Identification of Amino Acid Residues in the Catalytic Domain of RNase E Essential for Survival of <i>Escherichia coli</i>: Functional Analysis of DNase I Subdomain

Shin, Eunkyoung; Go, Hayoung; Yeom, Ji-Hyun; Won, Miae; Bae, Jang–Ho; Han, Seung Hyun; Han, Kyu‐Sung; Lee, Younghoon; Ha, Nam‐Chul; Moore, Christopher; Sohlberg, Björn; Cohen, Stanley N.; Lee, Kangseok

doi:10.1534/genetics.108.088492

Cited by 12 publications

(9 citation statements)

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“…To determine whether the ability of the N-Rne mutant (N-Rne-Q36R) to support cell viability at lower levels of IPTG-induced expression than wild-type N-Rne (N-Rne-wt) resulted from an increase in ribonucleolytic activity, we analyzed the steady-state level of RNA I in KSL2000 cells conditionally expressing N-Rne-Q36R in the absence of fulllength RNase E. RNase E cleaves RNA I, which is an antisense regulator of ColE1-type plasmid DNA replication; 25 this function has been used to assess the ribonucleolytic activity of RNase E in vivo by measuring ColE1-type plasmid copy number relative to an RNase E independent plasmid. 22,26,27 Transiently induced expression of the N-Rne-Q36R mutant by 100 μM IPTG resulted in an approximately 2.6-fold greater copy number of the pNRNE4-Q36R plasmid than was observed in KSL2000 cells containing the similarly induced wild-type N-Rne ( Fig. 2A).…”

Section: ©2 0 1 1 L a N D E S B I O S C I E N C E D O N O T D I S Tmentioning

confidence: 89%

“…The strains and plasmids used in this study are listed in Table 1. Random mutations were introduced into the DNA fragment encoding amino acid residues 1-398 of RNase E using error-prone PCR, 22 and the PCR products were then digested with NotI and DraIII (NEB, #R0189S and #R0510S) enzymes and ligated into sites of pNRNE4 generated by cleavage with these enzymes. The primers used were Nrne-5' (5'-GAATTGTGAGCGGATAAC-3') and Nrne-3' (5'-CTACCATCGGCGCTACGT-3').…”

Section: Methodsmentioning

confidence: 99%

“…To isolate hyperactive RNase E mutants, we used a genetic approach analogous to that used to screen for loss-of-function mutants of RNase E; 22 here, we introduced random mutations in the coding region of the minimal catalytic domain of Rne in pNRNE4. The DNA segment encoding amino acids 1-398 of Rne was amplified using error-prone PCR, ligated into pNRNE4 by replacing the corresponding wild-type segment of N-rne, and the resulting ligation product was introduced by transformation into E. coli strain KSL2000.…”

Section: Isolation Of Hyperactive N-rne Mutantsmentioning

confidence: 99%

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Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Moore

Shin

et al. 2011

RNA Biology

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Escherichia coli RNase e contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase e, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5' termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. studies of cleavage of fluorogenic substrate and eMsA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. however, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart. Two substrate-bound tryptic peptides, 65 hGFLpLK 71 -which includes amino acids previously implicated in substrate binding and catalysisand 24 LYDLDIespGheQK 37 -which includes the Q36 locus-were identified in wild-type enzyme complexes, whereas only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase e locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects.Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

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Section: ©2 0 1 1 L a N D E S B I O S C I E N C E D O N O T D I S Tmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Isolation Of Hyperactive N-rne Mutantsmentioning

confidence: 99%

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Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Moore

Shin

et al. 2011

RNA Biology

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“…Conserved amino acid residues essential for catalytic function have been identified by mutagenesis in the N-terminal half of RNase E (4,8,9,34). Several of these essential residues are very close or next to residues mutated in the suppressors of mutant EF-Tu.…”

Section: Resultsmentioning

confidence: 99%

Temperature-Sensitive Mutants of RNase E in Salmonella enterica

Hammarlöf

Liljas

Hughes

2011

Journal of Bacteriology

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RNase E has an important role in mRNA turnover and stable RNA processing, although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium, an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (TS) for growth. Four different TS mutations were identified, all in the N-terminal domain of RNase E: Gly663Cys, Ile2073Ser, Ile2073Asn, and Ala3273Pro. We also selected second-site mutations in RNase E that reversed temperature sensitivity. The complete set of RNase E mutations (53 primary mutations including the TS mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E by using the available three-dimensional (3-D) structures. Most single mutations were predicted to destabilize the structure, while second-site mutations that reversed the TS phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (TS, and TS plus second-site mutation) were tested for their effects on the degradation, accumulation, and processing of mRNA, rRNA, and tRNA. The greatest defect was observed on rne mRNA autoregulation, and this correlated with the ability to rescue the tufA499-associated slow-growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.RNase E (18) is a key component of the RNA degradosome (5, 20), a multienzyme complex that plays a central role in mRNA turnover and the processing of stable RNA in eubacteria (2,10,17,24,26). The degradosome is composed of a tetramer of RNase E molecules interacting via their N-terminal regions, with the C-terminal end of each RNase E molecule complexed with a monomer of RhlB, a dimer of enolase, and a trimer of PNPase (reviewed in reference 5). RNase E endonuclease activity can also be directed to specific mRNAs by the combined activity of Hfq protein and mRNA-specific small RNAs (13,25,31). The catalytic activity of RNase E is largely contained within the N-terminal half of the protein, the structure of which has been determined in Escherichia coli (4,10,15,22). The catalytic half of RNase E is divided into the small and big domains, with the latter including several folds related to RNase H, S1, and DNase I structures (4).In a previous study (11), mutations in RNase E were isolated in a selection for suppression of an extreme slow-growth phenotype caused by a mutant form of EF-Tu (tufA499, EF-Tu Gln125Arg) in Salmonella enterica serovar Typhimurium, and the question of how these mutations in RNase E suppress the tufA499 growth defect was raised. EF-Tu brings aminoacyltRNAs (aa-tRNAs), as part of the ternary complex EFTu ⅐ GTP ⅐ aa-tRN...

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“…The HSR2 region encodes for the DNase I subdomain of endoribonuclease RNase G [34], which participates in mRNA turnover of adhE (alcohol dehydrogenase) and in the maturation of the 5Ј terminal end of 16s rRNA [15,[37][38][39]. Deletion of rng HSR2 enabled the resulting strain, RM10, to complete 75 g l ¡1 xylose (and arabinose) fermentation in 72-84 h, with an 84% increased ethanol titer (35 g l ¡1 ) compared to the parent SZ470 (19 g l ¡1 ).…”

Section: Introductionmentioning

confidence: 99%

Partial deletion ofrng(RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenicEscherichia coliRM10

Manow

Wang

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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Previously, a native homoethanol pathway was engineered in Escherichia coli B by deletions of competing pathway genes and anaerobic expression of pyruvate dehydrogenase (PDH encoded by aceEF-lpd). The resulting ethanol pathway involves glycolysis, PDH, and alcohol dehydrogenase (AdhE). The E. coli B-derived ethanologenic strain SZ420 was then further improved for ethanol tolerance (up to 40 g l−1 ethanol) through adaptive evolution. However, the resulting ethanol tolerant mutant, SZ470, was still unable to complete fermentation of 75 g l−1 xylose, even though the theoretical maximum ethanol titer would have been less than 40 g l−1 should the fermentation have reached completion. In this study, the cra (encoding for a catabolite repressor activator) and the HSR2 region of rng (encoding for RNase G) were deleted from SZ470 in order to improve xylose fermentation. Deletion of the HSR2 domain resulted in significantly increased mRNA levels (47-fold to 409-fold) of multiple glycolytic genes (pgi, tpiA, gapA, eno), as well as the engineered ethanol pathway genes (aceEF-lpd, adhE) and the transcriptional regulator Fnr (fnr). The higher adhE mRNA level resulted in increased AdhE activity (>twofold). Although not measured, the increase of other mRNAs might also enhance expressions of their encoding proteins. The increased enzymes would then enable the resulting strain, RM10, to achieve increased cell growth and complete fermentation of 75 g l−1 xylose with an 84% improved ethanol titer (35 g l−1), compared to that (19 g l−1) obtained by the parent, SZ470. However, deletion of cra resulted in a negative impact on cell growth and xylose fermentation, suggesting that Cra is important for long-term fermentative cell growth.

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Identification of Amino Acid Residues in the Catalytic Domain of RNase E Essential for Survival of Escherichia coli: Functional Analysis of DNase I Subdomain

Cited by 12 publications

References 51 publications

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

Temperature-Sensitive Mutants of RNase E in Salmonella enterica

Partial deletion ofrng(RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenicEscherichia coliRM10

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