Two papers by the Frank Lab have just been accepted by Cell and Biopolymers journals.
Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29
Yaser Hashem, Amedee des Georges, Vidya Dhote, Robert Langlois, Hstau Y. Liao, Robert A. Grassucci, Christopher U.T. Hellen, Tatyana V. Pestova and Joachim Frank
Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAiMet), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6 Å resolution. It reveals that eIF2 interacts with the 40S subunit via its α subunit and supports Met-tRNAiMet in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.
Molecular machines are workshops in the cell that bring molecules and substrates together in a coordinated, processive way.1‐2. The molecular complexes acting as molecular machines are often spatially localized and assembled in an ad‐hoc fashion, in response to the demands of the cell’s metabolism. Products of such binding reactions can be RNA or DNA polymers, ATP, or other compounds needed at the site. Well‐known examples are RNA and DNA polymerases, ATP synthase, the ribosome, and the proteasome. Visualizing molecular machines in their various states of processing poses challenges which traditional techniques of structure research find hard to meet. The complexes formed in the course of the work cycle are typically large, relatively unstable, and flexible ‐‐ all properties that disfavor X‐ray crystallography as a means of visualization. In addition, molecular machines can have multiple binding partners and can go through numerous states, characterized by different conformations and binding configurations. Purification of such complexes can be done through two different routes: either from a cell extract or from an in vitro reconstituted system. In either case, it is difficult to isolate the complex by biochemical means in one of its many states with the purity required for structural research. Cryo‐electron microscopy (cryo‐EM) is a fairly new technique for obtaining a 3D image of a molecule from a large set of projections, harvested from micrographs of a field of molecules captured in random orientations.3 In some cases, resolutions in the range of 4 to 5.5 Å have already been achieved for the ribosome, an RNA‐containing molecular machine.4‐6 Purity of the sample in terms of conformation and binding state of the molecule is evidently an absolute requirement for obtaining a meaningful 3D image from the “single particle” projection data set. In certain cases, high purity can be achieved by affinity imaging techniques.7‐9 However, because of inherent difficulties of finding biochemical methods that guarantee 100% purity, it has been a goal in the development of cryo‐EM to accomplish the purification of the molecules after the imaging, at the data processing step. Cryo‐EM projection data have a typical signal‐to‐noise ratio in the order of 0.1 or even less.10 The challenge posed by classification of such noisy data is compounded by the fact that variability stemming from a change in viewing direction is intermingled with variability due to heterogeneity of composition or conformation.