We have generated probes of metal binding to zinc fingers (ZFs) that provide tools to study zinc trafficking in vivo. In this study, we used these probes to examine zinc binding by the Zap1 transcription factor of Saccharomyces cerevisiae. Zap1 contains two zincregulated activation domains (ADs), AD1 and AD2. AD2 is located within two C 2H2 ZFs, ZF1 and ZF2. Studies have indicated that apoAD2 activates transcription and zinc binding to ZF1 and that ZF2 forms an interacting-finger-pair structure that is necessary to inhibit AD function. A related structural finger pair, ZF3 and ZF4, is found in the Zap1 DNA binding domain. In vitro studies indicated that, although the ZF1͞2 and ZF3͞4 finger pairs bind zinc with similar affinities, zinc that was bound to ZF1͞2 was much more labile. We examined the properties of Zap1 ZFs in vivo by FRET. ZF pairs were flanked by enhanced yellow fluorescent protein and enhanced cyan fluorescent protein, allowing detection of zincinduced conformation changes by FRET. By using these reporters, we found that ZF1͞2 and ZF3͞4 showed similar responses to zinc under steady-state conditions in vivo. In contrast, ZF1͞2 zinc binding was significantly more labile than was ZF3͞4. Also, ZF1͞2 accumulated in an apo form that could rapidly bind zinc, whereas the ZF3͞4 pair did not. Last, we show that these properties are evolutionarily conserved indicating their importance to Zap1 function. These results indicate that the kinetic lability of ZF1͞2 in vivo is a key component of Zap1 zinc responsiveness.regulation ͉ transcription ͉ zinc finger

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SummaryThe Zap1 transcription factor is a central player in zinc homeostasis in yeast. This protein regulates the expression of genes involved in zinc accumulation and storage. For most of its target genes, Zap1 activates expression in zinc-limited cells and this function is inhibited in replete cells. Zap1 has two activation domains, AD1 and AD2, which are independently regulated by zinc status. In this study, we characterized AD1 and its regulation by zinc. AD1 was mapped using deletions to residues 332-402 of Zap1. The region required for the zinc responsiveness of this activation domain, designated 'ZRD AD1, was mapped to residues 182-502. Thus, AD1 is embedded within its larger zinc-responsive domain. Using a combination of in silico analysis, random mutagenesis and site-directed mutagenesis, we identified key residues within ZRD AD1 required for its regulation by zinc. Most of these residues are cysteines and histidines that could potentially serve as Zn(II) ligands. These results suggest that ZRD AD1 senses zinc by direct Zn(II) binding. Consistent with this hypothesis, purified ZRD AD1 bound multiple Zn(II) ions. Finally, our results indicate that, in the context of the full-length Zap1 protein, AD1 and AD2 are both critical to the full control of gene expression in response to zinc.

In an earlier contribution from this laboratory (Walker and Winslow, 1932) it was shown that toward the end of the initial lag period in either peptone or lactose-peptone water, there is manifest an enormous increase in metabolic activity, particularly with respect to anmnonia production. Formation of CO2 per cell per hour is increased thirty to seventy fold and formation of NH3 nitrogen fifty to one-hundred-and-fifty fold as compared with the peak stability rates. There is, in these increases, a very clear demonstration of the physiological youth of the bacterial cells as postulated by Sherman and Albus (1923).These phenomena seemed so important as to warrant further and more detailed study under different growth conditions and with observations made at more frequent intervals during the course of the growth cycle. METHODSThe culture employed was the same strain of Esch. coli used in previous studies in this laboratory. The media for the growth and metabolism experiments were peptone-water (1 per cent Difco Bacto peptone) and glucose-peptone water (0.5 per cent Baker's c.p. glucose plus 1 per cent Difco Bacto peptone). Starting at a

Escherichia coli has been cultivated in a peptone water medium saturated continuously with nitrogen by use of a gas train so as to produce anaerobic conditions. Under these circumstances growth was greatly inhibited. Cultures which originally contained 11 million bacteria per cc. showed on the average only 32 million after 5 hours (as compared with 655 million in similar cultures saturated with air). The metabolic activity of the cells in such a culture was greatly reduced by the anaerobic conditions. It actually fell off from 42 mg. x 10–11 per cell per hour during the 1st hour to 27 mg. during the 2nd hour and rose only to a maximum of 68 during the 3rd hour. Similar cultures saturated with air showed a rise from 37 mg. x 10–11 during the 1st hour to 123 during the 2nd hour. The addition of glucose to the medium, under aerobic conditions, has been shown in previous studies to cause only a slight increase in bacterial numbers (861 instead of 655 million after the 5th hour). In the cultures aerated with nitrogen, the addition of glucose has no effect during the first hours. There is again a long lag period and a reduced metabolic rate. After the 2nd hour, however, a wholly different phenomenon manifests itself. The bacterial population increases more rapidly than in the anaerobic peptone medium (reaching a maximum of 142 million after 5 hours). This growth is accompanied by an enormous increase in the rate of CO2 yield, which reaches 211 mg. x 10–11 per cell per hour during the 4th hour (nearly double the maximum values recorded under aerobic conditions). The same phenomenon is, of course, illustrated by the enormous yield of CO2 produced by the action of fermenting organisms in carbohydrate media recorded by Anderson (1924) and other students of the obligate anaerobes. We have here, however, a somewhat striking illustration of the distinct type of metabolic activity manifested by a facultative organism under anaerobic conditions in the presence of sugar measured on a cell-per-hour basis. This is a quantitative illustration of the "life without air" described by Pasteur.