Mechanism of the in vitro breakdown of guanosine 5'-diphosphate 3'-diphosphate in Escherichia coli.

Abstract

Degradation of guanosine tetraphosphate (ppGpp) involves an enzyme associated with the ribosomal fraction from spoT'strains of Escheric4ia coli. Double Auxotrophic strains of Escherichia coli are known to accumulate rapidly the unusual nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) when deprived of an essential amino acid (for reviews, see refs. 1 and 2). It has been proposed that (p)ppGpp functions as a pluripotent regulator molecule that controls a number of biosynthetic and catabolic pathways; it restricts synthesis of stable RNA (3), of purine nucleotides (4), of glycolytic esters (5), and of phospholipids (6).In order to elucidate the function of (p)ppGpp as a major regulator molecule in bacterial cells, it is essential to understand the precise mechanism of its synthesis and degradation as well. The mode of synthesis of (p)ppGpp has been discovered by Haseltine et al. (7) who showed that these compounds are synthesized on ribosomes by an enzyme named "stringent factor." Despite attempts by a number of groups, the mechanism of ppGpp breakdown has remained unresolved until recently (8, 9). Although, in vivo, ppGpp is rapidly degraded when E. coli cells are resupplemented with the lacking amino acid (10), attempts to demonstrate degradation in vitro have failed (11)(12)(13)(14)(15). Genetic studies have shown that in vivo breakdown of ppGpp is associated with the spoT+ allele; in spoTmutants, ppGpp breakdown is significantly slower than in wild-type strains (16,17). This may indicate that spoT-mutants either have a similar but less active degradation device or utilize an alternative pathway. The enzyme involved and the mechanism of ppGpp breakdown are the subject of this communication. MATERIALS AND METHODSThe ppGpp degrading enzyme was prepared from the stringent E. coli strains CGSC 2834/a (relA +) and CP78 (relA +, CCA+, his-, leu-, arg-, thr-, B1-) or from the relaxed strain CP79 (relA-, CCA-, his-, leu-, arg-, thr-, BR -); CP78 and CP79 were kindly provided by M. Deutscher (Farmington, CT); K10 (spoT-, relA-, tonA22) were from A. Bock (Regensburg, Germany). [8-3H]GTP (New England Nuclear, Boston, MA) was used for preparation of pp[3H]Gpp (specific activity, 2

The RelA/SpoT Homolog (RSH) Superfamily: Distribution and Functional Evolution of ppGpp Synthetases and Hydrolases across the Tree of Life

“…SpoT additionally interacts with ppGpp in its hydrolytic site [19], the acyl carrier protein ACP [9] and the Obg/Gtc GTPase [92]. However, the scarcity of high-resolution structural data (especially the absence of crystal structures for complexes or cryoEM reconstructions of the ribosome-bond RSH) hinders domain- or site-specific prediction of function.…”

Control ofspoT-dependent ppGpp Synthesis and Degradation inEscherichia coli

“…The purified ppGpp hydrolase is inhibited by uncharged, but not charged tRNA (Heinemeyer & Richter, 1978;Richter, 1980). The results to be reported here suggest that uncharged tRNA does, indeed, inhibit ppGpp degradation activity in vivo, and that this is the basis of the often-studied ppGpp accumulation (a) and (b) A culture of the B/r strain RL331T (relA + phe) was grown in glucose minimal medium plus phenylalanine, then cells were sedimented by centrifugation, washed free of phenylalanine (see Materials and Methods) and resuspended in pre-warmed glucose minimal medium without phenylalanine (phenylalanine starvation).…”

Fusidic acid‐resistant EF‐G perturbs the accumulation of ppGpp

“…In vivo evidence suggests a negative correlation of PSII activity with the size of the free amino acid pool (Ryals et al, 1982;Hernandez and Bremer, 1990). The spoT ppGpp hydrolase activity, in vivo and in vitro, is inhibited by uncharged tRNAs (Heinemeyer and Richter, 1978;Murray and Bremer, 1996). In rich media, in which free amino acid pools are large, PSII synthetase activity will be low and spoT hydrolase activity will be high.…”