In vitro DNA-binding assays demonstrate that the heat shock transcription factor (HSF) from the yeast Saccharomyces cerevisiae can adopt an altered conformation when stressed. This conformation, reflected in a change in electrophoretic mobility, requires that two HSF trimers be bound to DNA. Single trimers do not show this change, which appears to represent an alteration in the cooperative interactions between trimers. HSF isolated from stressed cells displays a higher propensity to adopt this altered conformation. Purified HSF can be stimulated in vitro to undergo the conformational change by elevating the temperature or by exposing HSF to superoxide anion. Mutational analysis maps a region critical for this conformational change to the flexible loop between the minimal DNA-binding domain and the flexible linker that joins the DNA-binding domain to the trimerization domain. The significance of these findings is discussed in the context of the induction of the heat shock response by ischemic stroke, hypoxia, and recovery from anoxia, all known to stimulate the production of superoxide. INTRODUCTIONSince its discovery in 1962 (Ritossa, 1962), the heat shock response has been the focus of intensive investigation, leading to significant insights into protein folding and global gene regulation. In virtually all organisms, the heat shock response is manifested as the stress-induced, rapid, and dramatic increase in synthesis rates of a small number of protein chaperones. The chaperones bind to partially unfolded proteins and act to prevent their aggregation and to facilitate their refolding. Thus, this highly conserved system serves as an intricate means to protect cells against damage resulting from environmental stress.The most common stresses that induce the heat shock response are elevated temperature and oxidative stress. The latter is particularly important medically, because it typically results from the production of superoxide anion that occurs during partial oxygen deprivation or during the recovery from anoxia that occurs upon reperfusion after ischemia. Induction of the heat shock system upon recovery from anoxia may be universal; it has been described not only in mammalian systems but in Drosophila (Ritossa, 1964) and yeast (Brazzell and Ingolia, 1984). It has been unclear, however, how reperfusion triggers the heat shock response. Superoxide is rapidly converted to hydrogen peroxide, which can stimulate the production of the highly reactive hydroxyl radical, which, in turn, causes considerable "reperfusion damage." Thus, the heat shock response could be induced directly via one or more of these reactive oxygen species, or it could be induced by the protein damage caused by the hydroxyl radical.In eukaryotes, the heat shock response depends on modulating the activity (rather than the concentration) of a transcription factor. The heat shock transcription factor (HSF) is synthesized constitutively; its activity is regulated posttranslationally. Despite this commonality, different species show remarkably dis...

The yeast heat shock transcription factor (HSF) is regulated by posttranslational modification. Heat and superoxide can induce the conformational change associated with the heat shock response. Interaction between HSF and the chaperone hsp70 is also thought to play a role in HSF regulation. Here, we show that the Ssb1/2p member of the hsp70 family can form a stable, ATP-sensitive complex with HSF-a surprising finding because Ssb1/2p is not induced by heat shock. Phosphorylation and the assembly of HSF into larger, ATP-sensitive complexes both occur when HSF activity decreases, whether during adaptation to a raised temperature or during growth at low glucose concentrations. These larger HSF complexes also form during recovery from heat shock. However, if HSF is assembled into ATP-sensitive complexes (during growth at a low glucose concentration), heat shock does not stimulate the dissociation of the complexes. Nor does induction of the conformational change induce their dissociation. Modulation of the in vivo concentrations of the SSA and SSB proteins by deletion or overexpression affects HSF activity in a manner that is consistent with these findings and suggests the model that the SSA and SSB proteins perform distinct roles in the regulation of HSF activity. INTRODUCTIONIt has long been known that in cells of many species, including Escherichia coli and Saccharomyces cerevisiae, cell division rates are tightly coupled with the steady-state levels and rates of synthesis of ribosomal proteins and rRNA. For example, cells in rich media, with a short generation time, display higher abundance of rRNA and higher rates of synthesis of ribosomal proteins than do cells in poor media, with a long generation time. In E. coli, some of this regulation is achieved by transcriptional attenuation and translational regulation via binding of ribosomal proteins to the RNAs of target operons (Freedman et al., 1985;Cole and Nomura, 1986). In yeast, regulation is partly transcriptional, via RAP1 and its binding site, the upstream activating sequence (UAS) rpg (Herruer et al., 1987;Moehle and Hinnebusch, 1991;Kraakman et al., 1993), although much of the regulation of ribosomal protein abundance is achieved by a competition between the assembly into ribosomes and the rapid degradation of unassembled ribosomal proteins (Warner et al., 1985;Maicas et al., 1988).Growth rate control clearly modulates the level of the translational machinery, which in turn influences the overall rate of protein synthesis. Changes in the rates of protein synthesis must, in turn, have profound implications for the protein chaperone system. Newly synthesized polypeptides typically do not adopt their mature conformation immediately but instead follow a complex folding pathway. For many proteins, the cytoplasmic chaperone system plays an integral role in helping these polypeptides adopt their mature conformations (for review, see Hendrick and Hartl, 1995). The hsp70 proteins are generally believed to bind efficiently to nascent or newly synthesized polypeptides...