:Restriction Mapping; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000378:metabolism
D012329:RNA, Bacterial; D011189:Potassium Chloride; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate