The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.

No abstract

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage A PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOScontrolled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UntuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli results in a marked increase in mutation rate (1-3). This mutagenesis is one consequence of an induced, multigene response to DNA damage termed the SOS pathway (2-5). Most SOS-induced mutations are targeted to the sites of the DNA lesions [e.g., the pyrimidinepyrimidone (6-4) photoproduct or cyclobutane pyrimidine dimer for UV mutagenesis] (6-9). Targeted mutagenesis requires the RecA, UmuD, and UmuC proteins (3-5, 10-15). The mutagenic events have been inferred to result from a functional (and possibly a physical) interaction between DNA polymerase III, RecA, UmuD, and UmuC that allows replication across the site of the DNA lesion (16-18). The direct requirement for RecA in mutagenesis is an additional activity to its regulatory role, cleavage of the LexA repressor protein, which induces SOS-controlled operons (4, 5).The biochemical analysis of SOS mutagenesis has been limited by the lack of the purified UmuD and UmuC proteins. The umuDC operon has been cloned into a plasmid vector, and the DNA sequence has been determined (19, 20); however, the cloned operon exhibits limited gene expression (19,20 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The Spl protein activates transcription from many eukaryotic promoters. Spl can act in vivo from enhancer sites that are distal to the promoter and exhibit synergistic interaction with promoter-proximal binding sites. To investigate possible protein-protein interactions between DNA-bound Spl molecules, we have used electron microscopy to visualize the DNA-protein complexes. At the SV40 promoter, we observed the expected localized interaction at the Spl sites; in addition, we found that DNA-bound Spl served to associate two or more DNA molecules. At a modified thymidine kinase promoter, we observed a localized interaction at each of two binding locations that were separated by 1.8 kbp; in addition, we noted a substantial fraction of DNA molecules in which the distant binding regions were joined by a DNA loop. As judged by studies with mutant Spl proteins, the distant interactions depended on the glutamine-rich regions of Spl required for transcriptional activation. We conclude that DNA-bound Spl can self-associate, bringing together distant DNA segments. From the correlation between DNA looping in vitro and synergistic activation of the modified thymidine kinase promoter shown previously in vivo, we suggest that Spl exerts its transcriptional synergism by a direct protein-protein association that loops the intervening DNA. Our experiments support the DNA-looping model for the function of transcriptional enhancers.[Key Words: Spl protein; DNA looping; DNA binding; transcription]Received December 26, 1990; revised version accepted January 23, 1991.The controlled initiation of transcription in prokaryotes and eukaryotes depends on the action of regulatory proteins from sites that are too distant for a direct interaction with promoter-bound proteins on linear DNA. Three principal models have been proposed for positive regulation from distant (enhancer) sites (Dynan and Tjian 1985;Echols 1986;Ptashne 1986). In the first, the regulatory protein (or RNA polymerase) associates with the DNA at the enhancer site and then traverses the DNA to the promoter site (scanning model). In the second, the enhancer-binding protein initiates a change in DNA structure that is propagated from the enhancer to the promoter, thereby activating transcription (structural transmission model). In the third, the enhancerbound regulatory protein activates transcription by a direct protein-protein interaction with RNA polymerase at the promoter (or other proteins that contact polymerase) (DNA-looping or nucleoprotein model).DNA looping is currently favored as the most likely mechanism for distant regulatory interactions. The interaction between DNA-bound proteins has been firmly established as the central structural feature for regulated 3Present address:

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD' to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD'. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD'. We have found that antibody to UmuC precipitates UmuD' from cell extracts, and antibody to UmuD and UmuD' precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD' in cells. For biochemical studies, we have purified the UmuC and UmuD' proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD', eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD' in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD'. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD' complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.