Increased Transcription Initiation Research Articles

Mutations in rpoD, the gene encoding the σ70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP

We have isolated a new class of mutations in rpoD, the gene encoding the σ 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and β-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the σ 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of σ 70.

リンコマイシンによるTEM型β-ラクタマーゼの産生増強メカニズム

The effect of lincomycin on the production of TEM β-lactamase by E. coli was studied to elucidate the mechanism of the stimulation by this antibiotic of the production of enterotoxins by certain bacteria. TEM β-lactamase was consistently measurable by a microiodometric assay. Lincomycin enhanced the activity of the enzyme encoded on the plasmid pBR322 2.4 times, while chromosomal β-galactosidase activity was inhibited. Lincomycin also stimulated the production of β-lactamase coded for by the bla gene integrated into the chromosome, but the antibiotic did not stimulate that of chloramphenicol acetyltransferase encoded on a ColEl-derived plasmid. The effect was independent of the copy number of the gene, suggesting that the effect takes place after transcription.The amount of bla mRNA was measured by [3H]-uridine pulse-labeling of mRNA followed by DNA-RNA hybridization. Lincomycin caused a 1.8 to 2.4-fold increase in the amount of bla mRNA. The possible presence of the repressor of the bla gene was examined by transducing the excess bla promoter region into E. coli 112-2, which carries Tn3 on its chromosome, but the repressor was not detected. The effect of bla promoters was examined by the construction of promoter deletion mutants of pBR322, each of which lacked one of two bla promoters. Lincomycin stimulated the production of β-lactamase coded by these plasmids at the same level as that coded by original pBR322, indicating that the effect was not due to increased transcription initiation. Preliminary examination of the degradation rate of bla transcripts showed that lincomycin increased half-life of bla mRNA.

Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene.

In Escherichia coli the product of the rpoH (htpR) gene, sigma 32, directs RNA polymerase to initiate transcription from heat shock promoters at all temperatures. Transcription of the heat shock genes is increased when cells are exposed to high temperatures because of increased transcription initiation by sigma 32-RNA polymerase. As a step toward understanding the regulation of the heat shock response we have examined the transcription of the rpoH gene. Using S1 mapping, promoter cloning, and in vitro transcription, we have identified the promoters and the terminator for the rpoH transcription unit. The rpoH transcripts are monocistronic and originate from at least three promoters. None of the promoters is recognized by sigma 32-RNA polymerase. Two are recognized by sigma 70-RNA polymerase and are active at both low and high growth temperatures. We do not know what form of RNA polymerase recognizes the third promoter. Transcripts from this promoter are abundant only at high temperature and are present after shift to the lethal temperature of 50 degrees C, even at times when there are no detectable transcripts from the other rpoH promoters. The amount of rpoH mRNA increases fivefold by 8 min after shift from 30 to 43.5 degrees C but rpoH mRNA synthesis increases by less than twofold, indicating that there is post-transcriptional control of the level of rpoH mRNA and presumably of sigma 32.