[Mutations in Escherichia coli pmp and htpR genes stabilize the products of foreign gene expression]. / Mutatsii v genakh pmp i htpR Escherichia coli stabiliziruiut produkty ékspressii chuzherodnykh genov.

The half lives of mRNA for Escherichia coli chloramphenicol-acetyltransferase, Bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in E. coli cells by molecular RNA.DNA hybridization. The effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mRNA was studied. The half life of interferon mRNA increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. The stability of interferon in E. coli cells depends on the htpR gene, controlling the heat shock response. The yields of leucocyte interferons alpha-2, alpha I-1 and fibroblast interferon beta increase ten times in htpR mutants. Thus, by using pnp and htpR mutants it is possible to enhance considerably the eukaryotic gene expression in bacterial cells.

[Stability of normal, abnormal and recombinant proteins in Escherichia coli strains deficient for intracellular proteinase La--the product of the lon gene]. / Izuchenie stabil'nosti normal'nykh, anomal'nykh i genno-inzhenernykh belkov v shtammakh Escherichia coli, defitsitnykh po vnutrikletochnoi proteinaze La--produkte gena lon.

Escherichia coli Lon-mutants deficient in intracellular protease La have been isolated. The rate of degradation of normal cellular proteins was 1.5-2-fold lower in Lon-mutants as compared with that of the wild type strain. The rate of degradation of canavanine-containing abnormal proteins, as well as foreign proteins was significantly higher in E. coli than that of normal proteins. Lon-mutants possessed 2-2.5-fold lower rates of degradation of abnormal proteins as compared with Lon+-strains. The rate of degradation of human interferon alpha-2 was 10-fold higher in E. coli than that of abnormal proteins. B. amyloliquefaciens alpha-amylase degraded in E. coli with the rate comparable with that of abnormal proteins, since chloramphenicolacetyltransferase from Tn9 was stable in E. coli. The rate of degradation of interferon alpha-2 was 2-fold lower in Lon-mutants (half-life 23-26 min) than in the initial strain (11-12 min). Lon-mutants were effectively used as recipient strains for constructing strains-producers of several human alpha- and beta-interferons.

[Transcription of ribosomal protein genes rplKAJL and RNA-polymerase genes rpoBC in Escherichia coli cells: metabolic regulation of attenuation and the effect of rifampicin]. / Transkriptsiia genov rplKAJL ribosomnykh belkov i genov rpoBC RNK-polimerazy v kletkakh Escherichia coli: metabolicheskaia reguliatsiia attenuatsii i deistvie rifampitsina.

The E. coli genes rplKAJL specifying ribosomal proteins L11, L1, L10, L7/L12 are co-transcribed with the genes rpoBC encoding the beta- and beta'-subunits of RNA polymerase, but are separated by the site of attenuation. The efficiency of attenuation within rplKAJL-rpoBC operon was determined as a ratio of rplKAJL transcription frequency to the same of rpoBC genes. The efficiency of attenuation was found to be a growth-rate dependent parameter of E. coli cells. At growth rate 1.2 doublings per hour the attenuation is rare and simultaneously increases with the increase in the growth rate (at mu = 1.2 doublings per hour the efficiency of attenuation is 4). Rifampicin (10-30 micrograms/ml) inhibits the transcription of both rplKAJL and rpoBC genes in fast growing cells but paradoxically stimulates their transcription in slowly growing cells. The stimulatory effect of rifampicin on rplKAJL genes transcription is supposed to be based on its ability to repress the ppGpp synthesis. The possible role of ppGpp in the regulation of transcription attenuation in rplKAJL-rpoBC operon is discussed.

[The role of ppGpp in the coordination of transcription of the ribosomal protein genes rplKAJL and RNA-polymerase rpoBC genes in Escherichia coli cells]. / Rol' ppGpp v koordinatsii transkriptsii genov ribosomnykh belkov rplKAJL i genov rpoBC RNK-polimerazy v kletkakh Escherichia coli.

Transcription of the ribosomal protein genes rplKAJL and of the RNA polymerase genes proBC in the E. coli cells depends on the level of regulatory nucleotide ppGpp. The ppGpp acts as a negative regulator of transcription of the rpoBC genes in conditions of moderate deficiency of amino acids (after the cells were shifted down from amino acid rich to minimal media) or after incomplete deacylation of tRNA exerted by addition of serine-hydroxamate, or by partial inactivation of valyl-tRNA synthetase. Rifampicin of low concentrations, which inhibit total transcription not more than to 50%, stimulates transcription of the genes rpoBC and rplKAJL. It was estimated that stimulatory effect of rifampicin results from the ability of this antibiotic to decrease synthesis of ppGpp--the negative regulator of transcription of genes rplKAJL and rpoBC.

[Stringent control of relA gene transcription in cells of Escherichia coli]. / "Strogii" kontrol' transkriptsii gena relA v kletkakh Escherichia coli.

Regulation of E. coli K12 relA gene transcription was studied. The rate of relA-RNA synthesis was measured by RNA-DNA-hybridization technique using cloned fragment of relA gene. Under seryl-tRNA deficiency the rate of relA-RNA synthesis was reduced six times in the strain CP78 (relA+) and only two times in its' relA-variant CP79. Chloramphenicol addition stimulated the rate of relA-RNA synthesis in starved relA+ cells. It was concluded that relA gene transcription is under "stringent control". The rate of relA-RNA synthesis increases proportionally with bacteria growth rate. Grown in glucose-minimal media strain with several copies of relA gene exhibits elevated ppGpp level (50 pmol/A450) and reduced rate of relA-RNA synthesis per one copy of relA gene. Thus, relA gene transcription is under negative regulation of ppGpp.

[Metabolic regulation of threonine operon transcription in E. coli cells]. / Metabolicheskaia reguliatsiia transkriptsii treoninovogo operona v kletkakh E. coli.

The threonine biosynthetic operon transcription in E. coli cells during balanced growth was studied. The rate of threonine-mRNA synthesis was measured by hybridization of impulse-labelled RNA with pYN 1107 DNA carrying the structural threonine genes A, B, C. It was shown that threonine-mRNA synthesis depends on bacterial growth rate being maximal at mu = 0.8 doublings per hour. The influence of the ppGpp on the bacterial growth rate and threonine-mRNA synthesis rate was demonstrated, using spoT-mutants and strains with several relA gene copies. The rate of threonine-mRNA synthesis is maximal at the ppGpp level of about 50-60 pmole/A450. The deviation from this ppGpp optimum level results in inhibition of the threonine-mRNA synthesis. Thus, ppGpp appears to be involved in metabolic regulation of operon transcription. A mechanism of negative regulation of threonine-mRNA synthesis by high concentrations of ppGpp is discussed.

[Selective inhibition of ribosomal RNA synthesis by rifampicin in E. coli cells]. / Izbiratel'noe podavlenie sinteza ribosomnoi RNK rifampitsinom v kletkakh E. Coli.

Under partial inhibition of total RNA synthesis by rifampicin the formation of beta- and beta'-subunits of RNA polymerase is stimulated and the rRNA synthesis is selectively repressed. The differential rate of synthesis of the beta- and beta'-subunits increases from 1,15% up to 2,88% in the presence of 30 micrograms rifampicin per ml. Simultaneously the differential rate of rRNA synthesis decreases from 41% down to 10%. The degree of inhibition of rRNA synthesis by rifampicin depends on the cell growth rate.

[The influence of chloramphenicol on synthesis of ribosomes and beta and beta' subunits of RNA polymerase]. / Vliianie khloramfenikola na sintez ribosom, beta-i beta -subbedinits RNK-polimerazy v kletkakh E. coli.

The effect of low chloramphenicol concentrations on the biosynthesis of RNA, ribosomal proteins and RNA polymerase in E. coli CP 78 cells was studied. When protein synthesis was decreased by 50--70%, 14C-uracil incorporation in DNA increased twice, the rRNA synthesis being stimulated preferentially. In the presence of antibiotic the RNA/DNA ratio increased from 5,7 to 13,3. The differential rate of r-protein synthesis increased simultaneously with the stimulation of rRNA synthesis, so that alphar rises from 0,083 (without antibiotic) to 0,122 and 0,161 at 5 and 10 microgram/ml of chloramphenicol, respectively. The inhibition of protein synthesis by chloramphenicol is accompanied also by the increase of differential rate of synthesis of beta and beta' subunits of RNA polymerase. In the presence of 5 and 10 microgram/ml of chloramphenicol, alphap increased from 0,90% to 1,44 and 1,57%, respectively. It is assumed that the genes for beta and beta' subunits of RNA polymerase as the ribosomal genes are negatively controlled by guanosine tetraphosphate which intracellular concentration decreased in the presence of chloramphenicol. The known data on the influence of streptolydigin and rifampicin on the RNA polymerase biosynthesis are discussed in view of proposed hypothesis.

[Ribosome stability of Escherichia coli cells in amino acid starvation]. / Stabil'nost' ribosom kletok Escherichia coli pri aminokislotnom golodanii

The dissociation of ribosomes from two isogenic pairs of Escherichia coli strains was studied during exponential growth, under amino acid starvation and subsequent chloramphenicol treatment. There were no significant differences in Mg2+-dependent dissociation of ribosomes from exponentially growing rel+ and rel- minus strains. The differences in dissociation of the ribosomes from rel+ and rel- minus cells were observed only upon amino acid starvation of these cultures. The dissociation of ribosomes from starved rel+ cells was more complete. After chloramphenicol treatment isolated ribosomes were more resistant to dissociation into subunits. Alterations of dissociation of the ribosomes in vitro correlated both with the amount of polysomes and the level of RNA synthesis in cells. It is proposed that rRNA synthesis in bacteria depends on the ratio of programmed and deprogrammed ribosomes.