Protein aggregation and inclusion body formation in Escherichia coli rpoH mutant defective in heat shock protein induction.

Mutations in the rpoH gene, encoding sigma 32, an alternative factor required for transcription of the heat shock genes, result in the extensive aggregation of virtually all cellular proteins and formation of inclusion bodies both under stress and non-stress conditions. Inhibitors of protein synthesis suppress this aggregation, suggesting that newly synthesized proteins preferentially aggregate in rpoH mutants. These data suggest that the heat shock proteins are involved in acquisition of the soluble state (i.e. correct conformation) of the bulk of intracellular proteins after their translation.

Neural network models for promoter recognition.

The problem of recognition of promoter sites in the DNA sequence has been treated with models of learning neural networks. The maximum network capacity admissible for this problem has been estimated on the basis of the total of experimental data available on the determined promoter sequences. The model of a block neural network has been constructed to satisfy this estimate and rules have been elaborated for its learning and testing. The learning process involves a small (of the order of 10%) part of the total set of promoter sequences. During this procedure the neural network develops a system of distinctive features (key words) to be used as a reference in identifying promoters against the background of random sequences. The learning quality is then tested with the whole set. The efficiency of promoter recognition has been found to amount to 94 to 99%. The probability of an arbitrary sequence being identified as a promoter is 2 to 6%.

Heat shock response in Escherichia coli promotes assembly of plasmid encoded RNA polymerase beta-subunit into RNA polymerase.

Escherichia coli cells, carrying a rifampicin sensitive RNA polymerase beta-subunit gene in the chromosome and a rifampicin resistant beta-subunit gene placed under the control of a strong promoter in a multicopy plasmid, are unable to grow in the presence of rifampicin, despite the accumulation of large quantities of the resistant subunit. A major portion of the overproduced subunit is found in an insoluble form. Conditions known to induce the heat shock proteins (hsps), e.g. elevated temperature or the presence of ethanol in the growth medium, increase the amount of the plasmid-borne beta-subunit which apparently assembles into active RNA polymerase and makes the plasmid bearing cells rifampicin resistant. Alternatively, plasmid-borne subunits assemble into RNA polymerase with low efficiency in rpoH mutant cells known to have reduced level of hsps. We suggest that the plasmid-borne subunit is poorly assembled into RNA polymerase and that hsps promote the assembly by interfering with beta-subunit aggregation.

Stability of the pBR322 plasmid as affected by the promoter region of the tetracycline-resistance gene.

A region affecting the pBR322 plasmid maintenance has been located within the region of the TcR gene promoter. On the basis of stability analysis of pBR322 derivatives comprising the modified region of the TcR gene, we deduced that it is the nucleotide sequence localized in the region of the HindIII site that causes destabilization of the plasmid and not the TcR gene product or active transcription of this region. The destabilizing effect is manifested both in cis and in trans.

[Effect of a fragment of pBR322 plasmid in the region of tetracycline resistance gene on the stability of this plasmid]. / Vliianie fragmenta DNK plazmidy pBR322 v oblasti gena ustoichivosti k tetratsiklinu na stabil'nost' étoi plazmidy.

A fragment destabilizing the pBR322 plasmid has been localized in the region of pBR322 tet gene promoter. On the basis of stability analysis of pBR322 derivatives comprising the modified region of tet gene, we deduced that it is the DNA sequence localized at the beginning of tet gene in the region of HindIII splitting site that ensures the plasmid destabilization, and not tet gene product or the active transcription of this region. The destabilizing effect of this fragment of the plasmid is manifested both in cis and in trans. Possible molecular mechanisms of the phenomenon are discussed.

[Effect of the heat shock reaction on the phenotypic manifestation of mutations in rifampicin resistance affecting the gene of the RNA polymerase beta-subunit under the control of the lactose promotor]. / Vliianie reaktsii teplovogo shoka na fenotipicheskoe proiavlenie mutatsii ustoichivosti k rifampitsinu, zatragivaiushchikh gen beta-sub''edinitsy RNK-polimerazy, postavlennoi pod kontrol' laktoznogo promotora.

Plasmids bearing the rifampicin-resistant RNA polymerase beta-subunit gene under control of the lac promoter failed to provide Escherichia coli cells with resistance to rifampicin, despite the accumulation of large quantities of the resistant subunit. The effect proved to be connected with the aggregation of the plasmid-borne subunit. Conditions that induce synthesis of heat-shock proteins make the plasmid-containing cells resistant to rifampicin. This finding suggests that heat-shock proteins prevent the formation of protein aggregates.

[Structure of the ARS element from telomeres of Drosophila melanogaster]. / Struktura ARS-élementa iz telomer Drosophila melanogaster.

We have determined the nucleotide sequence of 0.7 kb Drosophila DNA fragment specifying autonomous replication of plasmids in yeast. As shown by deletion mapping, the ARS consists of at least two domains: the core possessing the classical 11-member sequence 5'-TAAATATAAAT and the enhancer of no more than 92 nucleotide pairs located at the 3' end of the core. While comparing the sequence of the enhancer with those of 14 different ARS, we failed to detect essential homology regions. ARS elements' flanks, adjacent to the core that serve as enhancer, most probably have no regions detectable as consensus. It is suggested that they are responsible for the peculiar features of DNA secondary structure (bending, for instance) which are necessary for the interaction of proteins with ARS elements.

An ARS element from Drosophila melanogaster telomeres contains the yeast ARS core and bent replication enhancer.

We have sequenced the 0.7-kb-long fragment of Drosophila DNA which ensures the autonomous replication of plasmids in yeast. Deletion mapping has shown the ARS element to consist of at least two domains: the core having the consensus 11-bp sequence TAAATATAAAT and the enhancer which is no more than 90 bp long and is located at the 3'-end of the A-rich core strand. Neither domain per se ensures plasmid replication in yeast. A comparison of the enhancer sequence with the sequences of 14 different ARS elements failed to reveal significant homology areas. Most probably the ARS flanks that are adjacent to the core and act as enhancer do not carry any consensus. They may determine a peculiar structural feature of DNA (for example bends) which are necessary for the protein-ARS interaction.

DNA rearrangements generating artificial promoters.

The promoter-cloning plasmid pBRH4 (a derivative of pBR322 with a partially deleted promoter of the tet gene) is shown to contain a sequence which is located near the EcoRI site and can operate as an effective Pribnow box, but is not the remainder of the deletion-inactivated tet promoter of pBR322. If there is a sequence homologous to the '-35' promoter region at the border of the DNA fragment inserted at the EcoRI site, then a compound promoter arises and activates the tet gene. Point mutations in the nonfunctional--35 region of pBRH4 also activate the cryptic Pribnow box. Several compound promoters were obtained through deleting small portions of DNA around the HindIII site of pBR322; the deletions moved various sequences that could operate as Pribnow boxes towards the -35 region of the tet promoter.

Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters.

Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P0) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts. Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter. We conclude that the two RNA polymerases have very similar structures of DNA binding centers. The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species.

RNA polymerase rifampicin resistance mutations in Escherichia coli: sequence changes and dominance.

Five recombinant plasmids, pBK2646, pBK611, pRC3, pRC4 and pRC5, carrying rpoB rifampicin-resistant RNA-polymerase genes were obtained. The sequence analysis of these plasmids revealed certain structural changes in the rpoB gene which specify corresponding alterations in the beta-subunit of RNA polymerase. Some functional properties of the corresponding mutant strains and their RNA polymerases have been investigated.

Curing of Escherichia coli K12 plasmids by coumermycin.

Low concentrations of the antibiotic coumermycin A1, the inhibitor of bacterial DNA gyrase, effectively eliminate pBR322, pMB9 and other ColE 1 related plasmids from E. coli K12 strains. The curing action of antibiotic seems to result from the plasmid degradation and not just from the inhibition of replication.

Cold-sensitive mutations in beta and beta' subunit gene affecting the interaction of RNA polymerase with promoters.

RNA polymerases with a cold-sensitive activity were purified from seven mutants of Escherichia coli. Subunit reconstitution experiments have shown that RNA polymerases from three mutants (Rpob262, RpoB264, and RpoB265) owed their cold sensitivity to alterations in the beta subunit. Three mutants (RpoC3, RpoC263, and RpoC267) were shown to be defective in the beta' subunit and one (RpoBC266) in both beta and beta' subunits. Two mutations (rpoC3 and rpoC263) reduced the level of RNA polymerase reconstitution. RNA polymerases from RpoC3 and RpoBC266 mutants are defective in RNA chain elongation at 6 degree C, while all the other mutants are defective in RNA polymerase-promoter interaction. most mutant RNA polymerases differ from the wild-type enzyme in transcription selectivity. The results obtained in this study indicate that both beta and beta' subunits are involved in RNA chain elongation and promoter binding and selection.

[Effect of DNA supercoiling on transcription performed by normal and mutant Escherichia coli RNA-polymerases]. / Vliianie sverkhspiralizatsii DNK na Transkriptsiiu, Osuchchestvliaemuiu, Normal'noi i mutantnymi RNK-polimerazami Escherichia coli.

A specific DNA-gyrase, inhibitor, coumermycin A1, causes differential changes in the spectrum of proteins synthesized in E. coli wild types cells, while protein spectrum in the mutant cells with coumermycin-resistant DNA-gyrase is left unaffected. The mutation of RNA-polymerase RpoB265 lowers the sensitivity of bacteria to coumermycin A1, whereas the RpoC3 enhances it. The differences between the normal and mutant RpoB265 RNA polymerases on interaction in vitro with ColE1 DNA plasmid depend on its supercoiling. No dependences of this kind were detected when comparing the normal and RpoC3-RNA polymerase. The obtained data indicate that the template supercoiling may significantly affect the transcription in vivo and that the properties of RNA polymerase may in some cases define this influence.

[Cold-sensitive mutation in the beta-subunit of Escherichia coli RNA polymerase affecting the opening of promoters]. / Knolodochuvstvitel'naia mutatsiia V beta-subdedinntse RNK-polimerazy Escherichia coli, vliiaiushchaia na otkryvanie promotorov.

A cold-sensitive mutation in Escherichia coli, affecting the beta-subunit of RNA polymerase and causing an increase in the temperature of promoter opening on T2 phage DNA, was obtained. The mutation also affects the stages preceding promoter opening by increasing the dissociation rate of RNA polymerase--DNA closed complexes. The affinity of RNA polymerase to T2- and lambda-DNA is differently changed by the mutation. The relative efficiency of transcription of these two templates is also changed. These results suggest a participation of the beta-subunit of RNA polymerase in the interaction with promoters.