Structures of ribosome-bound initiation factor 2 reveal mechanism of subunit association

Throughout the four phases of protein biosynthesis—initiation, elongation, termination, and recycling—the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAiMet in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation

Molecular architecture of the ribosome-bound Hepatitis C Virus internal ribosomal entry site RNA

Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation

Streamlined structure determination by cryo-electron tomography and subtomogram averaging using TomoBEAR

Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to the structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR—a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages with reasonable presets to provide a transparent (“white box”) approach for data management and processing. We demonstrate applications of TomoBEAR to two data sets of purified macromolecular targets, to an ion channel RyR1 in a membrane, and the tomograms of plasma FIB-milled lamellae and demonstrate the ability to produce high-resolution structures. TomoBEAR speeds up data processing, minimizes human interventions, and will help accelerate the adoption of in situ structural biology by cryo-ET. The source code and the documentation are freely available

Cryo-electron tomography reveals structural insights into the membrane remodeling mode of dynamin-like EHD filaments

Eps15-homology domain containing proteins (EHDs) are eukaryotic, dynamin-related ATPases involved in cellular membrane trafficking. They oligomerize on membranes into filaments that induce membrane tubulation. While EHD crystal structures in open and closed conformations were previously reported, little structural information is available for the membrane-bound oligomeric form. Consequently, mechanistic insights into the membrane remodeling mechanism have remained sparse. Here, by using cryo-electron tomography and subtomogram averaging, we determined structures of nucleotide-bound EHD4 filaments on membrane tubes of various diameters at an average resolution of 7.6 Å. Assembly of EHD4 is mediated via interfaces in the G-domain and the helical domain. The oligomerized EHD4 structure resembles the closed conformation, where the tips of the helical domains protrude into the membrane. The variation in filament geometry and tube radius suggests a spontaneous filament curvature of approximately 1/70 nm(−1). Combining the available structural and functional data, we suggest a model for EHD-mediated membrane remodeling

The architecture of protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

Protein synthesis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopmental gene expression, and dominant pathology in neurological disease. However, the architecture of ribosomal complexes in the developing mammalian brain has not been analyzed at high resolution. This study investigates the architecture of ribosomes ex vivo from the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, with in vivo embryonic Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal point of gene expression control in the molecular specification of neuronal morphology

Genome-edited foods

Genome editing can transform agriculture and shape the future of food by improving crop yields and animal productivity, which in turn can help to achieve food security for the growing world population. CRISPR–Cas-based technologies are powerful gene editing tools that are applied to various food products. In this Review, we discuss the applications of CRISPR–Cas aimed at increasing the nutritional value of crops through macronutrient engineering and biofortification or the reduction of the amount of antinutrients. We examine the role of CRISPR–Cas in improving the flavour of crops and reducing post-harvest losses to increase consumer acceptance and decrease food waste. We also highlight the gene editing of animal food products and probiotics. We summarize the regulations for approval of gene-edited foods worldwide and the progressively evolving public view. Finally, we explore the strategies that can help to enhance the efficiency of genome editing techniques and the acceptance of genome-edited foods in the global market, and extend the technology to low-resource settings

Plant genome editing by novel tools: TALEN and other sequence specific nucleases.

Genome editing technologies using sequence specific nucleases (SSNs) became a tremendously powerful and precise tool for reverse genetic approaches and applied biology. Transcription activator-like effector nucleases (TALENs) in particular, consisting of a free designable DNA binding domain and a nuclease, have been exploited today by a huge number of approaches in many different organisms. The convenience of designing the DNA binding domain and straightforward protocols for their assembly, as well as the broad number of applications in different scientific fields made it Natures method of the year 2011. TALENs act as molecular scissors by introducing double strand breaks (DSBs) to the DNA at a given location. The DSBs are subsequently repaired by the cell itself using different repair pathways such as non-homologous end joining (NHEJ) or homologous recombination (HR). These mechanisms can lead to deletions, insertions, replacements or larger chromosomal rearrangements. By offering a template DNA it is possible to channel the repair in direction of HR. In this article we review the recent findings in the field of SSN approaches with emphasis on plants