All cells have stress response pathways that maintain homeostasis in each cellular compartment. In the Gram-negative bacterium Escherichia coli, the E pathway responds to protein misfolding in the envelope. The stress signal is transduced across the inner membrane to the cytoplasm via the inner membrane protein RseA, the anti-sigma factor that inhibits the transcriptional activity of E . Stress-induced activation of the pathway requires the regulated proteolysis of RseA. In this report we show that RseA is degraded by sequential proteolytic events controlled by the inner membrane-anchored protease DegS and the membrane-embedded metalloprotease YaeL, an ortholog of mammalian Site-2 protease (S2P). This is consistent with the mechanism of activation of ATF6, the mammalian unfolded protein response transcription factor by Site-1 protease and S2P. Thus, mammalian and bacterial cells employ a conserved proteolytic mechanism to activate membrane-associated transcription factors that initiate intercompartmental cellular stress responses.

The E2F transcription factors play a role in regulating the expression of genes required for cell proliferation. Their activity appears to be regulated by association with the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130. In vivo, pRb is found in complex with a subset of E2F components-namely, E2F-1, E2F-2, and E2F-3. Here we describe the characterization of cDNAs encoding two unusual E2Fs, E2F-4 and E2F-5, each identified by the ability of their gene product to interact with p130 in a yeast two-hybrid system. E2F-4 and -5 share common sequences with E2F-1, E2F-2, and E2F-3 and, like these other E2Fs, the ability to heterodimerize with DP-1, thereby acquiring the ability to bind an E2F DNA recognition sequence with high affinity. However, in contrast to E2F-1, E2F-4 and E2F-5 fail to bind pRb in a two-hybrid assay. Moreover, they show a unique pattern of expression in synchronized human keratinocytes: E2F-4 and E2F-5 mRNA expression is maximal in mid-G1 phase before E2F-1 expression is detectable. These findings suggest that E2F-4 and E2F-5 may contribute to the regulation of early GI events including the Go/Gl transition. E2F/DP heterodimeric transcription factors are likely to be required for regulation of a large number of genes involved in cell proliferation (1, 2). An E2F consensus binding site has been demonstrated to be critical for the control of promoters activated at various different points in the cell cycle including the promoters of the c-myc (3, 4), DHFR (5), and cdc2 (6) genes. This wide spectrum of action may reflect the activities of several distinct E2F heterodimers, whose expression and function are regulated differentially, following distinct, cell cycle-specific schedules.A number of observations support this model. Cellular E2F activity is associated with several different protein species. Three distinct genes coding for E2Fs (7-11) and three for DPs (refs. 1 and 12; C. L. Wu and E. Harlow, personal communication) have already been identified. Moreover, the expression of the various E2Fs has been reported to be cell cycle dependent. For example, E2F-1 is expressed in the late G1 phase of the cell cycle (8, 13), clearly later than the induction of some E2F-responsive genes such as c-myc (3, 4). Finally, E2F activity appears to be directly and tightly regulated at several successive levels by the cell cycle machinery (14). For example, the E2F-1/DP-1 heterodimer appears to be inactivated through its binding to hypophosphorylated pRb in G, (15,16). Subsequently, phosphorylation of both pRb and E2F-1 (17) in late G1 results in the release of active E2F-1/ DP-1 transcription factors and in the transient expression of E2F-1-dependent genes. During the S and G2 phases that follow, the direct phosphorylation of E2F-1/DP-1 by cdk2/ cyclin A may then cause inactivation (14, 18). These processes may well explain the regulation of the three E2F subtypes (E2F-1, -2, and -3) that associate with pRb (11), but they do not address yet other aspects of E2F behavior. Thus, ...

In Escherichia coli, E regulon functions are required for envelope homeostasis during stress and are essential for viability under all growth conditions. The E. coli genome encodes approximately 100 lipoproteins, and 6 of these are regulated by E . Phenotypes associated with deletion of each of these lipoproteins are the subject of this report. One lipoprotein, YfiO, is essential for cellular viability. However, overexpression of this protein is not sufficient to alleviate the requirement of E for viability, suggesting that the E regulon provides more than one essential function. The remaining five lipoproteins in the E regulon are nonessential; cells are viable even when all five are removed simultaneously. Deletion of three nonessential lipoprotein genes (nlpB, yraP, ygfL) results in the exhibition of phenotypes that suggest they are important for maintenance of the integrity of the cell envelope. ⌬nlpB cells are selectively sensitive to rifampin; ⌬yraP cells are selectively sensitive to sodium dodecyl sulfate. Such selective sensitivity has not been previously reported. Both ⌬yraP and ⌬nlpB are synthetically lethal with surA::Cm, which encodes a periplasmic chaperone and PPIase, suggesting that NlpB and YraP play roles in a periplasmic folding pathway that functions in parallel with that of SurA. Finally, the ⌬yfgL mutant exhibits a broad range of envelope defects, including sensitivity to several membrane-impermeable agents, an altered outer membrane protein profile, synthetic lethality with both surA::Cm and ⌬fkpA::Cm strains, and sensitivity to a bactericidal permeability-increasing peptide. We suggest that this lipoprotein performs a very important but as-yet-unknown function in maintaining the integrity of the cell envelope.

In yeast, the SNF/SWI complex is involved in transcriptional activation of several inducible promoters, possibly by causing a local modification of the chromatin structure. Recently, two human homologues of the SNF2/SWI2 protein have been isolated, hbrm and BRG-1. In addition, a complex containing one of the SNF2/SWI2 homologues and having an in vitro activity similar to the yeast complex has been partially purified from HeLa cells. Here we describe the characterization of a cDNA encoding a human nuclear protein containing a large domain of homology with SNF5, another member of the yeast SNF/SWI complex. This protein can be co-immunoprecipitated with hbrm and the interaction between the two proteins is dependent on the region conserved between the human and the yeast SNF5. These findings suggest that the cDNA we have cloned encodes one of the members of the human SNF/SWI complex.

Mammalian RNA polymerase II complexes and coactivators containing homologs of yeast Srb/Med proteins have been isolated recently from tissue culture cells. The yeast Srb/Med complex is involved in global gene expression and is essential, but itis not yet known if its mammalian counterparts are broadly expressed in tissues or if they are essential. We have isolated the murine gene encoding Srb7, an Srb/Med complex protein whose sequence and function is highly conserved between yeast and humans. The mouse Srb7 gene is single copy, and Northern analysis showed that it is expressed in all tissues examined. Disruption of the gene in embryonic stem cells revealed that it is essential for cell viability and murine embryonic development. These results, together with evidence that murine Srb7 is associated exclusively with high molecular weight forms of RNA polymerase II in extracts, suggest that Srb7-containing polymerase complexes occur in most tissues and have essential roles in expression of protein coding genes.Transcription initiation in eukaryotes involves the recruitment of RNA polymerase II and associated factors to promoters by gene-specific activators (Orphanides et al. 1996;Roeder 1996;Ptashne and Gann 1997;Hampsey 1998;Myer and Young 1998). Attempts to reconstitute transcription in vitro led to the identification of one set of initiation factors that are associated with RNA polymerase II at promoters, and these were called basal or general transcription factors (GTFs). The GTFs include TFIID, TFIIB, TFIIE, TFIIF, and TFIIH. Genetic and biochemical studies have identified additional factors that are necessary for appropriate regulated expression in vivo or for reconstitution of more physiologically relevant activities in vitro, among which are the yeast Srb/Med complex.We and others have proposed that the form of RNA polymerase II that is generally recruited to promoters by transcriptional activators in yeast cells contains an Srb/ med complex (for review, see Koleske and Young 1995;Ptashne and Gann 1997;Hampsey 1998;Myer and Young 1998). The genes encoding the yeast Srb proteins were discovered through a genetic screen designed to identify components of the transcription apparatus, which are involved in the response to transcriptional regulators (Nonet and Young 1989). Attempts to purify Srb proteins led to the isolation of a large complex containing core RNA polymerase II, a subset of the general transcription factors, and various regulatory proteins (Thompson et al. 1993;Koleske and Young 1994). This holoenzyme complex had the capacity to initiate transcription and respond to activators when supplemented with additional purified general transcription factors in vitro. A subcomplex that dissociated from the holoenzyme with anti-CTD antibodies was found to reconstitute the response to activators in vitro; this subcomplex contains Srb and additional proteins called Meds (Kim et al. 1994). The response to activators is critical because in vitro systems reconstituted with yeast GTFs and polymerase alone were no...