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C Georgopoulos

Publications and source records attributed to C Georgopoulos.

At least 127 records · Page 7Linked to original sources
PubMed1986

Purification and properties of the groES morphogenetic protein of Escherichia coli.

The morphogenesis of lambda proheads is governed by the products of at least four bacteriophage-coded genes (B, C, E and Nu3) and two host-coded genes (groES (mopB) and groEL (mopA)). Earlier genetic experiments indicated that the phenotypes of some of the groES- mutations could be suppressed by mutations in the groEL gene, suggesting an interaction between the two groE proteins in vivo (Tilly, K., and Georgopoulos, C. P. (1982) J. Bacteriol. 149, 1082-1088). The Mr 15,000 groES protein was overproduced and purified to homogeneity by monitoring its presence after polyacrylamide gel electrophoresis. Both gel filtration on an AcA34 sizing column and glycerol gradient centrifugation indicate that the groES protein possesses an oligomeric structure of Mr 80,000. In agreement, electron microscopic pictures of the purified groES protein show that it possesses a symmetrical ring-like structure. The sequence of the first five amino acids and the overall composition of the purified protein match those predicted by the nucleotide sequence of the groES gene. The following results implicate a physical association between the groES and groEL proteins in vitro. The groES protein inhibits the weak ATPase activity of the groEL protein, with a maximal effect seen at a 1:1 molar ratio; the two proteins cosediment during glycerol gradient centrifugation in the presence of ATP and Mg2+; and the groES protein binds specifically to a groEL-affinity column. These results help explain why mutations in either of the groE genes exhibit similar phenotypes with respect to both lambda and bacterial growth.

Adenosine Triphosphatases↗
PubMed1986

The nucleotide sequence of the Escherichia coli K12 dnaJ+ gene. A gene that encodes a heat shock protein.

The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures. It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth. We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions. Here we present the primary DNA sequence of the dnaJ gene. It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973. The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein. We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product.

Amino Acid Sequence↗
PubMed1986

Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes.

A lambda hybrid phage (lambda Sda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA+-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation.

Alleles↗
PubMed1986

Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature.

We have identified the grpE gene product as the B25.3 heat shock protein of Escherichia coli on the following evidence: (i) a protein similar in size and isoelectric point to B25.3 was induced after infection of UV-irradiated bacteria by lambda grpE+ transducing phage, (ii) mutant phage lambda grpE40, isolated by its inability to propagate on grpE280 bacteria, failed to induce the synthesis of the B25.3 protein, and (iii) lambda grpE+ revertants, derived from phage grpE40 as able to propagate on grpE280 bacteria, simultaneously recovered the ability to induce synthesis of the B25.3 protein. In addition, we show that E. coli bacteria carrying the grpE280 mutation are temperature sensitive for bacterial growth at 43.5 degrees C. Through transductional analysis and temperature reversion experiments, it was demonstrated that the grpE280 mutation is responsible for both the inability of lambda to replicate at any temperature tested and the lack of colony formation at high temperature. At the nonpermissive temperature the rates of synthesis of DNA and RNA were reduced in grpE280 bacteria.

Bacterial Proteins↗
PubMed1986

Escherichia coli DnaK protein possesses a 5'-nucleotidase activity that is inhibited by AppppA.

AppppA and the DnaK protein have both been hypothesized to function in regulating the heat shock response of Escherichia coli. The proposals are that AppppA serves as a signal (alarmone) to turn on the heat shock response, whereas the DnaK protein is necessary to turn off the heat shock response. A simple model would be that the DnaK protein turns off the response by degrading AppppA. We disproved this model by demonstrating that the DnaK protein possesses a 5'-nucleotidase activity capable of degrading many cellular nucleotides but not AppppA. Although AppppA was not a substrate, it did inhibit the 5'-nucleotidase activity of the DnaK protein. This inhibition may be specific and have biological function since the mutant DnaK756 protein, which is defective in turning off the heat shock response, is partially desensitized to AppppA inhibition. These findings led us to consider other possible mechanisms for AppppA and the DnaK protein in heat shock regulation.

5'-Nucleotidase↗
PubMed1986

Heat shock regulatory gene rpoH mRNA level increases after heat shock in Escherichia coli.

The Escherichia coli rpoH gene product sigma 32 is essential for the increase in heat shock gene transcription found after exposure of the bacteria to a sudden temperature increase. It is not known how the concentration of active sigma 32 is modulated. We showed that rpoH transcript levels increased after heat shock and that the magnitude of the increase in the level of mRNA was correlated with the magnitude of the temperature shift. The increase in the level of rpoH mRNA was still found in rpoH mutants so the mechanism of induction differed from that of the set of previously identified heat shock genes. The increased concentration of rpoH mRNA should result in a higher level of sigma 32, which is likely to be important for increasing heat shock gene transcription.

Bacterial Proteins↗
PubMed1985

Purification and properties of the dnaJ replication protein of Escherichia coli.

The Escherichia coli dnaJ gene was originally discovered because mutations in it blocked bacteriophage lambda DNA replication. Some of these mutations were subsequently shown to interfere with bacterial growth at high temperature, suggesting that dnaJ is an essential protein for the host as well. The first step in purifying the dnaJ protein was to overproduce it at least 50-fold by subcloning its gene into the pMOB45 runaway plasmid. The second step was the development of an in vitro system to assay for its activity. A Fraction II extract from dnaJ259 mutant bacteria was shown to be unable to replicate lambda dv DNA unless supplemented with an exogenous source of wild-type dnaJ protein. Using this complementation assay we purified the dnaJ protein to homogeneity from the membrane fraction of an overproducing strain of bacteria. The purified dnaJ protein was shown to be a basic (pI 8.5), yet hydrophobic, protein of Mr 37,000 and 76,000 under denaturing and native conditions, respectively, and to exhibit affinity for both single- and double-stranded DNA. Using a partially purified lambda dv replication system dependent on the presence of the lambda O and P initiator proteins and at least the host dnaB, dnaG, dnaJ, dnaK, single-stranded DNA-binding protein, gyrase, RNA polymerase holoenzyme, and DNA polymerase III holoenzyme, we have shown that the dnaJ protein is required at a very early step in the DNA replication process.

Amino Acids↗
PubMed1985

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.

Bacterial Proteins↗
PubMed1984

Purification and properties of the Escherichia coli dnaK replication protein.

The Escherichia coli dnaK+ gene was cloned into the "runaway" plasmid vector pMOB45 resulting in a large overproduction of the dnaK protein. The dnaK protein was purified by following its ability to complement the replication of single-stranded M13 bacteriophage DNA in a reaction system dependent on the presence of the lambda O and P DNA replication proteins. The DNA replication activity of the dnaK protein is also essential for lambda dv DNA replication in vitro, since antibodies against it were shown to inhibit the reaction. Purified dnaK protein preparations possess a weak ATPase activity and an autophosphorylating activity which copurify with its DNA replication activity throughout all purification steps. The dnaK protein is an acidic largely monomeric protein of Mr = 72,000 and 78,400 under denaturing and native conditions, respectively. The amino acid composition and N-terminal amino acid sequence match those predicted from the DNA sequence of the dnaK gene (Bardwell, J.C.A., and Craig, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 848-852).

Amino Acid Sequence↗
PubMed1984

The nucleotide sequence of the Escherichia coli K12 nusB (groNB) gene.

The nusB (groNB) gene product of Escherichia coli plays a pivotal role in allowing bacteriophage lambda N protein to function as an antiterminator of mRNA transcription and in modulating host gene expression. In addition it is essential for bacterial viability since mutations in it result in a cold-sensitivity phenotype for growth. We have previously cloned the nusB gene and shown it to code for a 14,500-Mr protein. Here we present the primary DNA sequence of the nusB gene. From the sequence we deduce that it codes for a slightly basic protein (21 basic as opposed to 20 acidic amino acids) composed of 139 amino acids with a cumulative 15,689-Mr. The predicted N-terminal amino acid sequence as well as the overall amino acid composition agrees well with that of the purified protein.

Amino Acid Sequence↗
PubMed1984

Bacteriophage lambda replication proteins: formation of a mixed oligomer and binding to the origin of lambda DNA.

The purified bacteriophage lambda replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers. Together they interact with each other forming an oligomer, composed of two molecules of lambda O and one molecule of lambda P. The lambda O-P oligomer is active in the in vitro replication of ori lambda-containing DNA. Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the lambda replication proteins and ori lambda DNA. It was found that the lambda P protein binds specifically to ori lambda-containing plasmid DNA only in the presence of lambda O protein. About 100 molecules of lambda O and 10 molecules of lambda P form a complex with the ori lambda DNA. The lambda DNA-lambda O-lambda P complex was shown to be active in an in vitro replication system. Since the physical interactions between ori lambda and lambda O and between lambda P and the Escherichia coli dnaB replication protein are well documented, the evidence for a lambda O-P interaction presented in this paper provides the missing link in the molecular mechanism that enables lambda to direct the host replication machinery to the replication of its own DNA.

Bacteriophage lambda↗
PubMed1983

The head genes of bacteriophage 21.

Physical and genetic maps of the head genes of lambdoid phage 21 have been made and compared with the head gene map of lambda. Because 21 and lambda have partial sequence homology throughout the head genes it was expected that the head genes of 21 would be analogous to those of lambda. Eight head genes of 21 have been identified and it was found that each of the genes is analogous in position, structure, and/or function to a lambda head gene. Phage 21 genes analogous to the lambda D and FI genes were not identified by mutation. Complementation studies between phage 21 and lambda mutants indicate that only gpFII (the protein product of a gene is referred to as gp (gene product] is fully interchangeable, gpW and gpD are partially interchangeable, and the rest of the head morphogenetic proteins are phage specific. In analogy with phage lambda, it is found that the gpNu3 analog (gp6) of phage 21 is synthesized from the same reading frame as the gpC analog (gp5), resulting in a protein identical to the carboxy terminus of gp5.

Bacteriophage lambda↗
PubMed1983

The dnaK protein modulates the heat-shock response of Escherichia coli.

E. coli bacteria respond to a sudden upward shift in temperature by transiently overproducing a small subset of their proteins, one of which is the product of the dnaK gene. Mutations in dnaK have been previously shown to affect both DNA and RNA synthesis in E. coli. Bacteria carrying the dnaK756 mutation fail to turn off the heat-shock response at 43 degrees C. Instead, they continue to synthesize the heat-shock proteins in large amounts and underproduce other proteins. Both reversion and P1 transduction analyses have shown that the failure to turn off the heat-shock response is the result of the dnaK756 mutation. In addition, bacteria that overproduce the dnaK protein at all temperatures undergo a drastically reduced heat-shock response at high temperature. We conclude that the dnaK protein is an inhibitor of the heat-shock response in E. coli.

Bacterial Proteins↗
PubMed1983

The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system.

The Escherichia coli dnaK gene product, originally defined by mutations that blocked lambda phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ gene relies primarily on the fact that the dnaK756 mutation results in the production of an ATPase activity with altered physical properties. The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction. The lambda O and P replication proteins were shown to interact in vitro with the dnaK protein. The ATPase activity of the dnaK protein was inhibited by purified lambda P protein and stimulated by purified lambda O protein. Moreover, the dnaK protein participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the O and P proteins. Anti-dnaK protein immunoglobulin specifically inhibited DNA synthesis in this system.

Adenosine Triphosphatases↗
PubMed1982

Mutants in the y region of bacteriophage lambda constitutive for repressor synthesis: their isolation and the characterization of the Hyp phenotype.

Bacteriophage lambdahyp mutants have been isolated as survivors of Escherichia coli K-12 bacteria lysogenic for lambda Nam7am53cI857. The hyp mutants are characterized by (i) their localization in the y region very close to the imm lambda/imm434 boundary, (ii) polarity on O gene expression, (iii) immediate recovery of lambda immunity at 30 degrees C after prolonged growth of lambda Nam7am53cI857 hyp lysogens at 42 degrees C even in the presence of an active cro gene product, (iv) ability of phage lambda v2v3vs326 but not lambda v1v2v3 to propagate on lambda cI+hyp lysogens, (v) inability to express lambda exonuclease activity after prophage induction, and (vi) inviability at any temperature of phage carrying the hyp mutation. All these properties are referred to collectively as the Hyp phenotype. We show that the Hyp phenotype is due to cII-independent constitutive cI-gene-product synthesis originating in the y region, which results in the synthesis of anti-cro RNA species, and constitutive levels of cro gene product present even in lambda cI+hyp lysogens. A model is presented which is consistent with all the experimental observations.

Antigens, Viral↗
PubMed1982

An IS4 transposition causes a 13-bp duplication of phage lambda DNA and results in the constitutive expression of the cI and cro gene products.

We have examined the nature of the additional DNA present in lambda hyp- mutants (Eisen et al., 1982). This DNA is an IS4 element in orientation I, in the y region of bacteriophage lambda at nucleotide position 39,139 (see Moore et al., 1979). Our assignment is based on (i) the similarity in size derived from the PstI, AvaI, and HindII restriction pattern and (ii) the DNA sequence of both the left and right lambda-IS4 DNA junctions in phage lambda hyp15rev4. The IS4 integration event resulted in the duplication of 13 bp of lambda DNA in contrast to the 11- and 12-bp duplications previously observed at the sites of IS4 integrations elsewhere (Klaer et al., 1981).

Bacteriophage lambda↗