scholarly journals Lysine acetylation of the housekeeping sigma factor enhances the activity of the RNA polymerase holoenzyme

2020 ◽  
Vol 48 (5) ◽  
pp. 2401-2411 ◽  
Author(s):  
Ji-Eun Kim ◽  
Joon-Sun Choi ◽  
Jong-Seo Kim ◽  
You-Hee Cho ◽  
Jung-Hye Roe
Keyword(s):  
Sigma Factor ◽  
Binding Activity ◽  
Lysine Acetylation ◽  
Label Free ◽  
Post Translational Modifications ◽  
Additional Layer ◽  
Bacterial Rna ◽  
Protein Lysine Acetylation ◽  
Rna Polymerase Holoenzyme

Abstract Protein lysine acetylation, one of the most abundant post-translational modifications in eukaryotes, occurs in prokaryotes as well. Despite the evidence of lysine acetylation in bacterial RNA polymerases (RNAPs), its function remains unknown. We found that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of an actinobacterium, Streptomyces venezuelae, and the acetylated HrdB was enriched in the RNAP holoenzyme complex. The lysine (K259) located between 1.2 and 2 regions of the sigma factor, was determined to be the acetylated residue of HrdB in vivo by LC–MS/MS analyses. Specifically, the label-free quantitative analysis revealed that the K259 residues of all the HrdB subunits were acetylated in the RNAP holoenzyme. Using mutations that mimic or block acetylation (K259Q and K259R), we found that K259 acetylation enhances the interaction of HrdB with the RNAP core enzyme as well as the binding activity of the RNAP holoenzyme to target promoters in vivo. Taken together, these findings provide a novel insight into an additional layer of modulation of bacterial RNAP activity.

scholarly journals Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ςS-Dependent Transcription in Pseudomonas putida

2000 ◽  
Vol 182 (23) ◽  
pp. 6707-6713 ◽  
Author(s):  
Eve-Ly Ojangu ◽  
Andres Tover ◽  
Riho Teras ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Deficient Mutant ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Silent Genes ◽  
Rna Polymerase Holoenzyme

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of ςS on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

scholarly journals Substitution of a Highly Conserved Histidine in the Escherichia coli Heat Shock Transcription Factor, σ32, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo

2007 ◽  
Vol 189 (23) ◽  
pp. 8430-8436 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Stable Complex ◽  
Tryptophan Residue ◽  
Specific Sequence ◽  
Bacterial Rna

ABSTRACT The heat shock sigma factor (σ32 in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of σ32 is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, σ70. This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing σ32, implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in σ32 impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.

scholarly journals OP0131 GUT DERIVED ACETATE PROMOTES REGULATORY B CELLS WITH ANTI-INFLAMMATORY EFFECTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 85.2-85
Author(s):  
C. Daien ◽  
J. Tan ◽  
R. Audo ◽  
J. Mielle ◽  
L. Macia
Keyword(s):  
Drinking Water ◽  
B Cells ◽  
Dietary Fiber ◽  
Lysine Acetylation ◽  
Regulatory B Cells ◽  
Acetyl Coa ◽  
Protein Lysine Acetylation ◽  
B10 Cells

Background:Regulatory B cells (Bregs) are defective in many auto-immune diseases, i.e. rheumatoid arthritis (RA). The short-chain fatty acid (SCFA) acetate, derived mostly from gut microbial fermentation of dietary fiber, promotes anti-inflammatory regulatory T cells and protects mice from type 1 diabetes and colitis. We hypothesized that acetate could be a good candidate to promote Bregs in auto-immune diseases.Objectives:To assess the effect of acetate on Breg number and function,in vitroandin vivoin mice and humans.Methods:Bregs were defined as IL-10 producing regulatory B cells (B10 cells). Their number was assessed after overnight exposure to acetate (Ac 10 mM) and 4 hours of CpG, ionomycin and PMA in mice and after 24 hours of acetate +/- CpG and 4 hours of ionomycin and PMA in humans. Acetate was given to mice either intraperitoneally (twice at a 12-hour interval) or in drinking water for 3 weeks. Acetate-treated B cells were transferred to mice with collagen-antibody -induced arthritis to assess their function. To decipher the mechanisms behind the effect of acetate, we used inhibitors of GPR43 (CATPB), ATP synthase (oligomycin), glycolysis (2-DG), ACSS2 and ACLY and assessed protein lysine acetylation by flow cytometry on human B cells. Acetate and B10 cells were also assessed before and after a 7-day high-fibre diet in 12 healthy volunteers.Results:In mice, acetate promoted B10 cell differentiation bothin vitro(medians [IQR] 3.1 [0.4-3.7] and 9.9 [5.9-17.6]% of B for CpG and CpG+Ac respectively, p=0.002) andin vivowhen intraperitoneal injected(22 [14-29] and 31 [25-37]% of B for PBS and acetate respectively,p=0.03) or added to drinking water (17 [6-25] and 39 [26-40]% of B for water or acetate respectively, p=0.02). Adoptive transfer of acetate-treated B cells protected mice from arthritis compared to non-exposed B cells (ANOVA p=0.008). Acetate also promoted B10 cells from human blood cells (2.5 [1.6-2.7] and 3.4 [2.6-4.5] for unstimulated [Un] and Ac respectively, p=0.0001). Conversely to CpG, acetate specifically promoted IL-10, with no impact or a decrease of proinflammatory cytokines (IL-6: 17 [5-29]; 12 [3-21] and 40 [20-47]% B cells for Un, Ac and CpG respectively, p<0.01 for all comparisons and TNF-a: 48 [29-61]; 41 [28-67] and 69 [64-78]% B cells for Un, Ac and CpG respectively, p<0.01 for CpG vs Un or Ac, NS for acetate vs Un). Inhibition of GPR43 and ACLY did not impact acetate response, while inhibition of glycolysis significantly decreased its effect. Blockade of ACSS2, converting acetate into acetyl-CoA, decreased acetate-induced B10 cells. Acetate was associated with an increase of protein lysine acetylation which was not observed in presence of CpG alone, suggesting a different mechanism of action (2.0 [1.3-3.4]; 3.3 [2.4-5.4] and 1.4 [0.5-1.7]% B cells for Un, Ac and CpG respectively, p=0.002 for Un vs Ac, NS with CpG). Conversion of acetate into acetyl-CoA could thus be used for the acetylation of cytoplasmic protein, a post-translational modification that regulates key cellular processes, including energy metabolism. In addition, B10 cells had significantly more lysine-acetylated proteins than IL-10negB cells or TNF+B cells (5.3[3.9-7.3]; 3.2 [2.4-5.4] and 3.9 [2.7-6.2] % of B for B10, IL-10negB cells or TNF+B cells respectively, p<0.01 for all comparisons). Finally, dietary fiber supplementation in healthy individuals was associated with increased acetate and B10 cells in the blood, which were significantly correlated (R2=0.20, p=0.02).Conclusion:Our results suggest that acetate induces functional Bregs, through its conversion into acetyl-CoA, used for cell metabolism and protein acetylation. Delivery of acetate or acetate producing diets or bacteria might be a promising approach to restore Bregs in non-communicable diseases such as RA in which they are defective.Disclosure of Interests:Claire DAIEN Grant/research support from: from Pfizer, Abbvie, Roche-Chugaï, Novartis, Abivax, Sandoz, Consultant of: Abbvie, Abivax, BMS, MSD, Roche-Chugaï, Lilly, Novartis, Speakers bureau: Abbvie, Abivax, BMS, MSD, Roche-Chugaï, Lilly, Novartis, Jian Tan: None declared, Rachel Audo: None declared, Julie Mielle: None declared, Laurence Macia: None declared

scholarly journals Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

2021 ◽  
Vol 8 ◽  
Author(s):  
Virtu Solano-Collado ◽  
Sofía Ruiz-Cruz ◽  
Fabián Lorenzo-Díaz ◽  
Radoslaw Pluta ◽  
Manuel Espinosa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Sigma Factor ◽  
Regulation Of Gene Expression ◽  
E Coli ◽  
Core Enzyme ◽  
Protein Architecture ◽  
Bacterial Rna ◽  
Promoter Recognition ◽  
Rna Polymerase Holoenzyme

Promoter recognition by RNA polymerase is a key step in the regulation of gene expression. The bacterial RNA polymerase core enzyme is a complex of five subunits that interacts transitory with one of a set of sigma factors forming the RNA polymerase holoenzyme. The sigma factor confers promoter specificity to the RNA polymerase. In the Gram-positive pathogenic bacterium Streptococcus pneumoniae, most promoters are likely recognized by SigA, a poorly studied housekeeping sigma factor. Here we present a sequence conservation analysis and show that SigA has similar protein architecture to Escherichia coli and Bacillus subtilis homologs, namely the poorly conserved N-terminal 100 residues and well-conserved rest of the protein (domains 2, 3, and 4). Further, we have purified the native (untagged) SigA protein encoded by the pneumococcal R6 strain and reconstituted an RNA polymerase holoenzyme composed of the E. coli core enzyme and the sigma factor SigA (RNAP-SigA). By in vitro transcription, we have found that RNAP-SigA was able to recognize particular promoters, not only from the pneumococcal chromosome but also from the S. agalactiae promiscuous antibiotic-resistance plasmid pMV158. Specifically, SigA was able to direct the RNA polymerase to transcribe genes involved in replication and conjugative mobilization of plasmid pMV158. Our results point to the versatility of SigA in promoter recognition and its contribution to the promiscuity of plasmid pMV158.

scholarly journals Construction of a Quantitative Acetylomic Tissue Atlas in Rice (Oryza sativa L.)

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2843 ◽  
Author(s):  
Zhiyong Li ◽  
Yifeng Wang ◽  
Babatunde Bello ◽  
Abolore Ajadi ◽  
Xiaohong Tong ◽  
...  
Keyword(s):  
Chloroplast Development ◽  
Oryza Sativa L ◽  
Protein Translation ◽  
Lysine Acetylation ◽  
Biological Processes ◽  
Label Free ◽  
Post Translational Modification ◽  
Rice Tissue ◽  
Protein Lysine Acetylation ◽  
Rice Tissues

PKA (protein lysine acetylation) is a key post-translational modification involved in the regulation of various biological processes in rice. So far, rice acetylome data is very limited due to the highly-dynamic pattern of protein expression and PKA modification. In this study, we performed a comprehensive quantitative acetylome profile on four typical rice tissues, i.e., the callus, root, leaf, and panicle, by using a mass spectrometry (MS)-based, label-free approach. The identification of 1536 acetylsites on 1454 acetylpeptides from 890 acetylproteins represented one of the largest acetylome datasets on rice. A total of 1445 peptides on 887 proteins were differentially acetylated, and are extensively involved in protein translation, chloroplast development, and photosynthesis, flowering and pollen fertility, and root meristem activity, indicating the important roles of PKA in rice tissue development and functions. The current study provides an overall view of the acetylation events in rice tissues, as well as clues to reveal the function of PKA proteins in physiologically-relevant tissues.

scholarly journals Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions

2019 ◽  
Author(s):  
Paula A. Agudelo Garcia ◽  
Prabakaran Nagarajan ◽  
Mark R. Parthun
Keyword(s):  
Affinity Purification ◽  
Mouse Embryonic Fibroblast ◽  
Mitochondrial Proteins ◽  
Lysine Acetylation ◽  
Label Free ◽  
Cellular Functions ◽  
Post Translational Modifications ◽  
Cell Extracts ◽  
Proteomics Approach ◽  
Regulatory Loop

ABSTRACTLysine acetylation has emerged as one of the most important post-translational modifications, regulating different biological processes. However, its regulation by lysine acetyltransferases is still unclear in most cases. Hat1 is a lysine acetyltransferase originally identified based on its ability to acetylate histones. Using an unbiased proteomics approach, we have determined how loss of Hat1 affects the mammalian acetylome. Hat1+/+ and Hat1−/− mouse embryonic fibroblast (MEF) cells lines were grown in both glucose- and galactose-containing media, as Hat1 is required for growth on galactose and Hat1−/− cells exhibit defects in mitochondrial function. Following trypsin digestion of whole cell extracts, acetylated peptides were enriched by acetyllysine affinity purification and acetylated peptides were identified and analyzed by label-free quantitation. Comparison of the acetylome from Hat1+/+ cells grown on galactose and glucose demonstrated that there are large carbon source-dependent changes in the mammalian acetylome where the acetylation of enzymes involved in glycolysis was the most affected. Comparisons of the acetylomes from Hat1+/+ and Hat1−/− cells identified 65 proteins whose acetylation decreased by at least 2.5-fold in cells lacking Hat1. In Hat1−/− cells, acetylation of the auto regulatory loop of CBP was the most highly affected, decreasing by up to 20-fold. In addition to proteins involved in chromatin structure, Hat1-dependent acetylation was also found in a number of transcriptional regulators, including p53, and mitochondrial proteins. Hat1 mitochondrial localization suggests that it may be directly involved in the acetylation of mitochondrial proteins.

scholarly journals The PspA Protein of Escherichia coli Is a Negative Regulator of ς54-Dependent Transcription

2000 ◽  
Vol 182 (2) ◽  
pp. 311-319 ◽  
Author(s):  
Jonathan Dworkin ◽  
Goran Jovanovic ◽  
Peter Model
Keyword(s):  
Escherichia Coli ◽  
Sigma Factor ◽  
Negative Regulator ◽  
Regulatory Domain ◽  
Expression Of Genes ◽  
The Family ◽  
Initiation Of Transcription ◽  
Rna Polymerase Holoenzyme

ABSTRACT In Eubacteria, expression of genes transcribed by an RNA polymerase holoenzyme containing the alternate sigma factor ς54 is positively regulated by proteins belonging to the family of enhancer-binding proteins (EBPs). These proteins bind to upstream activation sequences and are required for the initiation of transcription at the ς54-dependent promoters. They are typically inactive until modified in their N-terminal regulatory domain either by specific phosphorylation or by the binding of a small effector molecule. EBPs lacking this domain, such as the PspF activator of the ς54-dependent pspA promoter, are constitutively active. We describe here the in vivo and in vitro properties of the PspA protein of Escherichia coli, which negatively regulates expression of the pspA promoter without binding DNA directly.

New Antimicrobials Targeting Bacterial RNA Polymerase Holoenzyme Assembly Identified with an in Vivo BRET-Based Discovery Platform

2019 ◽  
Vol 14 (8) ◽  
pp. 1727-1736 ◽  
Author(s):  
Sara Sartini ◽  
Elisabetta Levati ◽  
Martina Maccesi ◽  
Matteo Guerra ◽  
Gilberto Spadoni ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Bacterial Rna ◽  
Rna Polymerase Holoenzyme

Analysis of Protein Lysine Acetylation In Vitro and In Vivo

2008 ◽  
Vol 54 (1) ◽  
Author(s):  
Nadine Pelletier ◽  
Serge Grégoire ◽  
Xiang‐Jiao Yang
Keyword(s):  
Lysine Acetylation ◽  
Protein Lysine Acetylation

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

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