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 ...

Ribonucleotide reductases (RRs, EC 1.17.4.1) form a family of allosterically regulated enzymes that cata-lyze the conversion of ribonucleotides to 2¢-deoxy-ribonucleotides and are essential for de novo DNA biosynthesis and repair, regulating other enzymes in the DNA synthesis pathway via control of the nucleotide pool [1]. Of the four known classes of RR (Ia, Ib, II and III) class Ia, which requires two different subunits R1 and R2 for activity and catalyzes the reduction of all four common NDPs, is the most widespread, comprising all eukaryotic RRs as well as some from eubacteria, bacteriophages and viruses. The R1 subunit contains the active site as well as allosteric sites. We have recently demonstrated that there are three such sites in murine R1 (mR1) (the specificity or s-site, the adenine or a-site, and the hexamerization or h-site) [2,3], leading to a complex pattern of regulation of enzymatic activity, the major features of which are summarized in Scheme 1, as follows: (a) ATP, dATP, dGTP, or dTTP binding to the s-site drives formation of R1 2 ; (b) ATP or dATP binding to the a-site drives formation of R1 4 , which exists in two conformations, R1 4a and R1 4b , with the latter predominating at equilibrium; (c) ATP binding to the rather low affinity (K d 1-4 mm) h-site, which occurs at physiologically significant concentrations, drives formation of R1 6-dATP does not bind to this site at physiologically significant concentrations; (d) the R2 2 complexes of R1 2 , R1 4a , and R1 6 are enzymatically active, whereas the R2 2 complex of mR1 4b has little, if any, activity; and (e) the sub-strate specificity of RR is determined by the ligand occupying the s-site: ATP and dATP stimulate the reduction of CDP and UDP, dTTP stimulates the Here we examine the enantioselectivity of the allosteric and substrate binding sites of murine ribonucleotide reductase (mRR). l-ADP binds to the active site and l-ATP binds to both the sand a-allosteric sites of mR1 with affinities that are only three-to 10-fold weaker than the values for the corresponding d-enantiomers. These results demonstrate the potential of l-nucleotides for interacting with and modulating the activity of mRR, a cancer chemotherapeutic and antiviral target. On the other hand, we detect no substrate activity for l-ADP and no inhibitory activity for N 3-l-dUDP, demonstrating the greater stereochemical stringency at the active site with respect to catalytic activity. Abbreviations mRR, mammalian ribonucleotide reductase; mR1, large subunit of mammalian ribonucleotide reductase; mR2, small subunit of mammalian ribonucleotide reductase; N 3-D

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