Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase

c N-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA-and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154.

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“…Similar to a previously reported procedure (36) Western immunoblot analysis. Purified proteins were separated by SDS-PAGE.…”

Section: Ltq-orbitrap Velos Liquid Chromatography (Lc)-tandem Ms (Ms/mentioning

confidence: 79%

“…In vitro phosphorylation was carried out by incubating lithium potassium AcP (Sigma) with purified, Histagged RcsB at 30°C for 30 min in buffer (40 mM Tris-HCl [pH 8.0], 10 mM MgCl 2 , 40 mM KCl, 1 mM DTT) as described previously (36).…”

Section: Ltq-orbitrap Velos Liquid Chromatography (Lc)-tandem Ms (Ms/mentioning

confidence: 99%

Acetylation of the Response Regulator RcsB Controls Transcription from a Small RNA Promoter

Khanh

Kuhn

et al. 2013

Journal of Bacteriology

108

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“…In Escherichia coli, the protein acetyltransferase (Pka) alters the fate of RNase R. Results from early studies suggested that the Pat homologue from E. coli, EcPka, acetylated the RNA polymerase alpha subunit; however, results from subsequent analyses failed to confirm this claim (180,181). A bona fide substrate of EcPka is the exoribonuclease RNase R, shown to degrade highly structured mRNA.…”

Section: Rla In Gram-negative Bacteriamentioning

confidence: 99%

Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

Hentchel

Escalante‐Semerena

2015

Microbiol Mol Biol Rev

169

175

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SUMMARY Acylation of biomolecules (e.g., proteins and small molecules) is a process that occurs in cells of all domains of life and has emerged as a critical mechanism for the control of many aspects of cellular physiology, including chromatin maintenance, transcriptional regulation, primary metabolism, cell structure, and likely other cellular processes. Although this review focuses on the use of acetyl moieties to modify a protein or small molecule, it is clear that cells can use many weak organic acids (e.g., short-, medium-, and long-chain mono- and dicarboxylic aliphatics and aromatics) to modify a large suite of targets. Acetylation of biomolecules has been studied for decades within the context of histone-dependent regulation of gene expression and antibiotic resistance. It was not until the early 2000s that the connection between metabolism, physiology, and protein acetylation was reported. This was the first instance of a metabolic enzyme (acetyl coenzyme A [acetyl-CoA] synthetase) whose activity was controlled by acetylation via a regulatory system responsive to physiological cues. The above-mentioned system was comprised of an acyltransferase and a partner deacylase. Given the reversibility of the acylation process, this system is also referred to as r eversible l ysine a cylation (RLA). A wealth of information has been obtained since the discovery of RLA in prokaryotes, and we are just beginning to visualize the extent of the impact that this regulatory system has on cell function.

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“…Large-scale quantitative studies later revealed that acetylation of K291 is acetylphosphate sensitive (59), likely representing the mechanism of acetylation, and this acetylation event is indeed glucose induced (106). The CTD of RpoA makes direct contacts with DNA elements and transcription factors (107,108), providing a potential mechanism by which acetylation can rapidly alter transcription in response to environmental cues (97,104,105,(107)(108)(109). In B. subtilis, an RNA helicase (CshA) that associates with RNAP is required for glucose induction of SigX-and SigM-dependent genes (110).…”

Section: Functional Consequences Of Protein Acetylationmentioning

confidence: 99%

“…In E. coli, the RNA polymerase (RNAP) is acetylated on multiple subunits (39,40,60,104,105), and two acetylation sites within the alpha subunit (RpoA) were shown to be important. Glucose induction of the CpxR regulon requires the YfiQ-mediated acetylation of K291 (105) and K298 in the C-terminal domain (CTD) of RpoA (104).…”

Section: Functional Consequences Of Protein Acetylationmentioning

confidence: 99%

Regulation, Function, and Detection of Protein Acetylation in Bacteria

2017

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N -Lysine acetylation is now recognized as an abundant posttranslational modification (PTM) that influences many essential biological pathways. Advancements in mass spectrometry-based proteomics have led to the discovery that bacteria contain hundreds of acetylated proteins, contrary to the prior notion of acetylation events being rare in bacteria. Although the mechanisms that regulate protein acetylation are still not fully defined, it is understood that this modification is finely tuned via both enzymatic and nonenzymatic mechanisms. The opposing actions of Gcn5-related N-acetyltransferases (GNATs) and deacetylases, including sirtuins, provide the enzymatic control of lysine acetylation. A nonenzymatic mechanism of acetylation has also been demonstrated and proven to be prominent in bacteria, as well as in mitochondria. The functional consequences of the vast majority of the identified acetylation sites remain unknown. From studies in mammalian systems, acetylation of critical lysine residues was shown to impact protein function by altering its structure, subcellular localization, and interactions. It is becoming apparent that the same diversity of functions can be found in bacteria. Here, we review current knowledge of the mechanisms and the functional consequences of acetylation in bacteria. Additionally, we discuss the methods available for detecting acetylation sites, including quantitative mass spectrometry-based methods, which promise to promote this field of research. We conclude with possible future directions and broader implications of the study of protein acetylation in bacteria.

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Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase

Cited by 58 publications

References 53 publications

Acetylation of the Response Regulator RcsB Controls Transcription from a Small RNA Promoter

Acetylation of the Response Regulator RcsB Controls Transcription from a Small RNA Promoter

Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

Regulation, Function, and Detection of Protein Acetylation in Bacteria

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