Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). ...Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3′ extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5′ end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.
Display omitted
•Cryo-EM snapshots of the transcription elongation, termination, and recycling states•After being copied, the template 3′ end rebinds the polymerase in a secondary site•Mechanism of viral mRNA poly(A) tail formation by stuttering elucidated•Efficient reformation of the promoter allows multiple transcripts from one RNP
Influenza polymerase transcribes the negative sense viral RNA genome into mRNA in the nucleus of infected cells. This work by Cusack and colleagues reports high-resolution cryo-EM structures of the polymerase at various stages of transcription providing a molecular basis for the complete transcription cycle, which should enable improved inhibitor design.
Long noncoding RNAs (lncRNAs) are recently discovered transcripts that regulate vital cellular processes, such as cellular differentiation and DNA replication, and are crucially connected to ...diseases. Although the 3D structures of lncRNAs are key determinants of their function, the unprecedented molecular complexity of lncRNAs has so far precluded their 3D structural characterization at high resolution. It is thus paramount to develop novel approaches for biochemical and biophysical characterization of these challenging targets. Here, we present a protocol that integrates non-denaturing lncRNA purification with in-solution hydrodynamic analysis and single-particle atomic force microscopy (AFM) imaging to produce highly homogeneous lncRNA preparations and visualize their 3D topology at ~15-Å resolution. Our protocol is suitable for imaging lncRNAs in biologically active conformations and for measuring structural defects of functionally inactive mutants that have been identified by cell-based functional assays. Once optimized for the specific target lncRNA of choice, our protocol leads from cloning to AFM imaging within 3-4 weeks and can be implemented using state-of-the-art biochemical and biophysical instrumentation by trained researchers familiar with RNA handling and supported by AFM and small-angle X-ray scattering (SAXS) experts.
Abstract
Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B ...to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition.
This study reports the levels of total arsenic and arsenic species in marine biota such as clams (Meretrix meretrix; N=21) and pearl oyster (Pinctada radiata; N=5) collected from nine costal sites in ...Jan 2014, and cuttlefish (Sepia pharaonis; N=8), shrimp (Penaeus semisulcatus; N=1), and seven commercially important finfish species (N=23) collected during Apr–May 2013 from seven offshore sites in the western Arabian Gulf. Total As and As species such as dimethylarsinic acid (DMA), arsenobetaine (AB), trimethylarsine oxide (TMAO), arsenocholine (AC), tetramethylarsonium ion (Tetra), arsenosugar-glycerol (As-Gly) and inorganic As (iAs) were determined by using ICPMS and HPLC/ICPMS. In bivalves, the total As concentrations ranged from 16 to 118mg/kg dry mass; the toxic iAs fraction contributed on average less than 0.8% of the total As, while the nontoxic AB fraction formed around 58%. Total As concentrations for the remaining seafood (cuttlefish, shrimp and finfish) ranged from 11 to 134mg/kg dry mass and the iAs and AB fractions contributed on average 0.03% and 81% respectively of the total As. There was no significant relationship between the tissue concentrations of total As and iAs in the samples. There was also no significant relationship between As levels in seafood and geographical location or salinity of the waters from which samples were collected. Based on our results, we recommend introducing a maximum permissible level of arsenic in seafood from the Gulf based on iAs content rather than based on total As. Our analyses of cancer risks and non-cancer hazards identified non-negligible risks and the potential for hazards; the greatest risks were identified for expatriate consumers of bivalves and high-end consumers of seafood. Despite this, many uncertainties remain that would be best addressed by further analyses.
Display omitted
•Arabian Gulf seafood contains relatively high concentrations of total arsenic.•Non-toxic arsenobetaine forms the major fraction (~70%) of total arsenic.•Toxic inorganic arsenic presents at only low levels (<1.84% of total As).•No significant relationship between the tissue concentrations of total As and iAs.•Health risk assessment shows non-negligible risks and the potential for hazards.
The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not ...inhibited by the macrolide rapamycin. Here, we present the structure of Saccharomyces cerevisiae TORC2 determined by electron cryo-microscopy. TORC2 contains six subunits assembling into a 1.4 MDa rhombohedron. Tor2 and Lst8 form the common core of both TOR complexes. Avo3/Rictor is unique to TORC2, but interacts with the same HEAT repeats of Tor2 that are engaged by Kog1/Raptor in mammalian TORC1, explaining the mutual exclusivity of these two proteins. Density, which we conclude is Avo3, occludes the FKBP12-rapamycin-binding site of Tor2's FRB domain rendering TORC2 rapamycin insensitive and recessing the kinase active site. Although mobile, Avo1/hSin1 further restricts access to the active site as its conserved-region-in-the-middle (CRIM) domain is positioned along an edge of the TORC2 active-site-cleft, consistent with a role for CRIM in substrate recruitment.
Target of Rapamycin (TOR) plays central roles in the regulation of eukaryote growth as the hub of two essential multiprotein complexes: TORC1, which is rapamycin-sensitive, and the lesser ...characterized TORC2, which is not. TORC2 is a key regulator of lipid biosynthesis and Akt-mediated survival signaling. In spite of its importance, its structure and the molecular basis of its rapamycin insensitivity are unknown. Using crosslinking-mass spectrometry and electron microscopy, we determined the architecture of TORC2. TORC2 displays a rhomboid shape with pseudo-2-fold symmetry and a prominent central cavity. Our data indicate that the C-terminal part of Avo3, a subunit unique to TORC2, is close to the FKBP12-rapamycin-binding domain of Tor2. Removal of this sequence generated a FKBP12-rapamycin-sensitive TORC2 variant, which provides a powerful tool for deciphering TORC2 function in vivo. Using this variant, we demonstrate a role for TORC2 in G2/M cell-cycle progression.
Display omitted
•EM structure of TORC2 reveals a rhomboid shape with pseudo-2-fold symmetry•Avo3 masks the FKBP12-rapamycin binding site in TORC2•Rapamycin-sensitive TORC2 provides a tool to dissect TORC2 signaling•TORC2 activity is required for G2/M cell-cycle progression
Rapamycin-sensitive TORC1 and rapamycin-insensitive TORC2 are multiprotein kinase complexes and key regulators of eukaryote physiology. Gaubitz et al. reveal the molecular architecture of TORC2 and why it is resistant to rapamycin, and generate a rapamycin-sensitive variant as a tool to dissect TORC2 signaling.
This study assessed the ecological health of waters within the Saudi Arabian Exclusive Economic Zone, by utilizing benthic biotic indices with a marine monitoring dataset covering the years 2013 to ...2018. This comprehensive evaluation covered a vast expanse, encompassing 67 distinctive sampling locations characterized by a wide range of depth and salinity gradients. The study examined spatial fluctuations in the benthic community and assessed potential correlations with environmental variables, including salinity, depth, sediment texture, total organic carbon, and other relevant factors. The macrobenthic density varied across the study sites, with an average density of 566 ± 120 ind.m−2. The Shannon diversity index ranged from 3.21 and 5.90, with an average of 4.70 ± 0.52. Based on the average AMBI values, all the locations were categorized as either slightly disturbed or undisturbed. Additionally, the M-AMBI analysis indicated that 95.5 % sites were in good or high ecological status.
•Studied 67 locations in the Arabian Gulf from 2013 to 2018, varying in depth and salinity, to assess benthic community changes.•Average macrobenthic density in the Arabian Gulf from 2013 to 2018 was 566 ± 120 ind.m², with a Shannon index of 4.70 ± 0.52.•AMBI values suggest slight disturbance or undisturbed conditions observed during the study period from 2013 to 2018.•M-AMBI analysis shows 95.5% of sites in the study areas are in good or high ecological status based on data from 2013 to 2018.•Continuous benthic monitoring is crucial due to the 'Shifting Baseline' trend, ensuring effective ecosystem management.
Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD ...effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1-8-9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking. UPF1 is recruited to the SMG-1 kinase domain and C-terminal insertion domain, inducing an opening of the head domain that provides access to the active site. SMG-8 and SMG-9 interact with the SMG-1 C-insertion and promote high-affinity UPF1 binding to SMG-1-8-9, as well as decelerated SMG-1 kinase activity and enhanced stringency of phosphorylation site selection. The presence of UPF2 destabilizes the SMG-1-8-9-UPF1 complex leading to substrate release. Our results suggest an intricate molecular network of SMG-8, SMG-9 and the SMG-1 C-insertion domain that governs UPF1 substrate recruitment and phosphorylation by SMG-1 kinase, an event that is central to trigger mRNA decay.
•Contamination of metals was studied in the surficial sediments of the Red Sea (Saudi Arabia).•Geochemical and ecological risk assessment indices were also estimated.•Enrichment of Ni, Zn, Fe, V, Cu ...and As in the deep stations was due to metalliferous nature.•Enrichment of Cr and Mn was due to the influence of brine pools and hydrothermal circulation.•Enrichment of Ni and Hg was due to anthropogenic activities.
The Gulf of Aqaba (hereafter ‘the Gulf’) is a narrow, semi-enclosed, warm, high saline, and oligotrophic water body. This baseline study provides the first quantitative data on deep-sea (207–1281 m ...depth) benthos of the Gulf. Fifty-five benthic species (predominantly polychaetes) with a density of 160–670 ind. m−2, species richness of 11–25, and Shannon-Wiener diversity (H′) of 3.14–4.17 bits. ind.−1 were recorded from nine stations. The density and H′ of benthos of the Gulf are comparable with those of the Red Sea, while both are lower than those reported from the Arabian Sea and the Mediterranean Sea. The good-high ecological status of benthic communities indicates the absence of major stress in the deep-sea habitats of the Gulf. As large-scale urbanization is proposed in the Saudi coastal areas of the Gulf, this study is expected to provide a baseline dataset for future environmental impact assessments.
•Deep-sea benthic community is composed mainly by polychaete species.•Density and diversity are comparable to those of the northern Red Sea.•Density is lower than that reported from the Arabian Sea and Mediterranean Sea.•Deep-sea benthic communities have good-high ecological status.•Factors affecting the benthos are organic matter and hydrographical parameters.
PDF
