First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary σ70 factor forms the RNAP holoenzyme to express housekeeping genes. The σ70 contains a large insertion between the conserved regions 1.2 and 2.1, the σ non-conserved region (σNCR), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP σ70 holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 Å resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between the σNCR and promoter DNA just upstream of the −10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography because of restraint of crystal packing effects. Disruption of the σNCR and DNA interaction by the amino acid substitutions (R157A/R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the σNCR and DNA interaction is conserved in proteobacteria, and RNAP in other bacteria replaces its role with a transcription factor. First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary σ70 factor forms the RNAP holoenzyme to express housekeeping genes. The σ70 contains a large insertion between the conserved regions 1.2 and 2.1, the σ non-conserved region (σNCR), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP σ70 holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 Å resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between the σNCR and promoter DNA just upstream of the −10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography because of restraint of crystal packing effects. Disruption of the σNCR and DNA interaction by the amino acid substitutions (R157A/R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the σNCR and DNA interaction is conserved in proteobacteria, and RNAP in other bacteria replaces its role with a transcription factor.
EpsG is the major pseudopilin protein of the Vibrio cholerae type II secretion system. An expression plasmid that encodes an N-terminally truncated form of EpsG with a C-terminal noncleavable His tag was constructed. Recombinant EpsG was expressed in Escherichia coli; the truncated protein was purified and crystallized by hanging-drop vapor diffusion against a reservoir containing 6 mM zinc sulfate, 60 mM MES pH 6.5, 15% PEG MME 550. The crystals diffracted X-rays to a resolution of 2.26 Å and belonged to space group P21, with unit-cell parameters a = 88.61, b = 70.02, c = 131.54 Å.
Abstract Adult individuals with Down syndrome (DS) develop Alzheimer disease (AD). Whether there is a difference between AD in DS and AD regarding the structure of amyloid-β (Aβ) and tau filaments is unknown. Here we report the structure of Aβ and tau filaments from two DS brains. We found two Aβ 40 filaments (types IIIa and IIIb) that differ from those previously reported in sporadic AD and two types of Aβ 42 filaments (I and II) identical to those found in sporadic and familial AD. Tau filaments (paired helical filaments and straight filaments) were identical to those in AD, supporting the notion of a common mechanism through which amyloids trigger aggregation of tau. This knowledge is important for understanding AD in DS and assessing whether adults with DS could be included in AD clinical trials.
Abstract In human neurodegenerative diseases associated with the intracellular aggregation of Tau protein, the ordered cores of Tau filaments adopt distinct folds. Here, we analyze Tau filaments isolated from the brain of individuals affected by Prion-Protein cerebral amyloid angiopathy (PrP-CAA) with a nonsense mutation in the PRNP gene that leads to early termination of translation of PrP (Q160Ter or Q160X), and Gerstmann–Sträussler–Scheinker (GSS) disease, with a missense mutation in the PRNP gene that leads to an amino acid substitution at residue 198 (F198S) of PrP. The clinical and neuropathologic phenotypes associated with these two mutations in PRNP are different; however, the neuropathologic analyses of these two genetic variants have consistently shown the presence of numerous neurofibrillary tangles (NFTs) made of filamentous Tau aggregates in neurons. We report that Tau filaments in PrP-CAA (Q160X) and GSS (F198S) are composed of 3-repeat and 4-repeat Tau isoforms, having a striking similarity to NFTs in Alzheimer disease (AD). In PrP-CAA (Q160X), Tau filaments are made of both paired helical filaments (PHFs) and straight filaments (SFs), while in GSS (F198S), only PHFs were found. Mass spectrometry analyses of Tau filaments extracted from PrP-CAA (Q160X) and GSS (F198S) brains show the presence of post-translational modifications that are comparable to those seen in Tau aggregates from AD. Cryo-EM analysis reveals that the atomic models of the Tau filaments obtained from PrP-CAA (Q160X) and GSS (F198S) are identical to those of the Tau filaments from AD, and are therefore distinct from those of Pick disease, chronic traumatic encephalopathy, and corticobasal degeneration. Our data support the hypothesis that in the presence of extracellular amyloid deposits and regardless of the primary amino acid sequence of the amyloid protein, similar molecular mechanisms are at play in the formation of identical Tau filaments.
EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.39, b = 71.11, c = 84.64 A. There were two protein molecules in the asymmetric unit, which gave a Matthews coefficient V(M) of 2.1 A(3) Da(-1), corresponding to 41.5% solvent content.
Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate (IP 3 ) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCɛ contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways downstream of receptor tyrosine kinases and G protein‐coupled receptors. PLCɛ shares a conserved core with other PLC enzymes, but the roles of individual domains in activity and membrane binding have not been established. Using biochemical assays, small‐angle X‐ray scattering (SAXS), and electron microscopy (EM), we have found that the PLCɛ PH domain is necessary for basal lipase activity, but is dispensable for stability. Importantly, we provide the first structural insights into domain organization of PLCɛ, and reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCɛ solution structure to that of the closely related PLCβ enzyme reveal that, in contrast to its crystal structure, the PLCβ PH domain is also mobile in solution. We also show the dynamic nature of the PH domain in these enzymes is functionally important, and may contribute to their regulation. These findings provide critical new insights into the solution structures of these signaling enzymes and their roles in cardiovascular disease and cancer. Support or Funding Information American Heart Association Scientist Development Grant 16SDG29920017; Purdue Center for Cancer Research; Showalter Foundation; all to A.M.L This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Significance T7 phage has been used as a model system to study dsDNA virus capsid assembly and maturation. Yet, atomic capsid models and details of capsid transformations are not elucidated. From our cryo-EM study we have derived near-atomic resolution reconstructions of the DNA-free procapsid, a DNA packaging intermediate, and the DNA-packaged, mature phage capsid. From these structures, we have derived the first near-atomic-level model of T7 capsid maturation. The structural knowledge obtained from this study can serve as a platform for analysis of other dsDNA viruses as well as a platform for the development of molecular tools such as improved phage display systems.
Abstract The COVID‐19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused considerable morbidity and mortality worldwide. Although authorized COVID‐19 vaccines have been shown highly effective, their significantly lower efficacy against heterologous variants, and the rapid decrease of vaccine‐elicited immunity raises serious concerns, calling for improved vaccine tactics. To this end, a p seudo v irus n ano p article (PVNP) displaying the receptor binding domains (RBDs) of SARS‐CoV‐2 spike, named S‐RBD, was generated and shown it as a promising COVID‐19 vaccine candidate. The S‐RBD PVNP was produced using both prokaryotic and eukaryotic systems. A 3D structural model of the S‐RBD PVNPs was built based on the known structures of the S 60 particle and RBDs, revealing an S 60 particle‐based icosahedral symmetry with multiple surface‐displayed RBDs that retain authentic conformations and receptor‐binding functions. The PVNP is highly immunogenic, eliciting high titers of RBD‐specific IgG and neutralizing antibodies in mice. The S‐RBD PVNP demonstrated exceptional protective efficacy, and fully (100%) protected K18‐hACE2 mice from mortality and weight loss after a lethal SARS‐CoV‐2 challenge, supporting the S‐RBD PVNPs as a potent COVID‐19 vaccine candidate. By contrast, a PVNP displaying the N‐terminal domain (NTD) of SARS‐CoV‐2 spike exhibited only 50% protective efficacy. Since the RBD antigens of our PVNP vaccine are adjustable as needed to address the emergence of future variants, and various S‐RBD PVNPs can be combined as a cocktail vaccine for broad efficacy, these non‐replicating PVNPs offer a flexible platform for a safe, effective COVID‐19 vaccine with minimal manufacturing cost and time.
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