Current perspectives of the Escherichia coli RNA degradosome

Burger, Adélle; Whiteley, Chris G.; Boshoff, Aileen

doi:10.1007/s10529-011-0713-6

Cited by 11 publications

(7 citation statements)

References 116 publications

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“…The brisk removal of S1 in our system provided a unique opportunity to detect any immediate changes in the abundance of mRNAs, with the intent of identifying direct influences of S1. We placed focus on several mRNAs: that which encodes S1 itself (rpsA), those encoding proteins that physically interact with S1 (S2 [rpsB], RNase E [rne], PNPase [pnp]) (1,10,17,37,38), messages known to be degraded by the degradosome and stabilized by S1 (CspE and S15 [rpsO]) (9,16,40), a collection of unrelated mRNAs with diverse 5= untranslated regions (UTRs) and translation initiation features (encoding LacI, LacZ, OmpT, and RecA), and two housekeeping transcripts (encoding GAPDH [gapA] [11,43,49,51] and CsdA [36,44]). …”

Section: Resultsmentioning

confidence: 99%

Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses

et al. 2012

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Targeted protein degradation is a powerful tool that can be used to create unique physiologies depleted of important factors. Current strategies involve modifying a gene of interest such that a degradation peptide is added to an expressed target protein and then conditionally activating proteolysis, either by expressing adapters, unmasking cryptic recognition determinants, or regulating protease affinities using small molecules. For each target, substantial optimization may be required to achieve a practical depletion, in that the target remains present at a normal level prior to induction and is then rapidly depleted to levels low enough to manifest a physiological response. Here, we describe a simplified targeted degradation system that rapidly depletes targets and that can be applied to a wide variety of proteins without optimizing target protease affinities. The depletion of the target is rapid enough that a primary physiological response manifests that is related to the function of the target. Using ribosomal protein S1 as an example, we show that the rapid depletion of this essential translation factor invokes concomitant changes to the levels of several mRNAs, even before appreciable cell division has occurred.T raditional approaches to unravel protein-coding gene function include making knockout or conditional mutant strains for comparative studies. In many cases, obtaining a conditional inactivation mutant is not tractable or there may be concerns that the inactivated protein is defective only in one of several traits. When investigating essential genes, knockout strains can be cultured for biochemical analyses when there is a complementing copy of the gene. By placing an essential gene under the control of a regulated promoter, so-called depletion-by-division experiments can be performed wherein the gene is turned off, and then cell division and normal protein turnover are used to deplete the encoded factor from the cell. Complications arise when the depletion of a factor induces downstream physiological responses that are not directly related to the factor's function, one example being the activation of multiple toxins when translation is inhibited (12,52,56). A solution to this problem is to specifically target the encoded gene product for conditional depletion (29). In doing so, the gene remains in its natural context, and the encoded product functions normally at the appropriate dose prior to its removal.Prior studies have demonstrated the utility of this approach for specific cases by reengineering target proteins such that they contain a peptide degradation signal that is recognized by a processive protease (7,15,20,24,29,53). In early examples, a degradation peptide that has a weakened affinity for the protease was selected, and then an adapter protein that increases the effective local concentration of the target near the protease to increase degradation was conditionally expressed (20,29). In another example, a cryptic degradation signal was used that was liberated by the conditional expressio...

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Section: Resultsmentioning

confidence: 99%

Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses

et al. 2012

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“…This assumption is also supported by previous studies, which showed that degradation of certain bacterial RNAs might involve endonucleolytic cleavages by RNase E at a single-stranded AU-rich region in the 3′UTR 30 33 34 . In E. coli , the degradation of many mRNAs is initiated by an endonucleolytic cleavage catalyzed by RNase E, which products serve as the substrates for additional RNase E cleavages as well as for digestion by the 3′→5′exonucleases, polynucleotide phosphorylase (PNPase) and RNase II 35 36 37 38 . Figure S4 indicates that the low mRNA stability seemed to result in reduction of the bmoF1 mRNA level in E. coli BL21(DE3), which in turn decreased total expression level of bmoF1 .…”

Section: Discussionmentioning

confidence: 99%

3′-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli

Song

Woo

Jung

et al. 2016

Sci Rep

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3′-Untranslated region (3′UTR)engineering was investigated to improve solubility of heterologous proteins (e.g., Baeyer-Villiger monooxygenases (BVMOs)) in Escherichia coli. Insertion of gene fragments containing putative RNase E recognition sites into the 3′UTR of the BVMO genes led to the reduction of mRNA levels in E. coli. Importantly, the amounts of soluble BVMOs were remarkably enhanced resulting in a proportional increase of in vivo catalytic activities. Notably, this increase in biocatalytic activity correlated to the number of putative RNase E endonucleolytic cleavage sites in the 3′UTR. For instance, the biotransformation activity of the BVMO BmoF1 (from Pseudomonas fluorescens DSM50106) in E. coli was linear to the number of RNase E cleavage sites in the 3′UTR. In summary, 3′UTR engineering can be used to improve the soluble expression of heterologous enzymes, thereby fine-tuning the enzyme activity in microbial cells.Synthetic biology and systems biology allow whole-cell biocatalysis and microbial fermentations to generate a variety of chemical products [1][2][3][4][5][6][7] . Not only small molecules but also large and complex metabolites (e.g., polyphenols, carotenoids, terpenoids, plant oxylipins) could be synthesized from renewable biomass. However, production of these molecules often remains low because of a number of factors including low expression level of the required enzymes in the microbial host. Most of all, oxygenases (e.g., P450 monooxygenases and Baeyer-Villiger monooxygenases (BVMOs)), which serve as one of the key enzymes in preparation of large and complex metabolites (e.g., polyphenols, carotenoids, terpenoids, plant oxylipins) from renewable biomass as well as oxyfunctionalization of hydrocarbons and fatty acids [8][9][10] , are usually difficult to overexpress in a functional form in bacterial cells [11][12][13] .Functional expression of heterologous proteins including oxygenases in bacterial cells can be improved by various methods. Optimization of not only the induction conditions for gene expression (e.g., cultivation temperature, type and concentration of inducer), but also the gene expression systems including the promoters, ribosome binding sites (RBSs), 5′ -untranslated region (5′ UTR), and codon usage have been largely investigated to enhance soluble expression of enzymes and proteins [14][15][16][17][18] . In addition, introduction of molecular chaperones 19,20 , the fusion of proteins with soluble peptides and proteins 21,22 , and other protein engineering methods (e.g., directed evolution) 23,24 often allowed or improved functional expression of foreign proteins and enzymes in bacterial host cells.3′ UTR has been found to mainly harbor a transcriptional terminator that contributes to RNA stabilization in prokaryotes 25,26 . Thereby, it was largely used to increase stability of translationally active mRNAs by inserting the specific sequence (e.g., repetitive extragenic palindromic (REP) sequence) into the 3′ UTRs 27 . For example, expression level of the MalE protein in...

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“…In response to cold stress, RhlB can be exchanged with DeaD (in vitro and in vivo) or RhlE (in vitro) to produce a cold stress degradosome 93 , 94 . Stoichiometry of subunits is not known, however a model in which PNPase:RNase E:enolase:RhlB associate in a ratio of 4:3:8:8 binding to the C-terminus of RNase E has been proposed 131 . Similar to E. coli , the B. subtilis and Staphylococcus aureus degradosomes assemble on RNase Y (RNase E equivalent) 105 and are composed of the RNA helicase CshA (RhlB equivalent), enolase and PnpA (PNPase equivalent).…”

Section: Rna Helicases In Rna Turnovermentioning

confidence: 99%

RNA helicases

Owttrim

2013

RNA Biology

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Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes.

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Current perspectives of the Escherichia coli RNA degradosome

Cited by 11 publications

References 116 publications

Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses

Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses

3′-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli

RNA helicases

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