Titration of the gene sites on DNA by DNA-RNA hybridization: II. The Escherichia coli chromosome

Abstract

Escherichia coli RNA, having the same specific radioactivity in all molecules (long-labeled RNA), or labeled by exposing bacteria to [ 3H]uracil for 30 seconds (pulse-labeled RNA), was hybridized to E. coli DNA over a wide range of RNA/ DNA inputs † † Definitions. Gene, DNA site, RNA/DNA input and titration of DNA, as used here, are defined in paper I of this series (Kennell & Kotoulas, 1968). : from ratios so low (less than 1 1000 ) that not all copies of any DNA site were filled, to RNA/DNA inputs so high (greater than 1000) that no more RNA could be bound. RNA with radioactivity only in stable components was also hybridized over a wide range of RNA/DNA inputs. (a) The maximum fraction of input RNA detectable as authentic hybrid was about 75%; it is difficult to exceed this figure because of simultaneous formation of partial hybrids subsequently removed by ribonuclease. (b) The first DNA sites to become saturated did so at RNA/DNA inputs of about 1 1000 . All sites for stable RNA were occupied at an RNA/DNA input of about 1 160 . (c) At any RNA/DNA input between about 1 4 and 1 160 , virtually all unstable RNA is hybridized, and the size of this fraction equals the difference between the fraction of radioactivity hybridized from total RNA and from stable RNA. Such measurements suggested that unstable RNA is 2.5 to 3% of the bacterial RNA while 50% of the synthesized RNA is unstable. Competition experiments with excess unlabeled rRNA gave the same figures. (d) There is a wide variation in the numbers of molecules of different mRNA species per cell; 10% of the active genes are homologous to 70% of the mRNA mass. (e) Over the entire range of abundances, the same frequency distribution was found for the radioactivity of pulse-labeled mRNA. This suggests that variable life-times do not contribute to the abundance distribution of mRNA species. (f) Only 10% of the DNA was observed to be complementary to virtually all (more than 99.85%) of the label from both pulse-labeled or long-labeled mRNA; this suggests that in these cultures most of the potential E. coli gene sites make extremely little if any RNA.