PrlA (SecY) and PrlG (SecE) interact directly and function sequentially during protein translocation in E. coli

Three strategies for genetic analysis show that two inner membrane components of the export machinery, PrlA (SecY) and PrlG (SecE), interact directly while catalyzing the translocation of secreted proteins across the cytoplasmic membrane of E. coli. The first, suppressor-directed inactivation (SDI), exploits the specific interaction between dominant prl suppressors of signal sequence mutations and mutant LacZ hybrid proteins. The second, Sec titration, extends SDI to allow the identification of various Sec proteins that are present in the translocation complex. The third uses the synthetic lethality of certain double-mutant strains to infer physical interactions between gene products. Biochemical data obtained with SDI strains allow the identification of two different secretory intermediates and indicate that PrlG functions before PrlA in the secretion pathway.     

SecA protein is required for secretory protein translocation into E. coli membrane vesicles

The soluble and membrane components of an E. coli in vitro protein translocation system prepared from a secA amber mutant, secA13[Am], contain reduced levels of SecA and are markedly defective in both the cotranslational and posttranslational translocation of OmpA and alkaline phosphatase into membrane vesicles. Moreover, the removal of SecA from soluble components prepared from a wild-type strain by passage through an anti-SecA antibody column similarly abolishes protein translocation. Translocation activity is completely restored by addition of submicrogram amounts of purified SecA protein, implying that the observed defects are solely related to loss of SecA function. Interestingly, the translocation defect can be overcome by reconstitution of SecA into SecA-depleted membranes, suggesting that SecA is an essential, membrane-associated translocation factor.     

The repair patch of E. coli (A)BC excinuclease.

The size of repair patch made by E. coli DNA polymerase I (Poll) following the removal of a thymine-psoralen monoadduct by E. coli (A)BC excinuclease was determined by using an M13mp19 DNA with a single psoralen monoadduct at the polylinker region. Incubation of this substrate with (A)BC excinuclease, Poll and a combination of 3 dnTP plus 1 dNTP(alpha S) for each nucleotide, and DNA ligase resulted in a repair patch with phosphorothioate linkages. The preferential hydrolysis of phosphorothioate bonds by heating in iodoethanol revealed a patch size--with minimal nick translation--equal in length to the 12 nucleotide gap generated by this excision nuclease.     

Atomic model of the E. coli membrane-bound protein translocation complex SecYEG

The Sec complex forms the core of a conserved machinery transporting proteins across or into membranes. In Escherichia coli SecYEG is active as an oligomer, but the structure predicts that the protein-conducting channel is formed by the monomer. A homology model of the E.coli complex was built using the atomic structure of Methanococcus jannaschii SecYEbeta. Another structure of the membrane-bound dimer was then determined by fitting the homology model to an 8A map of SecYEG determined by electron microscopy. We found that the substrate-binding site of the dimer has opened slightly and the plug domain moved toward the outside. This new position retains the channel in a closed state. These differences partially reflect the movements that have been proposed to occur during channel gating. Further opening of the substrate-binding pocket to bind and release bound substrate and displacement of the plug during secretion, presumably rely on the action of the partner proteins. The contacts arising at the dimer interface in the environment of the lipid bilayer may have activated the assembly.     

DNA translocation by the restriction enzyme from E. coli K

The restriction endonuclease Eco K binds to a host specificity site and then proceeds to cleave the DNA at sites that may to several thousand bases away. It does this by translocating the DNA past the enzyme in an ATP-dependent reaction that results in the formation of highly twisted loop intermediates. DNA cleavage can occur on either side of the host specificity site.     

Energy transfer in complexes of E. coli single-stranded DNA-binding protein with single-stranded poly-(2-thiouridylic acid)

The complexes of point-mutated Escherichia coli single-stranded DNA-binding protein (Eco SSB) with poly-(2-thiouridylic acid) (poly S2U) have been studied by optical detection of magnetic resonance spectroscopy (ODMR). Previous work has determined that two of four tryptophan (Trp) residues in Eco SSB undergo stacking interactions with nucleic acid bases. Selective photoexcitation of S2U bases was performed and subsequent triplet----triplet energy transfer from S2U to nearby Trp residues in the protein took place. The zero-field splitting (ZFS) parameters and sublevel kinetics were determined for each Trp residue sensitized by S2U. The sublevel lifetimes of the two sensitized residues are similar to those of normal Trp. The ZFS parameters, on the other hand, show a dramatic reduction relative to those of the uncomplexed protein, implying a more polarizable environment for the sensitized Trp residues and/or charge transfer interactions with the S2U bases.     

The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation

We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles. We now report the isolation of the SecY/E protein, the integral membrane protein component of the E. coli preprotein translocase. The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A. This translocation requires ATP and is strongly stimulated by the protonmotive force. The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable. The SecY/E protein consists of SecY, SecE, and an additional polypeptide. Antiserum against SecY immunoprecipitates all three components of the SecY/E protein.     

Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein

Summary We have used a sensitive gel electrophoresis assay to detect the products of Escherichia coli RecA protein catalysed strand exchange reactions between gapped and duplex DNA molecules. We identify structures that correspond to joint molecules formed by homologous pairing, and show that joint molecules are converted by RecA protein into heteroduplex monomers by reciprocal strand exchanges. However, strand exchanges only occur when there is a 3-terminus complementary to the single stranded DNA in the gap. In the absence of a complementary free end, the two DNA molecules pair and short heteroduplex regions are formed by localised interwinding.     

DNA repair in E. coli strains deficient in single-strand DNA binding protein

Summary Weigle reactivation and mutagenesis have been found to be defective in strains of E. coli deficient in single-strand DNA binding protein (SSB). These defects parallel those previously found in prophage induction and amplification of recA protein synthesis in ssb strains. Together, these results demonstrate a role for SSB in the induction of SOS responses. UV survival studies of ssb ^- recA^- and ssb ^- uvr^- strains are presented which also suggest a role for SSB in recombinational repair processes but not in excision repair. Studies of host cell reactivation support this latter conclusion.     

ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle

We have isolated large amounts of E. coli outer-membrane protein A precursor (proOmpA). Purified proOmpA is active in membrane assembly, and this assembly is saturable with respect to the precursor protein. A proOmpA-Sepharose matrix allows affinity isolation of trigger factor, a soluble, 63,000 dalton monomeric protein that stabilizes proOmpA in assembly competent form. Comparison of trigger factor's amino-terminal sequence with those in a computer data bank and with those encoded by sec genes, as well as groEL and heat shock gene dnaK, suggests that trigger factor is encoded by a previously undescribed gene. Trigger factor and proOmpA form a 1:1 complex that can be isolated by gel filtration. Purified canine signal recognition particle (SRP) can also stabilize proOmpA for membrane insertion. This postribosomal activity of SRP suggests a unifying theme in protein translocation mechanisms.     

Identification of a protein coded by pR plasmid and involved in SOS repair in E. coli.

The TP120 plasmid is known to determine enhanced UV survival in E. coli wild type an uvrB and PolA mutants but not in RecA mutant. In order to analyze the function involved in the SOS repair, we have constructed a new plasmid named pR derived by cleavage of TP120 with Hind III endonuclease. This new plasmid maintains the Ap and UV resistance. The insertion of Tn5 transposon in the plasmid allows to select several pR::Tn5 plasmids whose UV resistance was inactivated by the transposition. The comparison of the protein synthesis in the minicells of the pR and pR::Tn5 shows that the pR codes for a 22.000 M.W. dalton protein which is absent in protein pattern of pR::Tn5.     

DNA repair mechanisms affecting cytotoxicity by streptozotocin in E. coli

Mechanisms underlying cytotoxicity by the monofunctional nitrosourea streptozotocin (STZ) were evaluated in DNA repair-deficient E. coli mutants. Strains not proficient in recombinational repair which lack either RecA protein or RecBC gene products were highly sensitive to STZ. In contrast, cells that constitutively synthesize RecA protein and cannot initiate SOS repair mechanisms because of uncleavable LexA repressor (recAo98 lexA3) were resistant to this drug compared to a lexA3 strain. Further, E. coli cells lacking both 3-methyladenine DNA glycosylases I (tag) and II (alkA) also were highly sensitive to STZ. DNA synthesis was most inhibited by STZ in recA and alkA tag E. coli mutants, but was suppressed less markedly in wild-type and recBC cells. DNA degradation was most extensive in recA E. coli after STZ treatment, while comparable in recBC, alkA tag, and wild-type cells. Although increased single-stranded DNA breaks were present after STZ treatment in recA and recBC mutants compared to the wild type, no significant increase in DNA single-stranded breaks was noted in alkA tag E. coli. Further, DNA breaks in recBC cells were repaired, while those present in recA cells were not. These findings establish the critical importance of both recombinational repair and 3-methyladenine DNA glycosylase in ameliorating cytotoxic effects and DNA damage caused by STZ in E. coli.