Transcription in bacteria at different DNA concentrations

G Churchward, H Bremer, R Young
1982 Journal of Bacteriology  
The effect of changing the DNA concentration on RNA synthesis, protein synthesis, and cell growth rate was studied in Escherichia coli B/r. The DNA concentration was varied by changing the replication velocity or by changing replication initiation in a thymine-requiring strain with a mutation in replication control. The results demonstrate that changes in DNA concentration (per mass) have no effect on the cell growth rate and the rates of synthesis (per mass) of stable RNA (rRNA, tRNA), bulk
more » ... RNA, tRNA), bulk mRNA, or protein or on the concentration of RNA polymerase (total RNA polymerase per mass). Thus, transcription in E. coli is not limited by the concentration of DNA, but rather by the concentration of functional RNA polymerase in the cytoplasm. Changing the DNA concentration does, however, affect fully induced lac gene activity, here used as a model for constitutive gene expression. The magnitude of the effect of DNA concentration on lac gene activity depends on the distribution of replication forks over the chromosome, which is a function of the replication velocity. Analysis of these data reinforces the conclusion that transcription is limited by the concentration of functional RNA polymerase in the cytoplasm. MATERLALS AND METHODS Bacterial strains and growth conditions. The bacteria used were E. coli B/r A (ATCC 12407; 22) and E. coli TJK16, which is a thyA deoB derivative of E. coli B/r A obtained from J. Kwoh (M.S. thesis, University of Texas at Dallas, 1975). NF955 ilv thr leu thi (A b515 b519 c1857 S7 A dilv5), used to prepare X dilv DNA for hybridization studies, was kindly provided by N. Fiil. Growth conditions were as described previously (23, 39). Determination of cell mass, DNA, protein, and RNA. Cell mass (units of optical density at 460 nm [OD4w] per 109 cells) and DNA (colorimetric assay) were determined as described previously (7, 12). There was no significant difference between the plating titer and the particle concentration for either strain used. To determine protein and RNA, 5-ml samples were precipitated with 1 ml of 3 M trichloroacetic acid, filtered through glass fiber filters (Reeve Angel; 984H), 572 on May 9, 2020 by guest http://jb.asm.org/ Downloaded from I Standard deviation, a (%) = NMT/£(n -1); A, deviation from average in percent; n, number of samples (10).
doi:10.1128/jb.150.2.572-581.1982 fatcat:fz3u6stp6zcffmbxbqgczzyhxe