Using a modified method that involves minimal manipulation of cells, we report new information about nucleotide pool sizes and changes throughout the Escherichia coli growth curve. Nucleotide pool sizes are critically dependent on sample manipulation and extraction methods. Centrifugation and even short (2 min) lapses in sample preparation can dramatically affect results. The measured ATP concentration at three different growth rates is at least 3 mM, well above the 0.8 mM needed to saturate the rRNA promoter P1 in vitro. Many of the pools, including ATP, GTP, and UTP, begin to decrease while the cells are still in mid-log growth. After an almost universal drop in nucleotide concentration as the cells transition from logarithmic to stationary phase, there is a "rebound" of certain nucleotides, most notably ATP, after the cells enter stationary phase, followed by a progressive decrease. UTP, in contrast, increases as the cells transition into stationary phase. The higher UTP values might be related to elevated UDP-glucose/galactose, which was found to be at higher concentrations than expected in stationary phase. dTTP is the most abundant deoxynucleoside triphosphate (dNTP) in the cell despite the fact that its precursors, UDP and UTP, are not. All dNTPs decrease through the growth curve but do not have the abrupt drop, as seen with other nucleotides when the cells transition into stationary phase.It is increasingly recognized that the relative concentrations of nucleotides play important roles in prokaryotic cell regulation. The classic example of this is the stringent response in which a hyperphosphorylated guanosine, ppGpp, is produced in response to stalled ribosomes in amino acid-starved cells (8,9,11). ppGpp is produced even under nutrient-rich growth conditions, however, and its presence probably ensures transcriptional balance for different promoters under a variety of physiological states (13,24,26). It is well accepted now that ppGpp is the primary, although not the exclusive, determinant of growth rate dependence: the observation that the amount of rRNA produced in Escherichia coli is proportional to the growth rate.In addition to alarmones such as ppGpp, the relative concentrations of even standard nucleotides such as ATP and GTP affect bacterial physiology. One theory suggests the concentration of initiating nucleotide (iNTP) for rRNA promoters changes under different growth conditions, which in turn affects the transcriptional rate from these promoters (15). Although it appears under different growth rates the concentration of ATP, the iNTP for rRNA promoter P1, does not change, it has been observed that the ATP concentration does decrease as cells enter stationary phase (21,24). Similarly, Fis, a nucleoid-associated protein that affects transcription, is controlled at the promoter level by concentrations of its iNTP, CTP (32). The ratio of the nucleotide triphosphates (NTPs) to diphosphates also has regulatory consequences. In Bacillus subtilis, the ratio of GTP to GDP plays a critical role in the ...