For many years, our lab has investigated the role of energy-dependent proteolysis in regulation of gene expression in bacteria. The ATP-dependent cytoplasmic proteases, akin to the eukaryotic proteasome, contain ATPase domains or subunits that recognize substrates and unfold them, feeding them to the proteolytic domains. Bacteria contain multiple ATP-dependent proteases;five of them have been characterized in E. coli. Substrates of these proteases that are involved in regulatory networks fall into two general classes: proteins that are always degraded, so that regulation of their abundance depends primarily on changes in synthesis, and proteins that show regulated proteolysis. In all cases, identifying how the substrate is recognized by the protease and how recognition is affected by growth conditions is important in understanding how regulation is carried out. In the past, our lab first showed that the Lon protease regulated capsular polysaccharide synthesis and cell division by degrading the RcsA and SulA proteins, discovered and characterized the two-component Clp proteases, ClpAP and ClpXP, and investigated the roles of these proteases in vivo and in vitro. In recent years, our focus has been on the regulated degradation of the RpoS sigma factor, a subunit of RNA polymerase that directs the polymerase to specific promoters. RpoS is important for cells to switch to a stationary or stress response gene expression program, and the cell regulates RpoS accumulation in a variety of ways, including at the level of translation via small RNA activators of translation, and by regulated proteolysis. We have been studying this proteolysis, one of the best examples of regulated protein turnover in E. coli. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments;the mode of stabilization was a mystery until recent work from our lab. A genetic screen for regulators of RpoS degradation led to discovery of a small, previously uncharacterized protein, now named IraP. Mutants of iraP have somewhat decreased stability of RpoS under normal growth conditions and totally abolish the stabilization of RpoS after phosphate starvation. IraP blocks RpoS turnover in a purified in vitro system, and directly interacts with RssB. In E. coli, phosphate starvation is sensed by an increase in the levels of the small molecule ppGpp, and the iraP promoter is positively regulated by ppGpp. IraP is only the first of a set of what we have called anti-adaptors, proteins that block the ability of the adaptor RssB to act. Two other small proteins also stabilize RpoS in a purified in vitro system, IraM, and IraD. These proteins are not similar in predicted structure to IraP. IraM is made in response to magnesium starvation, dependent on the PhoP and PhoQ regulators;IraD is important after DNA damage. In collaborative studies, a model for IraP structure has been determined, and residues on the surface of IraP mutated to identify critical contacts for IraP function. Results thus far suggest the existence of two patches of surface residues important for IraP function. The anti-adaptors define a new level of regulatory control, interacting with the RssB adaptor protein and blocking its ability to act;environmental signals regulate RpoS turnover by regulating expression of different anti-adaptors. RssB structure and function are being further investigated using a genetic selection to identify mutations in RssB resistant to the anti-adaptors, and a bacterial two-hybrid system to investigate the interaction of wild-type and mutant derivatives of RssB and its domains with the anti-adaptors and other components of the system. All three anti-adaptors interact with RssB in this system, and mutations in RssB resistant to IraP lose or decrease the interaction with IraP and with IraD. However, IraM is unaffected by these mutations, suggesting it acts somewhat differently. Consistent with this, different domains of RssB interact with IraP and with IraM. In vitro analysis of some of these RssB derivatives suggest the existence of an allosteric switch in RssB;the mutations may prejudice the form of RssB to a form that is either unable to interact with the anti-adaptors or interacts but is no longer inhibited. Other resistant mutants may reflect the direct sites of interaction with RssB We also have identified AppY, a transcriptional regulator, as involved in RpoS stabilization;when it is overproduced, RpoS is stable, independent of IraP, IraM, or IraD. We believe it acts by regulating synthesis of a novel anti-adaptor. Deletions of the H-NS protein also stabilize RpoS. Because H-NS represses many genes, we asked if it might repress expression of the anti-adaptors. We find that IraD and IraM expression is repressed by H-NS, and in the absence of these two anti-adaptors, much of the stabilizing effect of H-NS is lost. The bacterial two-hybrid system has also been used to screen a library of E. coli proteins for other proteins that interact with RssB. These may provide yet additional anti-adaptors, giving us a sense of how broad this family of regulators is;alternatively, we may find other substrates for RssB, which has previously been believed to be specific to RpoS. A number of interesting regulatory proteins have been identified and found to affect RpoS turnover, and thus may be acting as anti-adaptors or competing substrates, both of which are of significant interest.