62 research outputs found

    Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates

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    <p>Abstract</p> <p>Background</p> <p>Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).</p> <p>Results</p> <p>Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species.</p> <p>Conclusions</p> <p>We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.</p

    Identification and Characterization of Two Functionally Unknown Genes Involved in Butanol Tolerance of Clostridium acetobutylicum

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    Solvents toxicity is a major limiting factor hampering the cost-effective biotechnological production of chemicals. In Clostridium acetobutylicum, a functionally unknown protein (encoded by SMB_G1518) with a hypothetical alcohol interacting domain was identified. Disruption of SMB_G1518 and/or its downstream gene SMB_G1519 resulted in increased butanol tolerance, while overexpression of SMB_G1518-1519 decreased butanol tolerance. In addition, SMB_G1518-1519 also influences the production of pyruvate:ferredoxin oxidoreductase (PFOR) and flagellar protein hag, the maintenance of cell motility. We conclude that the system of SMB_G1518-1519 protein plays a role in the butanol sensitivity/tolerance phenotype of C. acetobutylicum, and can be considered as potential targets for engineering alcohol tolerance

    Function of the Bacillus subtilis transcription elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader

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    NusA and NusG are transcription elongation factors that bind to RNA polymerase (RNAP) after σ subunit release. Escherichia coli NusA (NusAEc) stimulates intrinsic termination and RNAPEc pausing, whereas NusGEc promotes Rho-dependent termination and pause escape. Both Nus factors also participate in the formation of RNAPEc antitermination complexes. We showed that Bacillus subtilis NusA (NusABs) stimulates intrinsic termination and RNAPBs pausing at U107 and U144 in the trpEDCFBA operon leader. Pausing at U107 and U144 participates in the transcription attenuation and translational control mechanisms, respectively, presumably by providing additional time for trp RNA-binding attenuation protein (TRAP) to bind to the nascent trp leader transcript. Here, we show that NusGBs causes modest pause stimulation at U107 and dramatic pause stimulation at U144. NusABs and NusGBs act synergistically to increase the U107 and U144 pause half-lives. NusGBs-stimulated pausing at U144 requires RNAPBs, whereas NusABs stimulates pausing of RNAPBs and RNAPEc at the U144 and E. coli his pause sites. Although NusGEc does not stimulate pausing at U144, it competes with NusGBs-stimulated pausing, suggesting that both proteins bind to the same surface of RNAPBs. Inactivation of nusG results in the loss of RNAP pausing at U144 in vivo and elevated trp operon expression, whereas plasmid-encoded NusG complements the mutant defects. Overexpression of nusG reduces trp operon expression to a larger extent than overexpression of nusA

    Global role of the bacterial post-transcriptional regulator CsrA revealed by integrated transcriptomics

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    The RNA-binding protein CsrA regulates the expression of hundreds of bacterial genes. Here, Potts et al. use several approaches to assess the contribution of CsrA to global gene expression in E. coli, revealing new binding targets and physiological roles such as in envelope function and iron homeostasis

    CsrA Represses Translation of sdiA, Which Encodes the N-Acylhomoserine-l-Lactone Receptor of Escherichia coli, by Binding Exclusively within the Coding Region of sdiA mRNA ▿ †

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    The RNA binding protein CsrA is the central component of a conserved global regulatory system that activates or represses gene expression posttranscriptionally. In every known example of CsrA-mediated translational control, CsrA binds to the 5′ untranslated region of target transcripts, thereby repressing translation initiation and/or altering the stability of the RNA. Furthermore, with few exceptions, repression by CsrA involves binding directly to the Shine-Dalgarno sequence and blocking ribosome binding. sdiA encodes the quorum-sensing receptor for N-acyl-l-homoserine lactone in Escherichia coli. Because sdiA indirectly stimulates transcription of csrB, which encodes a small RNA (sRNA) antagonist of CsrA, we further explored the relationship between sdiA and the Csr system. Primer extension analysis revealed four putative transcription start sites within 85 nucleotides of the sdiA initiation codon. Potential σ70-dependent promoters were identified for each of these primer extension products. In addition, two CsrA binding sites were predicted in the initially translated region of sdiA. Expression of chromosomally integrated sdiA′-′lacZ translational fusions containing the entire promoter and CsrA binding site regions indicates that CsrA represses sdiA expression. The results from gel shift and footprint studies demonstrate that tight binding of CsrA requires both of these sites. Furthermore, the results from toeprint and in vitro translation experiments indicate that CsrA represses translation of sdiA by directly competing with 30S ribosomal subunit binding. Thus, this represents the first example of CsrA preventing translation by interacting solely within the coding region of an mRNA target

    An incoherent feedforward loop formed by SirA/BarA, HilE and HilD is involved in controlling the growth cost of virulence factor expression by Salmonella Typhimurium.

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    An intricate regulatory network controls the expression of Salmonella virulence genes. The transcriptional regulator HilD plays a central role in this network by controlling the expression of tens of genes mainly required for intestinal colonization. Accordingly, the expression/activity of HilD is highly regulated by multiple factors, such as the SirA/BarA two-component system and the Hcp-like protein HilE. SirA/BarA positively regulates translation of hilD mRNA through a regulatory cascade involving the small RNAs CsrB and CsrC, and the RNA-binding protein CsrA, whereas HilE inhibits HilD activity by protein-protein interaction. In this study, we show that SirA/BarA also positively regulates translation of hilE mRNA through the same mentioned regulatory cascade. Thus, our results reveal a paradoxical regulation exerted by SirA/BarA-Csr on HilD, which involves simultaneous opposite effects, direct positive control and indirect negative control through HilE. This kind of regulation is called an incoherent type-1 feedforward loop (I1-FFL), which is a motif present in certain regulatory networks and represents a complex biological problem to decipher. Interestingly, our results, together with those from a previous study, indicate that HilE, the repressor component of the I1-FFL reported here (I1-FFLSirA/BarA-HilE-HilD), is required to reduce the growth cost imposed by the expression of the genes regulated by HilD. Moreover, we and others found that HilE is necessary for successful intestinal colonization by Salmonella. Thus, these findings support that I1-FFLSirA/BarA-HilE-HilD cooperates to control the precise amount and activity of HilD, for an appropriate balance between the growth cost and the virulence benefit generated by the expression of the genes induced by this regulator. I1-FFLSirA/BarA-HilE-HilD represents a complex regulatory I1-FFL that involves multiple regulators acting at distinct levels of gene expression, as well as showing different connections to the rest of the regulatory network governing Salmonella virulence

    Translational Repression of NhaR, a Novel Pathway for Multi-Tier Regulation of Biofilm Circuitry by CsrA

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    The RNA binding protein CsrA (RsmA) represses biofilm formation in several proteobacterial species. In Escherichia coli, it represses the production of the polysaccharide adhesin poly-β-1,6-N-acetyl-d-glucosamine (PGA) by binding to the pgaABCD mRNA leader, inhibiting pgaA translation, and destabilizing this transcript. In addition, CsrA represses genes responsible for the synthesis of cyclic di-GMP, an activator of PGA production. Here we determined that CsrA also represses NhaR, a LysR-type transcriptional regulator which responds to elevated [Na+] and alkaline pH and activates the transcription of the pgaABCD operon. Gel shift studies revealed that CsrA binds at two sites in the 5′ untranslated segment of nhaR, one of which overlaps the Shine-Dalgarno sequence. An epitope-tagged NhaR protein, expressed from the nhaR chromosomal locus, and an nhaR posttranscriptional reporter fusion (PlacUV5-nhaR′-′lacZ) both showed robust repression by CsrA. Northern blotting revealed a complex transcription pattern for the nhaAR locus. Nevertheless, CsrA did not repress nhaR mRNA levels. Toeprinting assays showed that CsrA competes effectively with the ribosome for binding to the translation initiation region of nhaR. Together, these findings indicate that CsrA blocks nhaR translation. Epistasis studies with a pgaA-lacZ transcriptional fusion confirmed a model in which CsrA indirectly represses pgaABCD transcription via NhaR. We conclude that CsrA regulates the horizontally acquired pgaABCD operon and PGA biosynthesis at multiple levels. Furthermore, nhaR repression exemplifies an expanding role for CsrA as a global regulator of stress response systems
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