(p)ppGpp directly regulates translation initiation during entry into quiescence

Abstract

12Many bacteria exist in a state of metabolic quiescence where they must minimize energy 13 consumption so as to maximize available resources over a potentially extended period of time. 14 As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be 15 an appropriate target for downregulation during the transition from growth to quiescence. We find 16 that when Bacillus subtilis exits growth, a subpopulation of cells emerges with very low levels of 17 protein synthesis dependent on synthesis of the nucleotides (p)ppGpp. We show that (p)ppGpp 18 inhibits protein synthesis in vivo and in vitro by preventing the allosteric activation of the essential 19 GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that IF2 is 20 an authentic in vivo target of (p)ppGpp during the entry into quiescence, thus providing a 21 mechanistic basis for the observed attenuation of protein synthesis. 22 al., 2016), DNA primase (Wang et al., 2007) and the GTP biosynthetic enzymes HprT and Gmk 51 (Kriel et al., 2012), respectively. 52 Overexpression of a truncated RelA protein that synthesizes (p)ppGpp in the absence of 53 amino acid limitation results in a rapid decrease in 35 S-methionine incorporation (Svitil et al., 54 1993), consistent with an inhibitory effect of (p)ppGpp on translation. However, further 55investigation of a direct effect of (p)ppGpp on translation has been complicated by several factors. 56 First, (p)ppGpp affects synthesis of ribosomal proteins in E. coli (Lindahl et al., 1976). Whether 57 this effect is direct has been difficult to assess given the well-established effect of (p)ppGpp on 58 transcription by E. coli polymerase. Second, studies examining the effect of (p)ppGpp produced 59 by RelA use conditions that produce uncharged tRNAs in order to stimulate RelA (Arenz et al., 60 2016; Brown et al., 2016; Haseltine and Block, 1973; Loveland et al., 2016; O'Farrell, 1978). Since 61 uncharged tRNAs directly arrest translation, it has been difficult to differentiate this effect from a 62 direct effect of (p)ppGpp on translation. 63 In vitro, (p)ppGpp inhibits translation in a manner similar to that observed with non-64 hydrolyzable GTP analogs (Wagner and Kurland, 1980), suggesting that (p)ppGpp is targeting a 65 translational GTPase. Consistently, (p)ppGpp inhibits the GTPase activity of IF2 and EF-Tu 66 (Hamel and Cashel, 1974) as well as of GTPases that mediate other aspects of translation such 67 as ribosome assembly (Corrigan et al., 2016; Pausch et al., 2018), by acting as competitive 68 inhibitors. (p)ppGpp is capable of binding translational GTPases including EF-Tu, EF-G (Rojas et 69 al., 1984), IF2 (Milon et al., 2006; Mitkevich et al., 2010) and the ribosome assembly GTPase 70 ObgE (Persky et al., 2009) at affinities that are commensurate with the in vivo levels of (p)ppGpp 71 observed following stringent response induction, consistent with the enzymes being in vivo 72 targets. Of note, the affinity of IF2 f...

The alarmones (p)ppGpp are part of the heat shock response of Bacillus subtilis

“…Nevertheless, the (p)ppGpp˚strain still displayed significantly higher translation rates compared to wild type and the Δrel B. subtilis strains (Fig 6A and 6B). The observed influence of (p)ppGpp on translation suggests that the most important impact of (p)ppGpp under heat stress appears not to be its effect on transcription (Fig 4 and S5 Fig), but the direct modulation of translation (Fig 6), possibly by directly interfering with the activity of different translational GTPases [39,40,68,69].…”

“…In addition to this protective function, (p)ppGpp, which is synthesized only as a pulse by Rel during relatively uninhibited growth at 50˚C, could modulate or inhibit translation, presumably by directly interfering with translation factors [38,39,41,69]. A set of interesting in vitro experiments suggested a modulating effect of ppGpp and (p)ppGpp on IF2 and translation initiation, which also depended on specific structured elements of translated mRNA, allowing translation of specific mRNA's in the presence of alarmones [41].…”

Anaerobiosis: A Surfactant Helps Bacteria Breathe a Sigh of Relief

“…Microbiologists have long investigated the mechanisms that facilitate survival under conditions where growth is nonoptimal. These mechanisms typically involve the synthesis of signaling molecules such as the nucleotide ppGpp that attenuate ATP-consuming processes of the cell such as protein synthesis [16]. Most of this work has focused on strategies relevant to nutrient limitation such as carbon or amino acid limitation.…”