Coordinated Regulation of Rsd and RMF for Simultaneous Hibernation of Transcription Apparatus and Translation Machinery in Stationary-Phase Escherichia coli

Abstract

Transcription and translation in growing phase of Escherichia coli, the best-studied model prokaryote, are coupled and regulated in coordinate fashion. Accordingly, the growth rate-dependent control of the synthesis of RNA polymerase (RNAP) core enzyme (the core component of transcription apparatus) and ribosomes (the core component of translation machinery) is tightly coordinated to keep the relative level of transcription apparatus and translation machinery constant for effective and efficient utilization of resources and energy. Upon entry into the stationary phase, transcription apparatus is modulated by replacing RNAP core-associated sigma (promoter recognition subunit) from growth-related RpoD to stationary-phase-specific RpoS. The anti-sigma factor Rsd participates for the efficient replacement of sigma, and the unused RpoD is stored silent as Rsd-RpoD complex. On the other hand, functional 70S ribosome is transformed into inactive 100S dimer by two regulators, ribosome modulation factor (RMF) and hibernation promoting factor (HPF). In this review article, we overview how we found these factors and what we know about the molecular mechanisms for silencing transcription apparatus and translation machinery by these factors. In addition, we provide our recent findings of promoter-specific transcription factor (PS-TF) screening of the transcription factors involved in regulation of the rsd and rmf genes. Results altogether indicate the coordinated regulation of Rsd and RMF for simultaneous hibernation of transcription apparatus and translation machinery.

Keywords: Escherichia coli K-12; RNA polymerase sigma factor; anti-sigma factor (Rsd); hibernation; ribosome; ribosome modulation factor; stationary phase.

Figure 1

Hibernation of transcription apparatus and translation machinery in Escherichia coli K-12. Upon entry of E. coli growth into the stationary phase, RNAP RpoD becomes silent through binding of anti-sigma factor Rsd onto the RpoD region-4 (promoter -35 recognition site) (Jishage and Ishihama, 1998; Jishage et al., 2001) while functional 70S ribosomes are converted to inactive 100S dimers through association with RMF (Wada et al., 1990; Wada, 1998) and HPF (Ueta et al., 2008; Yoshida and Wada, 2014). Here, we describe the coordinated regulation of two key regulators, Rsd and RMF, in E. coli K-12. The binding targets and binding sites of these two regulators on RNAP and ribosomes are described in text and also in Figure 6. Other factors involved in these processes are also described in text. RNAP, RNA polymerase; RMF, ribosome modulation factor; HPF, hibernation promoting factor.

Figure 2

Growth phase-dependent discontinuous increase of cell buoyant density of Escherichia coli K-12. (A) E. coli W3110 was grown in LB medium at 37°C with shaking. At various times, an aliquot of cell suspension was subjected to Percoll gradient centrifugation for 1 h at 20,000 rpm at 4°C in a Beckman SW40Ti rotor (Makinoshima et al., 2002; Makinoshima et al., 2003). The location of marker beads is indicated on the left: a, 1.035 g/ml; b, 1.074 g/ml; c, 1.087 g/ml; d, 1.102 g/ml; e, 1.119 g/ml. (B)E. coli wild-type BW25113 and its single-gene knockout mutants were grown in LB for 4 (L) or 24 h (S) and subjected to Percoll gradient centrifugation. The increase in cell buoyant density was interfered for these mutants, remaining at specific positions as indicated on the right. LB, lysogeny broth.

Figure 3

Growth phase-dependent synthesis of 18 representative stationary proteins in Escherichia coli K-12. E. coli K-12 AD202 was grown in minimal medium E (Vogel and Bonner, 1956) containing 2% peptone at 37°C. The cell growth was monitored for 10 days by measuring the turbidity at 660 nm and by counting viable cells as shown in the inset. Aliquots of the culture were harvested at the indicated time (X-axis), and the cell lysates were fractionated into CD (insoluble cell debris), CE (cell extract supernatant), CR (crude ribosome), and PRS (post ribosomal supernatant) fractions. All these fractions prepared at each time point was subjected to RFHR 2D gel system, and the stained protein spots were measured by densitometry. The relative levels (Y-axis) are shown at each culture time (X-axis) for a total of 18 representative stationary proteins. The proteins shown under purple background indicate those involved in the hibernation of ribosomes.

Figure 4

Growth phase-dependent expression patterns of a total of 65 stationary proteins in Escherichia coli K-12. The growth phase-dependent synthesis was measured for a total 65 stationary proteins. The relative level of synthesis from log phase (3-h culture) to day 8 is shown for all 65 proteins. The maximum level is shown by filling the day column with full red color. Spot numbers listed in Table 1 are shown on the horizontal axis, with colors indicating fraction type (green: PRS, orange: CD, and magenta: CR). The protein products so far identified are shown in red below the corresponding spot numbers.

Figure 5

Intracellular levels of sigma factors and anti-RpoD sigma (Rsd). (A) Intracellular levels of all seven sigma factors in exponential phase E. coli K-12 was determined by Western blot analysis with use of specific antibodies (Jishage and Ishihama, 1995; Jishage et al., 1996). (B) Intracellular levels of growth-related RpoD sigma, stationary-phase-specific RpoS sigma, and anti-RpoD sigma Rsd were determined at various growth phases of E. coli K-12 (Jishage and Ishihama, 1998; Jishage and Ishihama, 1999). (C) The contact site of anti-sigma factor Rsd on the growth-related RpoD sigma was determined to be located within RpoD region-4 (promoter -35 recognition site) by using the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA by analysis of the complex formation between Ala-substituted σ⁷⁰ and Rsd (Jishage and Ishihama, 2001). Rsd-binding to RpoD region-3 leads to silencing RpoD function.

Figure 6

Growth phase-coupled alteration of ribosomes in Escherichia coli K-12. (A) In exponentially growing bacterial cells, most ribosomes are involved in the functional cycle of protein synthesis, consisting of initiation, elongation, termination, and recycling. For initiation, 30S and 50S ribosomes bind to mRNA, forming functional 70S ribosomes on mRNA and ultimately leading to form polysomes. After termination, 70S ribosomes are dissociated into 30S and 50S subparticles for reutilization. (B) Upon entry into stationary phase, unused ribosomes are converted into functionally inactive 100S dimeric ribosomes by sequential binding of RMF and HPF in E. coli K-12, one of Gram-negative bacteria (Wada, 1998; Yoshida and Wada, 2014). We designated this process as “hibernation.” Formation of 100S dimers is interfered by RaiA (renamed YfiA) (Ueta et al., 2005). The location of RMF on 30S ribosome is based on the recent cryo-electron micrography structure of 100S ribosome dimer (Beckert et al., 2018). By biochemical analyses, however, RMF was also indicated to bind 23S rRNA (Yoshida et al., 2004) and the peptidyl transferase center (Yoshida et al., 2002).

Figure 7

PS-TF screening was performed for search of TFs involved in regulation of the rsd and rmf genes. A total of 74 TF species were found to bind to both the rsd and rmf promoter probes, although the binding affinity appeared different between these TFs (Yoshida et al., 2018). Besides these 74 TFs, some other TFs have been identified to bind only the rsd gene or the rmf gene, indicating independent regulation of the two genes under as yet unidentified conditions. Detailed analysis of the regulatory roles in vitro and in vivo was performed for the five representative stress-response TFs (ArcA, McbR, RcdA, SdiA, and SlyA) (Yoshida et al., 2018). ArcA was indicated to repress transcription of both rsd and rmf genes, while other four were suggested to activate both genes. gSELEX indicated that all these TFs regulate not only the rsd and rmf genes but also regulate a number of genes supposedly required for survival under stressful conditions. PS-TF, promoter-specific transcription factor.