Cellular localization of the Escherichia coli SpoT protein

Gentry, Daniel R.; Cashel, Michael

doi:10.1128/jb.177.13.3890-3893.1995

Cited by 40 publications

(38 citation statements)

References 28 publications

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“…Intrigued by the controversial results regarding the ribosome association of SpoT (15,20,48), we decided to reinvestigate the localization of SpoT by using sucrose density centrifugation of ribosomal particles. Several experimental details were taken into consideration.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, the hydrolase activity of SpoT is inhibited in the presence of uncharged tRNA and more severely inhibited in the presence of ribosomes (42), suggesting that the activity of SpoT may be controlled by tRNA on the ribosome, as seen with RelA. SpoT was not, however, found associated with the ribosomal particles by sucrose density centrifugation (15).…”

mentioning

confidence: 87%

“…RelA is a (p)ppGpp synthetase and a well-established ribosome-associated protein (15,40,55). The (p)ppGpp synthetase activity of RelA is activated by uncharged tRNA at the ribosome A site (19) and is partially dependent on the large ribosomal protein L11 (55,58).…”

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confidence: 99%

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G-Protein Control of the Ribosome-Associated Stress Response Protein SpoT

et al. 2007

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The bacterial response to stress is controlled by two proteins, RelA and SpoT. RelA generates the alarmone (p)ppGpp under amino acid starvation, whereas SpoT is responsible for (p)ppGpp hydrolysis and for synthesis of (p)ppGpp under a variety of cellular stress conditions. It is widely accepted that RelA is associated with translating ribosomes. The cellular location of SpoT, however, has been controversial. SpoT physically interacts with the ribosome-associated GTPase CgtA, and we show here that, under an optimized salt condition, SpoT is also associated with a pre-50S particle. Analysis of spoT and cgtA mutants and strains overexpressing CgtA suggests that the ribosome associations of SpoT and CgtA are mutually independent. The steady-state level of (p)ppGpp is increased in a cgtA mutant, but the accumulation of (p)ppGpp during amino acid starvation is not affected, providing strong evidence that CgtA regulates the (p)ppGpp level during exponential growth but not during the stringent response. We show that CgtA is not associated with pre-50S particles during amino acid starvation, indicating that under these conditions in which (p)ppGpp accumulates, CgtA is not bound either to the pre-50S particle or to SpoT. We propose that, in addition to its role as a 50S assembly factor, CgtA promotes SpoT (p)ppGpp degradation activity on the ribosome and that the loss of CgtA from the ribosome is necessary for maximal (p)ppGpp accumulation under stress conditions. Intriguingly, we found that in the absence of spoT and relA, cgtA is still an essential gene in Escherichia coli.

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Section: Resultsmentioning

confidence: 99%

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confidence: 87%

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G-Protein Control of the Ribosome-Associated Stress Response Protein SpoT

et al. 2007

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“…SpoT purifies with crude ribosomal fractions (21, 52, 59) but is not found associated with ribosomes separated by sucrose density centrifugation (15). Given that SpoT interacts with CgtA E , a ribosome-associated protein, we predicted that SpoT is also ribosome associated but not detected on sucrose gradients under the conditions used previously (15). Recent work from our laboratory reveals that SpoT is, in fact, associated with ribosomes (P. Wout, M. J. Jiang, and J. Maddock, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

TheEscherichia coliGTPase CgtA_ECofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase

et al. 2004

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CgtA E /Obg E /YhbZ is an Escherichia coli guanine nucleotide binding protein of the Obg/GTP1 subfamily whose members have been implicated in a number of cellular functions including GTP-GDP sensing, sporulation initiation, and translation. Here we describe a kinetic analysis of CgtA E with guanine nucleotides and show that its properties are similar to those of the Caulobacter crescentus homolog CgtA C . CgtA E binds both GTP and GDP with moderate affinity, shows high guanine nucleotide exchange rate constants for both nucleotides, and has a relatively low GTP hydrolysis rate. We show that CgtA E is associated predominantly with the 50S ribosomal subunit. Interestingly, CgtA E copurifies with SpoT, a ribosome-associated ppGpp hydrolase/synthetase involved in the stress response. The interaction between CgtA E and SpoT was confirmed by reciprocal coprecipitation experiments and by two-hybrid assays. These studies raise the possibility that the ribosome-associated CgtA E is involved in the SpoT-mediated stress response.

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“…The production of ribosomal proteins is also post-transcriptionally feedback-regulated to match the rRNA production (reviewed in 5). Two different ppGpp synthetases (PS) exist in E. coli, the ribosome-associated PS I, encoded by relA, and the cytoplasmic PS II (6), encoded by spoT (7,8). The protein PS II is also responsible for ppGpp hydrolysis (1).…”

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confidence: 99%

Underproduction of ς70 Mimics a Stringent Response

Magnusson

Nyström

Farewell

2003

Journal of Biological Chemistry

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When Escherichia coli cells enter stationary phase due to carbon starvation the synthesis of ribosomal proteins is rapidly repressed. In a ⌬relA ⌬spoT mutant, defective in the production of the alarmone guanosine tetraphosphate (ppGpp), this regulation of the levels of the protein synthesizing system is abolished. Using a proteomic approach we demonstrate that the production of the vast majority of detected E. coli proteins are decontrolled during carbon starvation in the ⌬relA ⌬spoT strain and that the starved cells behave as if they were growing exponentially. In addition we show that the inhibition of ribosome synthesis by the stringent response can be qualitatively mimicked by artificially lowering the levels of the housekeeping factor, 70 . In other words, genes encoding the protein-synthesizing system are especially sensitive to reduced availability of 70 programmed RNA polymerase. This effect is not dependent on ppGpp since lowering the levels of 70 gives a similar but less pronounced effect in a ppGpp 0 strain. The data is discussed in view of the models advocating for a passive control of gene expression during stringency based on alterations in RNA polymerase availability.The alarmone guanosine tetraphosphate (ppGpp) 1 of the stringent response network in Escherichia coli affects ribosome production by specifically lowering the transcription of ribosomal RNA (rRNA) (1) and some of the genes encoding ribosomal proteins (2-4). The production of ribosomal proteins is also post-transcriptionally feedback-regulated to match the rRNA production (reviewed in 5). Two different ppGpp synthetases (PS) exist in E. coli, the ribosome-associated PS I, encoded by relA, and the cytoplasmic PS II (6), encoded by spoT (7,8). The protein PS II is also responsible for ppGpp hydrolysis (1).Increased levels of ppGpp not only down-regulate ribosome production but also induce transcription from several promoters (9 -14). As suggested by Schreiber et al. (9), the E. coli promoters can be divided into three groups depending on their response to ppGpp: promoters specifically induced or repressed by the stringent response and promoters that are unaffected by the rise in ppGpp levels. Promoters dependent on the housekeeping factor, 70 , exist in at least two of these groups; one group is positively regulated by ppGpp, e.g. PuspA (11) and Phis (8), whereas the other group is repressed during stringency, e.g. rrnP1. Attempts to explain this dual effect of ppGpp have involved considerations of RNAP (RNA polymerase) availability and intrinsic differences in the kinetic properties of the promoters affected (15). Several lines of evidence suggest that the availability of transcriptional and/or translational machinery play a role in global gene regulation in concert with classical activators and repressors (13-17). One well known example of this type of regulation, where the level of RNAP is involved, is factor competition in both Bacillus subtilis (18) and E. coli (19,20).Whether or not RNAP availability is involved in the actual m...

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Cellular localization of the Escherichia coli SpoT protein

Cited by 40 publications

References 28 publications

G-Protein Control of the Ribosome-Associated Stress Response Protein SpoT

G-Protein Control of the Ribosome-Associated Stress Response Protein SpoT

TheEscherichia coliGTPase CgtA_ECofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase

Underproduction of ς70 Mimics a Stringent Response

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Cellular localization of the Escherichia coli SpoT protein

Cited by 40 publications

References 28 publications

G-Protein Control of the Ribosome-Associated Stress Response Protein SpoT

G-Protein Control of the Ribosome-Associated Stress Response Protein SpoT

TheEscherichia coliGTPase CgtAECofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase

Underproduction of ς70 Mimics a Stringent Response

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TheEscherichia coliGTPase CgtA_ECofractionates with the 50S Ribosomal Subunit and Interacts with SpoT, a ppGpp Synthetase/Hydrolase