Synthesis of the β and β′ subunits of Escherichia coli RNA polymerase is autogenously regulated in vivo by both transcriptional and translational mechanisms

Abstract

Numerous experiments have indicated that the synthesis of RNA polymerase (beta beta' alpha 2 sigma 70) in Escherichia coli is autogenously regulated. In the present study, we have examined expression of the rpoB and rpoC genes which encode the beta and beta' subunits of RNA polymerase. These genes are the distal cistrons of the rplKAJLrpoBC ribosomal protein-RNA polymerase transcription unit. Both transcriptional (operon) and translational (gene) fusions of either rpoB or rpoC to the lacZ reporter were used to monitor their in vivo expression by inserting single copies of these fusions into the bacterial chromosome on integration-proficient lambda vectors. In order to examine the expression of the rpoBC genes in response to varied intracellular concentrations of the RNA polymerase subunits, a series of expression plasmids carrying the rpoB, rpoC, rpoA (alpha) and rpoD (sigma 70) genes in different combinations were then introduced into these cells. Elevated concentrations of either beta or beta' were found to repress the expression of both rpoB and rpoC at the translational level. However, the simultaneous increase in the concentration of all the subunits that comprise holoenzyme repressed the transcription of rpoBC. To determine the site of this repression, additional operon fusions were utilized which placed lacZ downstream of each of the transcriptional regulatory sites of this gene cluster, including two promoters, rplKp and rplJp, and a transcriptional attenuator in the rplL-rpoB intercistronic region. Expression from these fusions showed that transcriptional repression is achieved primarily by reducing initiation at both upstream promoters, coupled with a small increase in termination at the attenuator.

Conserved Gene Clusters in Bacterial Genomes Provide Further Support for the Primacy of RNA

“…In E. coli the L10 operon lies immediately downstream of the L11 operon and the genes for the ␤ and ␤Ј subunits of RNA polymerase adjoin it, forming a single transcription unit. The regulation of this operon is complex, involving both transcriptional and translational mechanisms (Keener and Nomura 1996;Dykxhoorn et al 1996). In E. coli, the expression levels of nusA and infB are modulated by several RNA secondary structures upstream from nusA and the NusA protein itself may be involved as well (Plumbridge et al 1984).…”

A Proximal Promoter Element Required for Positive Transcriptional Control by Guanosine Tetraphosphate and DksA Protein during the Stringent Response

“…All promoter-lacZ constructs in this work were incorporated into bacteriophage RS45 and integrated in the Escherichia coli chromosomal att site as described previously (42). Transformation of the E 70 overproduction plasmids (43) and transductions of the ⌬dksA, ⌬relA, and ⌬spoT alleles were introduced into the different strains by standard methods. Cultures were grown in Erlenmeyer flasks in M9 defined medium supplemented with a limiting concentration of glucose (0.08%), thiamine (10 M), and all the amino acids in excess (44) at 37°C.…”

Involvement of AlgQ in Transcriptional Regulation of Pyoverdine Genes in Pseudomonas aeruginosa PAO1

“…We tested this hypothesis by introducing either rpoD or pvdS on a high-copy-number plasmid into PAO1 and PAO1⌬algQ. As far as expression of pvd biosynthetic genes is concerned, multicopy rpoD mimicked and even exacerbated the ⌬algQ phenotype in wild-type and algQ mutant strains, respectively, without altering the levels of RNAPc (13). Accordingly, multicopy pvdS suppressed the ⌬algQ phenotype.…”