scholarly journals Ubiquitous mRNA decay fragments in E. coli redefine the functional transcriptome

2021 ◽  
Author(s):  
Lydia Herzel ◽  
Julian A Stanley ◽  
James C Taggart ◽  
Gene-Wei LI
Keyword(s):  
Large Fraction ◽  
Life Cycles ◽  
Full Length ◽  
Rnase E ◽  
Short Life ◽  
E Coli ◽  
Rna Fragments ◽  
Large Heterogeneity ◽  
State Growth

Bacterial mRNAs have short life cycles, in which transcription is rapidly followed by translation and degradation within seconds to minutes. The resulting diversity of mRNAs impacts their functionality but has remained unresolved. Here we quantitatively map the 3' status of cellular RNAs in Escherichia coli during steady-state growth and report a large fraction of molecules (median>60%) that are fragments of canonical full-length mRNAs. The majority of RNA fragments are decay intermediates following endonuclease cleavage by RNase E and yet-unknown nucleases, whereas nascent RNAs contribute to a smaller fraction. Despite the prevalence of decay intermediates in total RNA, they are underrepresented in the pool of ribosome-associated transcripts and can thus distort quantifications for the abundance of full-length, functional mRNAs. The large heterogeneity within mRNA molecules in vivo highlights the importance in discerning functional transcripts and provides a lens for studying the dynamic life cycle of mRNAs.

scholarly journals Multiple layers of control govern expression of the Escherichia coli ibpAB heat-shock operon

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 66-76 ◽  
Author(s):  
Lena C. Gaubig ◽  
Torsten Waldminghaus ◽  
Franz Narberhaus
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Transcriptional Control ◽  
Rnase E ◽  
Translation Control ◽  
E Coli ◽  
Complex Process ◽  
Shock Proteins

The Escherichia coli ibpAB operon encodes two small heat-shock proteins, the inclusion-body-binding proteins IbpA and IbpB. Here, we report that expression of ibpAB is a complex process involving at least four different layers of control, namely transcriptional control, RNA processing, translation control and protein stability. As a typical member of the heat-shock regulon, transcription of the ibpAB operon is controlled by the alternative sigma factor σ 32 (RpoH). Heat-induced transcription of the bicistronic operon is followed by RNase E-mediated processing events, resulting in monocistronic ibpA and ibpB transcripts and short 3′-terminal ibpB fragments. Translation of ibpA is controlled by an RNA thermometer in its 5′ untranslated region, forming a secondary structure that blocks entry of the ribosome at low temperatures. A similar structure upstream of ibpB is functional in vitro but not in vivo, suggesting downregulation of ibpB expression in the presence of IbpA. The recently reported degradation of IbpA and IbpB by the Lon protease and differential regulation of IbpA and IbpB levels in E. coli are discussed.

Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Anna Scherhag ◽  
Martina Rüger ◽  
Katrin Gerbracht ◽  
Jaqueline Rehner ◽  
Susanne Zehner ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Model Organism ◽  
Functional Complementation ◽  
Full Length ◽  
Protein Activity ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Ggdef Domain

<p>The molecule c-di-GMP is a bacterial second messenger that controls various processes such as motility or biofilm formation in bacteria [1]. To synthesize and degrade c-di-GMP, enzymes called diguanylate cyclases (DGC) containing a GGDEF-domain and phosphodiesterases (PDE) containing an EAL-domain or HD-GYP-domain are important [1, 2].<em> Pseudomonas aeruginosa</em>, a model organism for biofilm formation and dispersion, encodes for 18 GGDEF, 5 EAL, 16 GGDEF / EAL, and 3 HD-GYP-domain-containing proteins [3].<br />One of the GGDEF / EAL-containing proteins is NbdA. This protein also harbors an N-terminal membrane anchored MHYT-domain, that is predicted to be a sensor for NO, CO or O<sub>2</sub> [4]. In this work, recombinant and affinity purified NbdA was tested for its PDE activity. Three different methods were used to measure the PDE activity of NbdA: a bis-pNPP-assay in which the conversion of the pseudosubstrate bis-pNPP into p-nitrophenol was detected spectroscopically, an HPLC-analysis of an enzymatic assay with the native substrate c-di-GMP, and a MANT-c-di-GMP-assay in which a fluorescently labeled form of the presumed substrate c-di-GMP was utilized.<br />To establish these methods, the two known phosphodiesterases, PdeH from <em>Escherichia coli</em> [5] and RocR from <em>P. aeruginosa</em> [6], were also produced and tested. Subsequently, three variants of NbdA were investigated: the full-length version and two truncated versions of the protein. Activity was further assessed using functional complementation of an <em>E. coli</em> phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity <em>in vivo</em>.</p> <p> </p> <p> </p> <p>[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.</p> <p>[2] Römling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629–639.</p> <p>[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547–12555.</p> <p>[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17–23.</p> <p>[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434–2446.</p> <p>[6] Chen et al. (2012) Chen, M.W., Kotaka, M., Vonrhein, C., Bricogne, G., Rao, F., Chuah, M.L.C., Svergun, D., Schneider, G., Liang, Z.-X., Lescar, J.  (2012). Signaling. J. Bacteriol. 194: 4837–4846</p> <p> </p>

scholarly journals Evidence for Multimerization of Neu Proteins Involved in Polysialic Acid Synthesis in Escherichia coliK1 Using Improved LexA-Based Vectors

2000 ◽  
Vol 182 (18) ◽  
pp. 5267-5270 ◽  
Author(s):  
Dayle A. Daines ◽  
Richard P. Silver
Keyword(s):  
Protein Interactions ◽  
Polysialic Acid ◽  
Genetic System ◽  
Acid Synthesis ◽  
Full Length ◽  
Protein Protein Interactions ◽  
Protein Fragment ◽  
Reading Frame ◽  
E Coli

ABSTRACT Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205–212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background. However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame. Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in E. coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.

scholarly journals The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

1993 ◽  
Vol 4 (11) ◽  
pp. 1189-1204 ◽  
Author(s):  
M A Heine ◽  
M L Rankin ◽  
P J DiMario
Keyword(s):  
Nucleic Acid ◽  
Binding Activity ◽  
Full Length ◽  
Nucleic Acid Binding ◽  
Binding Assays ◽  
Nucleic Acid Probes ◽  
E Coli ◽  
Indirect Immunofluorescence Staining

Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.

scholarly journals Single-step purification of full-length human androgen receptor

2005 ◽  
Vol 3 (1) ◽  
pp. nrs.03001 ◽  
Author(s):  
Dalia Juzumiene ◽  
Ching-yi Chang ◽  
Daju Fan ◽  
Tanya Hartney ◽  
John D. Norris ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Affinity Chromatography ◽  
Spodoptera Frugiperda ◽  
Single Step ◽  
Full Length ◽  
Structural Studies ◽  
Site Specific ◽  
E Coli ◽  
Human Androgen Receptor

The full-length human androgen receptor with an N-terminal biotin acceptor peptide tag was overexpressed in Spodoptera frugiperda cells in the presence of 1 μM dihydrotestosterone. Site-specific biotinylation of BAP was achieved in vivo by co-expression of E. coli biotin holoenzyme synthetase. The androgen receptor was purified by single-step affinity chromatography using Streptavidin Mutein Matrix under native conditions. The resultant protein was active, stable, 95% homogeneous, and we obtained sufficient yield for use in functional and structural studies.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine

2020 ◽  
Vol 21 (4) ◽  
pp. 316-324
Author(s):  
Manica Negahdaripour ◽  
Navid Nezafat ◽  
Reza Heidari ◽  
Nasrollah Erfani ◽  
Nasim Hajighahramani ◽  
...  
Keyword(s):  
In Silico ◽  
Group Versus ◽  
Tlr Agonists ◽  
E Coli ◽  
Metal Affinity ◽  
Th1 And Th2 Cytokines ◽  
Ifn Γ ◽  
Humoral Responses ◽  
Future Work

Background: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. Methods: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. Results: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund’s adjuvant group. Conclusion: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

Comparison of the Effects of Silver in Nanostructured and Ultrahigh Diluted Form on Growth and Volatile Compounds Produced by Escherichia coli and Staphylococcus aureus

2020 ◽  
Vol 10 (3) ◽  
pp. 316-329
Author(s):  
Fateme Mirzajani ◽  
Amin Hamidi
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Life Cycles ◽  
Gas Chromatography Mass Spectrometry ◽  
Untreated Sample ◽  
E Coli ◽  
Volatile Metabolites ◽  
Octyl Acetate ◽  
Volatile Organic ◽  
And Staphylococcus Aureus

Introduction: In this project, the growth and volatile metabolites profiles of Escherichia coli (E. coli ) and Staphylococcus aureus were monitored under the influence of silver base chemical, nanoparticle and ultra-highly diluted compounds. Materials & Methods: The treatments were done for 12000 life cycles using silver nanoparticles (AgNPs) as well as ultra-highly diluted Argentum nitricum (Arg-n). Volatile organic metabolites analysis was performed using gas chromatography mass spectrometry (GC-MS). The results indicated that AgNPs treatment made the bacteria resistant and adapted to growth in the nanoparticle condition. The use of ultra-highly diluted Arg-n initially increased growth but it decreased later. Also, with the continuous usage of these materials, no more bacterial growth was observed. Results: The most important compounds produced by E. coli are Acetophenone, Octyl acetate, Styrene, 1,8-cineole, 4-t-butyl-2-(1-methyl-2-nitroethyl)cyclohexane, hexadecane and 2-Undecanol. The main compounds derived from S. aureus are Acetophenone,1,8-cineole, Benzaldehyde, 2-Hexan-1-ol, Tridecanol, Dimethyl Octenal and tetradecane. Acetophenone and 1,8-cineole were common and produced by both organisms. Conclusion: Based on the origin of the produced volatiles, main volatiles percentage of untreated sample is hydrocarbon (>50%), while bacteria treatments convert the ratio in to aldehydes, ketones and alcohols in the case of AgNPs, (>80%) and aldehydes, ketones and terpenes in the case of Arg-n (>70%).

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