Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. This facilitates tRNA binding, allows scanning of mRNA, and maintains fidelity of start codon recognition. Using cryo-EM, we have obtained 3D reconstructions of 40S bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that together, eIF1 and eIF1A stabilize a conformational change that opens the mRNA binding channel. Biochemical data reveal that both factors accelerate the rate of ternary complex (eIF2*GTP*Met-tRNA(i)(Met)) binding to 40S but only eIF1A stabilizes this interaction. Our results suggest that eIF1 and eIF1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF1 release to stabilize ternary complex binding and clamp down on mRNA.

Despite recent advances, the structures of many proteins cannot be determined by electron cryomicroscopy because the individual proteins move during irradiation. This blurs the images so they cannot be aligned with each other to calculate a 3D density. Much of this movement stems from instabilities in the carbon substrates used to support frozen samples in the microscope. Here we demonstrate a new gold specimen support that nearly eliminates substrate motion during irradiation. This increases the sub-nanometer image contrast such that α-helices of individual proteins are resolved. With this improvement we determine the structure of apoferritin, a smooth, octahedral shell of α-helical subunits that is particularly difficult to solve by electron microscopy.This advance in substrate design will enable the solution of currently intractable protein structures.Recent developments in electron cryomicroscopy (cryo-EM) have allowed structure determination to near-atomic resolution for some macromolecular complexes (1-3). Still, many small and challenging structures cannot be determined by current cryo-EM methods to the resolutions required for accurate modeling of atomic structure. This is because electron micrograph quality still falls short of the physical limits imposed by radiation damage to the macromolecules (4-6). Reduced image quality likely has two primary origins: Inefficient detection of imaging electrons (6, 7), and motion and blurring of the particles during irradiation (6,(8)(9)(10)(11). Recent developments in electron detectors have addressed the first constraint (12,13), and have enabled the development of motion correction algorithms to ameliorate the effects of the second (14-16). Direct electron detectors can acquire images using fractions of the electron dose previously required, thus allowing the measurement of single molecule positions in time. This allows accurate tracking of large ensembles of particles, and offers a way to determine the physical origins of radiation-induced particle movement (14-17).Conventional amorphous carbon (am-C) substrates undergo bending and deformation during irradiation (9,11), which includes movement both parallel and perpendicular to the substrate plane. Incorporation of titanium-silicon, doped silicon carbide or graphene into substrate designs reduced radiation-induced specimen motion (17)(18)(19). Still these designs did not stop substrate movement and are challenging to manufacture and use. Here, we demonstrate a new cryo-EM support that nearly eliminates the radiation-induced deformation of thin, iceembedded specimens at cryogenic temperatures. This curtails the perpendicular and in-plane * To whom correspondence should be addressed; passmore@mrc-lmb.cam.ac.uk. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts components of motion during imaging, and thus improves image quality for all radiationsensitive cryogenic specimens.The support comprises a regular array of micron sized holes in a ≃500 Å thick foil ...

SummaryTranslation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases—Ccr4 and Caf1/Pop2—that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes. Surprisingly, Pab1 release relies on Ccr4 activity. In agreement with this, in vivo experiments in budding yeast show that Ccr4 is a general deadenylase that acts on all mRNAs. In contrast, Caf1 only trims poly(A) not bound by Pab1. As a consequence, Caf1 is a specialized deadenylase required for the selective deadenylation of transcripts with lower rates of translation elongation and reduced Pab1 occupancy. These findings reveal a coupling between the rates of translation and deadenylation that is dependent on Pab1 and Ccr4-Not.

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.

Newly transcribed eukaryotic precursor messenger RNAs (pre-mRNAs) are processed at their 3′ ends by the ~1-megadalton multiprotein cleavage and polyadenylation factor (CPF). CPF cleaves pre-mRNAs, adds a polyadenylate tail, and triggers transcription termination, but it is unclear how its various enzymes are coordinated and assembled. Here, we show that the nuclease, polymerase, and phosphatase activities of yeast CPF are organized into three modules. Using electron cryomicroscopy, we determined a 3.5-angstrom-resolution structure of the ~200-kilodalton polymerase module. This revealed four β propellers, in an assembly markedly similar to those of other protein complexes that bind nucleic acid. Combined with in vitro reconstitution experiments, our data show that the polymerase module brings together factors required for specific and efficient polyadenylation, to help coordinate mRNA 3′-end processing.

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The role of protein ubiquitylation in the control of diverse cellular pathways has recently gained widespread attention. Ubiquitylation not only directs the targeted destruction of tagged proteins by the 26 S proteasome, but it also modulates protein activities, protein-protein interactions and subcellular localization. An understanding of the components involved in protein ubiquitylation (E1s, E2s and E3s) is essential to understand how specificity and regulation are conferred upon these pathways. Much of what we know about the catalytic mechanisms of protein ubiquitylation comes from structural studies of the proteins involved in this process. Indeed, structures of ubiquitin-activating enzymes (E1s) and ubiquitin-conjugating enzymes (E2s) have provided insight into their mechanistic details. E3s (ubiquitin ligases) contain most of the substrate specificity and regulatory elements required for protein ubiquitylation. Although several E3 structures are available, the specific mechanistic role of E3s is still unclear. This review will discuss the different types of ubiquitin signals and how they are generated. Recent advances in the field of protein ubiquitylation will be examined, including the mechanisms of E1, E2 and E3. In particular, we discuss the complexity of molecular recognition required to impose selectivity on substrate selection and topology of poly-ubiquitin chains.

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H-and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester-and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol-and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerolenriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.