Differential stringent control of Escherichia coli rRNA promoters: effects of ppGpp, DksA and the initiating nucleotides

Abstract

Transcription of rRNAs in Escherichia coli is directed from seven redundant rRNA operons, which are mainly regulated by their P1 promoters. Here we demonstrate by in vivo measurements that the amounts of individual rRNAs transcribed from the different operons under normal growth vary noticeably although the structures of all the P1 promoters are very similar. Moreover, we show that starvation for amino acids does not affect the seven P1 promoters in the same way. Notably, reduction of transcription from rrnD P1 was significantly lower compared to the other P1 promoters. The presence of DksA was shown to be crucial for the ppGpp-dependent downregulation of all P1 promoters. Because rrnD P1 is the only rrn promoter starting with GTP instead of ATP, we performed studies with a mutant rrnD promoter, where the initiating G+1 is replaced by A+1. These analyses demonstrated that the ppGpp sensitivity of rrn P1 promoters depends on the nature and concentration of initiating nucleoside triphosphates (iNTPs). Our results support the notion that the seven rRNA operons are differentially regulated and underline the importance of a concerted activity between ppGpp, DksA and an adequate concentration of the respective iNTP. INTRODUCTIONThe rapid adaptation of cell growth in response to changing environmental conditions is central for the fitness and survival of bacteria. Bacterial growth rate is largely determined by the number of ribosomes. Hence, regulation of the translational components plays a central role in adaptation. The biogenesis of ribosomes, representing one of the cell's most energy-intensive activities, is governed by a complex network of regulation (Schneider et al., 2003;Wagner, 2009). The key molecules in this network are rRNAs. In contrast, the synthesis of ribosomal proteins, despite having similar regulatory mechanisms to those for rRNA, is largely linked to rRNA transcription by a translational feedback mechanism, which couples ribosomal protein translation to the amount of available rRNAs (Keener & Nomura, 1996;Lemke et al., 2009;Nomura et al., 1984). Therefore, the precise adjustment of rRNA synthesis rate to the nutritional quality and supply from the environment ensures that an appropriate number of ribosomes will be produced and energy resources are not wasted (Condon et al., 1995b;Wagner, 1994Wagner, , 2009).In Escherichia coli, rRNA genes (16S, 23S and 5S rRNA), together with at least one tRNA gene, are organized in a redundant fashion in seven different transcriptional units (the rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH operons). Although the seven ribosomal operons exhibit a high structural similarity, there are several striking sequence micro-heterogeneities, which could potentially contribute to functionally different ribosome populations (Wagner, 2009). Evidence for such specialized ribosomes in various organisms has been presented (Gunderson et al., 1987;Kim et al., 2007; Ló pez-Ló pez et al., 2007). If the different rRNA operons encode functionally distinct molecules one would expect tha...

Nutrient Transitions Are a Source of Persisters in Escherichia coli Biofilms

“…The seven rRNA operons are regulated by the stringent response, and, when levels of ppGpp are high, transcription of the rRNA is repressed. [41] However, in ΔrelA this inhibition does not occur and levels of stable rRNA have been shown to be ∼2- to 10-fold higher than that of a relA + strain in similar conditions [46]–[50]. Within colony biofilms, we found that both 23 S and 16 S RNA expression was ∼2-fold higher in ΔrelA than wild-type during carbon source transitions at FC OD600  = 14 (Figure S4, Table S5).…”

Co‐ordinated regulation of the extracytoplasmic stress factor, sigmaE, with other Escherichia coli sigma factors by (p)ppGpp and DksA may be achieved by specific regulation of individual holoenzymes

“…The explanation for why (p)ppGpp is not needed may lie in the observation that nucleotide levels drop significantly when phosphate is depleted from the growth medium. At the rrnB P1 promoter and other stringently regulated rRNA promoters, Eσ 70 forms unstable open complexes, which are stabilized by binding of initiating nucleotides (iNTPs) and destabilized by DksA and (p)ppGpp (Gaal et al ., 1997; Barker et al ., 2001; Murray et al ., 2003; Murray and Gourse, 2004; Paul et al ., 2004b; Kolmsee et al ., 2011). A higher concentration of iNTP is required for transcription initiation at rrnB P1 in the presence of DksA than in its absence (Paul et al ., 2004b).…”

“…Because nucleotide levels drop during phosphate starvation, the decreased availability of nucleotides in combination with destabilization of open complexes by DksA may reduce transcription initiation, eliminating the requirement for (p)ppGpp. Because most other rrn promoters form similarly unstable open complexes limited by iNTP concentrations, this mechanism of regulation is likely to be shared and not particular to rrnB P1 (Gaal et al ., 1997; Barker et al ., 2001; Murray et al ., 2003; Murray and Gourse, 2004; Paul et al ., 2004a; Kolmsee et al ., 2011). …”

The dual role of DksA protein in the regulation ofEscherichia colipArgX promoter

“…This is manifested in the inhibition of production of the protein-synthesis system that is translation components: rRNA, r-protein and tRNA (1,2). The promoter initiating the expression of one of the seven rRNA operons in E. coli, rrnB P1, has been extensively studied and is currently one of the best characterized bacterial promoters (23–25). Promoters initiating the synthesis of tRNA (another class of stringently controlled RNA) are far less studied in comparison to rrnB P1; however the mechanism underlying their inhibition during the stringent response was proposed to be similar (26,27).…”