Small molecules generally activate or inhibit gene transcription as externally added substrates or as internally accumulated endproducts, respectively. Rarely has a connection been made that links an intracellular intermediary metabolite as a signal of gene expression. We report that a perturbation in the critical step of a metabolic pathway-the D-galactose amphibolic pathwaychanges the dynamics of the pathways leading to accumulation of the intermediary metabolite UDP-galactose. This accumulation causes cell stress and transduces signals that alter gene expression so as to cope with the stress by restoring balance in the metabolite pool. This underscores the importance of studying the global effects of alterations in the level of intermediary metabolites in causing stress and coping with it by transducing signals to genes to reach a stable state of equilibrium (homeostasis). Such studies are an essential component in the integration of metabolomics, proteomics, and transcriptomics.galactose metabolism ͉ gene signals ͉ intracellular stress ͉ tiling arrays A cell is capable of carrying out thousands of chemical reactions to make or break compounds of different kinds (metabolites). To achieve proper balance in its chemical constituents, these reactions are programmed in time. If there is either a lack or an excess of an important metabolite, it could create stress. The cell is expected to take care of such a stress in two ways: (i) The first is a quick fix, which occurs at a biochemical level by activation or inhibition of the catalytic activities of enzymes by metabolites so as to restore homeostasis to the chemical milieu of the cell. (ii) The second is a long term solution in which the synthesis of enzymes or proteins whose presence or absence causes the chemical imbalance is turned off or on. These control mechanisms occur at different levels, but the primary level of a metabolite signal is perceived at the level of transcription. So far, the literature describes two classes of metabolites that signal gene transcription: (i) a substrate of an enzymatic pathway induces the synthesis of the enzymes of that pathway (usually catabolic), or (ii) the end-product of a pathway represses the synthesis of the enzymes of the pathway (usually anabolic). However, except for protein-modifying metabolites, e.g., acetylCoA (1, 2) or acetyl-phosphate (3), intermediary metabolites signaling even a specific gene transcription have been reported only in very few cases (4-6). We propose that gene regulation mediated by intermediary metabolites may be more widespread than previously perceived, particularly with respect to the regulation of genes encoding enzymes of amphibolic pathways, in which catabolic pathways overlap with or connect to anabolic pathways. Knowledge of the nature and extent of this type of regulation in an organism is needed for a complete understanding of the genetic regulatory network of cells. We explored one such metabolic intermediate, UDP-galactose, which we found not only sends signals to specific genes to...

The prophage state of bacteriophage λ is extremely stable and is maintained by a highly regulated level of λ repressor protein, CI, which represses lytic functions. CI regulates its own synthesis in a lysogen by activating and repressing its promoter, P RM . CI participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening DNA. We investigated the roles of each individual site under conditions that permitted DNA loop formation by using in vitro transcription assays for the first time on supercoiled DNA that mimics in vivo situation. We confirmed that DNA loops generated by oligomerization of CI bound to its operators influence the autoactivation and autorepression of P RM regulation. We additionally report that different configurations of DNA loops are central to this regulation-one configuration further enhances autoactivation and another is essential for autorepression of P RM .activation | cooperativity | repression B acteriophage lambda (λ) of Escherichia coli can grow either in a lytic or lysogenic mode. In a lysogenic cell, the phageencoded λ repressor protein (CI) prevents lytic growth by directly repressing two promoters needed to express lytic functions, P L and P R (1-3). Each promoter is associated with a CI recognition site or operator,. O R is associated with promoter P RM (promoter for maintenance of repressor synthesis), which directs transcription of the cI gene in the prophage state (4-6). Each subsite binds a CI dimer (7).The CI protein autoregulates its synthesis. At low cellular CI concentration, CI enhances its own synthesis from P RM ; when high, CI represses P RM (1,8,9). It was originally believed that both positive and negative autoregulations are achieved exclusively by the action of CI dimers at the P RM -O R -P R sequence of the phage genome (1, 10) (Fig. 1A), based on the following observations. (i) There is a hierarchy of intrinsic binding affinities of a CI dimer to individual operators sites: (11)(12)(13)(14)(15)(16)(17); (ii) CI bound to the intrinsically weak O R 2 site is strengthened by cooperative interactions with CI bound to the stronger adjacent O R 1 site, and the ensemble of two CI dimers at the O R 1 ∼ O R 2 sites represses P R and activates P RM (13, 16) (Fig. 1A); (iii) at high CI concentrations, a CI dimer can bind to the weakest operator site, O R 3, repressing P RM (1, 2) (Fig. 1C). Incidentally, a second pair of CI dimers binds cooperatively to O L 1 ∼ O L 2, and represses P L (7).This model was recently modified on the basis of physical and genetic experiments showing that a CI tetramer cooperatively bound to O R 1 ∼ O R 2 interacts with a tetramer cooperatively bound to O L 1 ∼ O L 2, located 2.3 kbp away (18-21), resulting in the looping out of the intervening DNA, as shown by electron and atomic force microscopy (18,22,23). DNA loop was also observed by cooperative interactions of CI at sites separated by only five and six helical turns (23, 24). The kinetic and thermodynamic properties ...

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