Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Polyadenylation helps regulate functional tRNA levels in Escherichia coli.

ABSTRACT: Here we demonstrate a new regulatory mechanism for tRNA processing in Escherichia coli whereby RNase T and RNase PH, the two primary 3' → 5' exonucleases involved in the final step of 3'-end maturation, compete with poly(A) polymerase I (PAP I) for tRNA precursors in wild-type cells. In the absence of both RNase T and RNase PH, there is a >30-fold increase of PAP I-dependent poly(A) tails that are ≤10 nt in length coupled with a 2.3- to 4.2-fold decrease in the level of aminoacylated tRNAs and a >2-fold decrease in growth rate. Only 7 out of 86 tRNAs are not regulated by this mechanism and are also not substrates for RNase T, RNase PH or PAP I. Surprisingly, neither PNPase nor RNase II has any effect on tRNA poly(A) tail length. Our data suggest that the polyadenylation of tRNAs by PAP I likely proceeds in a distributive fashion unlike what is observed with mRNAs.

SUBMITTER: Mohanty BK

PROVIDER: S-EPMC3378859 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Polyadenylation helps regulate functional tRNA levels in Escherichia coli.

Mohanty Bijoy K BK Maples Valerie F VF Kushner Sidney R SR

Nucleic acids research 20120128 10

Here we demonstrate a new regulatory mechanism for tRNA processing in Escherichia coli whereby RNase T and RNase PH, the two primary 3' → 5' exonucleases involved in the final step of 3'-end maturation, compete with poly(A) polymerase I (PAP I) for tRNA precursors in wild-type cells. In the absence of both RNase T and RNase PH, there is a >30-fold increase of PAP I-dependent poly(A) tails that are ≤10 nt in length coupled with a 2.3- to 4.2-fold decrease in the level of aminoacylated tRNAs and a ...[more]

PMID: 22287637

Similar Datasets

RNA polyadenylation and cellular ATP levels regulate the stability of RNAs in Escherichia coli

Project description:To investigate the extent of the effect of poly(A) polymerase (PAP I)-mediated polyadenylation on RNA stability, we performed the first genome-wide study of RNA stability in the absence of PAP I activity. Inactivation of the pcnB gene coding for PAP I led to a global stabilization of E. coli RNAs, with 1403 stabilized transcripts and only 4 destabilized. Stabilized RNAs were involved in essential cellular functions such as DNA replication and repair, translation, RNA degradation, central carbon metabolism but also in stress responses. Because PAP I is an ATP-consuming enzyme we wondered whether the RNA stabilization observed after inactivation of PAP I could also be related to changes in intracellular ATP levels. We demonstrated for the first time in E. coli, that lowering intracellular ATP levels below 1 µM/OD stabilizes RNAs. Although the ATP level was reduced by 20 % in the pcnB mutant on glucose, the ATP level was still too high to have any role in the observed RNA stabilization. However, in experiments where the ATP level was artificially strongly decreased, inactivation of PAP I by substrate availability was implicated in the stabilization mechanism of certain RNAs. This study clearly demonstrates that PAP I is at the crossroads of the regulation of RNA stability by energy status in E. coli cells.

2024-11-27 | GSE248472 | GEO

Zebrafish nephrosin helps host defence against Escherichia coli infection.

Project description:Neutrophils play important roles in innate immunity and are mainly dependent on various enzyme-containing granules to kill engulfed microorganisms. Zebrafish nephrosin (npsn) is specifically expressed in neutrophils; however, its function is largely unknown. Here, we generated an npsn mutant (npsnsmu5 ) via CRISPR/Cas9 to investigate the in vivo function of Npsn. The overall development and number of neutrophils remained unchanged in npsn-deficient mutants, whereas neutrophil antibacterial function was defective. Upon infection with Escherichia coli, the npsnsmu5 mutants exhibited a lower survival rate and more severe bacterial burden, as well as augmented inflammatory response to challenge with infection when compared with wild-type embryos, whereas npsn-overexpressing zebrafish exhibited enhanced host defence against E. coli infection. These findings demonstrated that zebrafish Npsn promotes host defence against bacterial infection. Furthermore, our findings suggested that npsn-deficient and -overexpressing zebrafish might serve as effective models of in vivo innate immunity.

| S-EPMC5577445 | biostudies-literature

Protein synthesis in Escherichia coli with mischarged tRNA.

Project description:Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA(Asn) are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.

| S-EPMC156233 | biostudies-literature

Escherichia coli Alanyl-tRNA synthetase oxidation

Project description:LC MS/MS analysis (Fred Hutchinson Cancer Research Center, Seattle, Washington)

2021-12-20 | MSV000088593 | MassIVE

RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase.

Project description:In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and T?C stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the T?C arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated.

| S-EPMC3234781 | biostudies-literature

How many initiator tRNA genes does Escherichia coli need?

Project description:Multiple copies of a gene require enhanced investment on the part of the cell and, as such, call for an explanation. The observation that Escherichia coli has four copies of initiator tRNA (tRNAi) genes, encoding a special tRNA (tRNA(fMet)) required to start protein synthesis, is puzzling particularly because the cell appears to be unaffected by the removal of one copy. However, the fitness of an organism has both absolute and relative connotations. Thus, we carried out growth competition experiments between E. coli strains that differ in the number of tRNAi genes they contain. This has enabled us to uncover an unexpected link between the number of tRNAi genes and protein synthesis, nutritional status, and fitness. Wild-type strains with the canonical four tRNAi genes are favored in nutrient-rich environments, and those carrying fewer are favored in nutrient-poor environments. Auxotrophs behave as if they have a nutritionally poor internal environment. A heuristic model that links tRNAi gene copy number, genetic stress, and growth rate accounts for the findings. Our observations provide strong evidence that natural selection can work through seemingly minor quantitative variations in gene copy number and thereby impact organismal fitness.

| S-EPMC4097586 | biostudies-literature

A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNA<sup>His</sup> pair.

Project description:BackgroundDevelopment of new aminoacyl-tRNA synthetase (aaRS)•tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNAHis recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNAHis pair is a good candidate for GCE, its use in C. crescentus is limited by the lack of established genetic selection methods and by the low transformation efficiency of C. crescentus.MethodsE. coli was genetically engineered to use a C. crescentus HisRS•tRNAHis pair. Super-folder green fluorescent protein (sfGFP) and chloramphenicol acetyltransferase (CAT) were used as reporters for read-through assays. A library of 313 ncAAs coupled with the sfGFP reporter system was employed to investigate the specificity of E. coli HisRS in vivo.ResultsA genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS•tRNAHis pair for growth, and displays a similar doubling time as the parental E. coli strain. sfGFP- and CAT-based assays showed that the E. coli HisRS•tRNAHis pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNAHisCUA elevated its suppression efficiency by 2-fold.ConclusionsThe C. crescentus HisRS•tRNAHis pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNAHis is orthogonal in MEOV1 cells. E. coli tRNAHisCUA is an efficient amber suppressor in MEOV1.General significanceWe developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.

| S-EPMC5592127 | biostudies-literature

Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli.

Project description:Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

| S-EPMC6209979 | biostudies-literature

Functional segregation of a predicted "hinge" site within the beta-strand linkers of Escherichia coli leucyl-tRNA synthetase.

Project description:Some aminoacyl-tRNA synthetases (AARSs) employ an editing mechanism to ensure the fidelity of protein synthesis. Leucyl-tRNA synthetase (LeuRS), isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase (ValRS) share a common insertion, called the CP1 domain, which is responsible for clearing misformed products. This discrete domain is connected to the main body of the enzyme via two beta-strand tethers. The CP1 hydrolytic editing active site is located approximately 30 A from the aminoacylation active site in the canonical core of the enzyme, requiring translocation of mischarged amino acids for editing. An ensemble of crystal and cocrystal structures for LeuRS, IleRS, and ValRS suggests that the CP1 domain rotates via its flexible beta-strand linkers relative to the main body along various steps in the enzyme's reaction pathway. Computational analysis suggested that the end of the N-terminal beta-strand acted as a hinge. We hypothesized that a molecular hinge could specifically direct movement of the CP1 domain relative to the main body. We introduced a series of mutations into both beta-strands in attempts to hinder movement and alter fidelity of LeuRS. Our results have identified specific residues within the beta-strand tethers that selectively impact enzyme activity, supporting the idea that beta-strand orientation is crucial for LeuRS canonical core and CP1 domain functions.

| S-EPMC2905381 | biostudies-literature

A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli.

Project description:tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2'-phosphotransferase to transfer the splice junction 2'-phosphate from ligated tRNA to NAD, producing ADP ribose 1"-2" cyclic phosphate (Appr>p). We show here that functional 2'-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2'-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2'-phosphate. Analysis of the database shows that likely members of the 2'-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2'-phosphotransferase into Eubacteria, suggesting that the 2'-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2'-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2'-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.

| S-EPMC24339 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data