The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like “stringent” RNA polymerases in Escherichia coli

Abstract

In Escherichia coli, stringently controlled genes are highly transcribed during rapid growth, but ''turned off'' under nutrient limiting conditions, a process called the stringent response. To understand how transcriptional initiation at these promoters is coordinately regulated, we analyzed the interactions between RNA polymerase (RNAP) (both wild type and mutants) and four stringently controlled promoters. Our results show that the interactions between RNAP and stringently controlled promoters are intrinsically unstable and can alternate between relatively stable and metastable states. The mutant RNAPs appear to specifically further weaken interactions with these promoters in vitro and behave like ''stringent'' RNAPs in the absence of the stringent response in vivo, constituting a novel class of mutant RNAPs. Consistently, these mutant RNAPs also activate the expression of other genes that normally require the response. We propose that the stability of initiation complexes is coupled to the transcription of stringently controlled promoters, and this unique feature coordinates the expression of genes positively and negatively regulated by the stringent response.Under optimal growth conditions, rapidly dividing Escherichia coli cells transcribe a set of genes at a very high rate. These genes engage most of the RNA polymerase (RNAP) molecules in the cell, although they constitute only a small fraction of the genome. In contrast, nutrition-limiting conditions, such as amino acid starvation, cause a rapid accumulation of the guanine derivative, ppGpp, and a dramatic reduction in expression of these genes, a process termed the stringent response (1). Because expression of these genes is negatively regulated during the stringent response in a manner dependent on the allelic state of the relA gene (2), they are called stringently controlled (or stringent) genes.Most of the stringent genes encode translational machinery, but some encode mRNAs. One such example is pyrBI, a pyrimidine biosynthetic operon, whose expression is inhibited during the stringent response (3). Thus, the expression of operons encoding rRNA, such as rrnB P1, and those encoding nucleotide biosynthetic enzymes, such as pyrBI, are coordinately regulated during the stringent response, even through each of these operons also has its own unique regulatory feature(s) (4-6). The stringent response serves as a global regulatory mechanism, coordinating the transcriptional activity of RNAP with the growth conditions of the cell.Despite the biological importance of the stringent response and extensive studies in the past decades, the mechanism(s) by which stringently controlled genes are coordinately regulated has been elusive (1). To further understand the regulation of stringently controlled genes, we decided to analyze RNAP mutants that appeared to have specifically altered interaction with stringently controlled (or stringent) promoters. Studying such RNAP mutants and defining the nature of their defects in interaction with this class of promoters...

Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis

“…The interaction between RNA polymerase and stringent promoters is easily modified by RNA polymerase missense mutations; consequently, suppressor mutations often arise in ppGpp 0 strains, and most of these strains are resistant to rifampicin (Murphy & Cashel, 2003;Zhou & Jin 1998). So, we used the primers BPrpoB-F and BPrpoB-R to sequence the region containing the sites in the rpoB gene in which suppressor mutations are detected.…”

RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media

“…Some RNAP mutants exhibit changes in initiation kinetics that resemble the effects of elevated (p)ppGpp on RNAP that occur during the stringent response (3). Further, (p)ppGpp is required for growth of E. coli in minimal media (9), which raised the possibility that the adaptive changes to RNAP might permanently change the enzyme in ways similar to that achieved transiently by (p)ppGpp binding.…”

“…kinetics | stringent response | transcription M utations to the primary RNAP genes in bacteria (rpoABCZ encoding the core α, β, β′, and ω subunits and rpoD encoding the housekeeping σ 70 subunit) exhibit a wide range of pleiotropic effects on bacterial phenotypes (1)(2)(3)(4)(5), and it has been said that mutations to RNAP genes can satisfy virtually any selection (6). We recently described the discovery of mutations in rpoB and rpoC following adaptive evolution of Escherichia coli K-12 MG1655 in glycerol M9 minimal medium (GMM) over a period of several weeks (7).…”

Translation quality control is critical for bacterial responses to amino acid stress

“…The initiation of the transcription of some ribosomal protein operons is regulated via ppGpp, although regulation of these genes largely occurs through translational feedback (17,18). At the same time the transcription of genes required for stress survival, virulence, and antibiotic resistance and those encoding metabolic enzymes, particularly glycolytic and amino acid biosynthesis enzymes, is up-regulated upon activation of the stringent response (19). Given that tRNA aminoacylation levels directly regulate both transcriptional attenuation of pheA and the stringent response, we investigated the possible role of misacylated tRNA editing in determining the responsiveness of cellular responses to amino acid stress.…”