A Borrelia burgdorferi homolog of the Escherichia coli rho gene.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
K Tilly
Publications and source records attributed to K Tilly.
Linear plasmids and chromosomes in bacteria.
Linear plasmids and chromosomes were unknown in prokaryotes until recently but have now been found in spirochaetes, Gram-positive bacteria, and Gram-negative bacteria. Two structural types of bacterial linear DNA have been characterized. Linear plasmids of the spirochaete Borrelia have a covalently closed hairpin loop at each end and linear plasmids of the Gram-positive filamentous Streptomyces have a covalently attached protein at each end. Replicons with similar structures are more frequent in eukaryotic cells than in prokaryotes. Linear genomic structures are probably more common in bacteria than previously recognized, however, and some replicons may interconvert between circular and linear isomers. The molecular biology of these widely dispersed elements provides clues to explain the origin of linear DNA in bacteria, including evidence for genetic exchange between prokaryotes and eukaryotes.
Bacteria↗
Isolation of dnaJ, dnaK, and grpE homologues from Borrelia burgdorferi and complementation of Escherichia coli mutants.
The heat-shock proteins DnaJ, DnaK, and GrpE are involved in the replication of various species of DNA in Escherichia coli, in addition to their roles in other processes, including protein disaggregation and export. We have cloned the Borrelia burgdorferi homologues of these genes. DNA sequence analysis revealed an open reading frame encoding a protein that is 62% identical to the E. coli DnaK protein. Genes homologous to the E. coli grpE and dnaJ genes, encoding products 28% and 39% identical to their homologues, are located up- and downstream, respectively, of the B. burgdorferi dnaK gene. No obvious promoters were detected in the sequenced DNA, although a potential transcription terminator was found downstream of the dnaJ gene, so these three genes may form an operon, perhaps with a fourth gene located upstream of the grpE gene. The grpE homologue complemented an E. coli grpE mutant and the dnaJ homologue complemented an E. coli dnaJ mutant, whereas the B. burgdorferi dnaK gene did not complement dnaK mutants.
Amino Acid Sequence↗
Independence of bacteriophage N15 lytic and linear plasmid replication from the heat shock proteins DnaJ, DnaK, and GrpE.
The chromosome of the temperate bacteriophage N15 replicates as a linear plasmid with covalently closed ends (or hairpins) when it forms a lysogen. I found that, in contrast to the cases for lambda and the low-copy-number plasmids F and P1, both phage and plasmid replication of N15 are independent of the heat shock proteins DnaJ, DnaK, and GrpE.
Bacterial Proteins↗
Participation of the Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in autorepression of the P1 plasmid repA promoter.
The replicon of the low copy number plasmid P1 uses the three Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE for the efficient initiation of its DNA replication. The only P1-encoded protein required for plasmid replication is the initiator, RepA. Binding of RepA to the origin also represses the promoter for the repA gene, which is located within the origin. We found that repression is incomplete in E. coli strains with mutations in the dnaJ, dnaK, or grpE genes. Since there is no decrease in RepA concentration in the mutant strains, the mutations are likely to affect the protein-DNA or protein-protein reactions required for repression, thereby decreasing RepA binding at its promoter. We also showed that the deficit in repression can be overcome by providing excess RepA, implying that the mechanism of repression is not altered in the mutant strains. Since repression requires RepA binding to the origin, a binding deficit might account for the replication defect in the heat shock mutants.
Bacterial Proteins↗
Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication.
Low-copy-number plasmids, such as P1 prophage and the fertility factor F, require a plasmid-encoded replication protein and several host products for replication. Stable maintenance also depends on active partitioning of plasmids into daughter cells. Mini-P1 par+ and par plasmids were found to be destabilized by mutations in the dnaJ, dnaK, and grpE genes of Escherichia coli. The transformation efficiency and stability of mini-F plasmids were also reduced in the mutant strains. These results indicate that heat shock proteins DnaJ, DnaK, and GrpE play roles in the replication of plasmid P1 and probably also in of F.
DNA Replication↗
Modulation of stability of the Escherichia coli heat shock regulatory factor sigma.
The heat shock response of Escherichia coli is under the positive control of the sigma 32 protein (the product of the rpoH gene). We found that overproduction of the sigma 32 protein led to concomitant overproduction of the heat shock proteins, suggesting that the intracellular sigma 32 levels limit heat shock gene expression. In support of this idea, the intracellular half-life of the sigma 32 protein synthesized from a multicopy plasmid was found to be extremely short, e.g., less than 1 min at 37 and 42 degrees C. The half-life increased progressively with a decrease in temperature, reaching 15 min at 22 degrees C. Finally, conditions known previously to increase the rate of synthesis of the heat shock proteins, i.e., a mutation in the dnaK gene or expression of phage lambda early proteins, were shown to simultaneously result in a three- to fivefold increase in the half-life of sigma 32.
Escherichia coli↗
Homologous plant and bacterial proteins chaperone oligomeric protein assembly.
An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.
Adenosine Triphosphatases↗
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↗
Fine structure genetic analysis of a beta-globin promoter.
A novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse beta-major globin gene. The effects of these promoter substitutions on transcription were determined by transfecting the cloned mutant genes into HeLa cells on plasmids containing an SV40 transcription enhancer, and measuring the levels of correctly initiated beta-globin transcripts after 2 days. Mutations in three regions of the promoter resulted in a significant decrease in the level of transcription: (i) the CACCC box, located between -87 and -95, (ii) the CCAAT box, located between -72 and -77, and (iii) the TATA box, located between -26 and -30 relative to the start site of transcription. In contrast, two different mutations in nucleotides immediately upstream from the CCAAT box resulted in a 3- to 3.5-fold increase in transcription. With two minor exceptions, single base substitutions in all other regions of the promoter had no effect on transcription. These results precisely delineate the cis-acting sequences required for accurate and efficient initiation of beta-globin transcription, and they establish a general approach for the fine structure genetic analysis of eukaryotic regulatory sequences.
Animals↗
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↗
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↗
Interactions of bacteriophage and host macromolecules in the growth of bacteriophage lambda.
Explore the source record for details and available documents.
Bacteriophage lambda↗
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↗
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↗
Identification of the heat-inducible protein C15.4 as the groES gene product in Escherichia coli.
The product of the Escherichia coli morphogenetic gene groES (mopB) was identified as the heat-inducible protein C15.4 by two-dimensional gel analysis of the products of wild-type and mutant alleles carried on the bacterial chromosome, on a hybrid plasmid, and on a transducing phage.
Bacterial Proteins↗
Evidence that the two Escherichia coli groE morphogenetic gene products interact in vivo.
The Escherichia coli groEL and groES gene products are essential for both phage morphogenesis and bacterial growth. Although the gene products have been identified, their exact roles in these processes are not known. We have isolated mutations in the groEL gene that suppress defects in the groES gene. These intergenic suppressors were shown to map in the groEL gene by a variety of genetic and biochemical analyses. These results suggest that the two morphogenetic gene products interact in vivo and help to explain why mutations in either gene exhibit the same phenotype with respect to lambda head assembly and bacterial growth.
Bacterial Proteins↗
The B66.0 protein of Escherichia coli is the product of the dnaK+ gene.
B66.0 is one of the most abundant proteins of Escherichia coli. Its relative rate of synthesis is highly regulated depending on temperature and the growth rate of the culture. We identified the B66.0 protein to be the dnaK+ structural gene product since dnaK756 mutant bacteria synthesized a B66.0 protein with a more acidic isoelectric point.
Bacterial Proteins↗