Jianlin Lei

Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome

A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.

Controlling Assembly of Paired Gold Clusters within Apoferritin Nanoreactor for in Vivo Kidney Targeting and Biomedical Imaging

Functional nanostructures with high biocompatibility and stability, low toxicity, and specificity of targeting to desired organs or cells are of great interest in nanobiology and medicine. However, the challenge is to integrate all of these desired features into a single nanobiostructure, which can be applied to biomedical applications and eventually in clinical settings. In this context, we designed a strategy to assemble two gold nanoclusters at the ferroxidase active sites of ferritin heavy chain. Our studies showed that the resulting nanostructures (Au-Ft) retain not only the intrinsic fluorescence properties of noble metal, but gain enhanced intensity, show a red-shift, and exhibit tunable emissions due to the coupling interaction between the paired Au clusters. Furthermore, Au-Ft possessed the well-defined nanostructure of native ferritin, showed organ-specific targeting ability, high biocompatibility, and low cytotoxicity. The current study demonstrates that an integrated multimodal assembly strategy is able to generate stable and effective biomolecule-noble metal complexes of controllable size and with desirable fluorescence emission characteristics. Such agents are ideal for targeted in vitro and in vivo imaging. These results thus open new opportunities for biomolecule-guided nanostructure assembly with great potential for biomedical applications.

Structure of the eukaryotic MCM complex at 3.8 Å

DNA replication in eukaryotes is strictly regulated by several mechanisms. A central step in this replication is the assembly of the heterohexameric minichromosome maintenance (MCM2–7) helicase complex at replication origins during G1 phase as an inactive double hexamer. Here, using cryo-electron microscopy, we report a near-atomic structure of the MCM2–7 double hexamer purified from yeast G1 chromatin. Our structure shows that two single hexamers, arranged in a tilted and twisted fashion through interdigitated amino-terminal domain interactions, form a kinked central channel. Four constricted rings consisting of conserved interior β-hairpins from the two single hexamers create a narrow passageway that tightly fits duplex DNA. This narrow passageway, reinforced by the offset of the two single hexamers at the double hexamer interface, is flanked by two pairs of gate-forming subunits, MCM2 and MCM5. These unusual features of the twisted and tilted single hexamers suggest a concerted mechanism for the melting of origin DNA that requires structural deformation of the intervening DNA.

The architecture of the mammalian respirasome

The respiratory chain complexes I, III and IV (CI, CIII and CIV) are present in the bacterial membrane or the inner mitochondrial membrane and have a role of transferring electrons and establishing the proton gradient for ATP synthesis by complex V. The respiratory chain complexes can assemble into supercomplexes (SCs), but their precise arrangement is unknown. Here we report a 5.4 Å cryo-electron microscopy structure of the major 1.7 megadalton SCI1III2IV1 respirasome purified from porcine heart. The CIII dimer and CIV bind at the same side of the L-shaped CI, with their transmembrane domains essentially aligned to form a transmembrane disk. Compared to free CI, the CI in the respirasome is more compact because of interactions with CIII and CIV. The NDUFA11 and NDUFB9 supernumerary subunits of CI contribute to the oligomerization of CI and CIII. The structure of the respirasome provides information on the precise arrangements of the respiratory chain complexes in mitochondria.

Structural insights into cognate versus near-cognate discrimination during decoding.

The structural basis of the tRNA selection process is investigated by cryo‐electron microscopy of ribosomes programmed with UGA codons and incubated with ternary complex (TC) containing the near‐cognate Trp‐tRNATrp in the presence of kirromycin. Going through more than 350 000 images and employing image classification procedures, we find ∼8% in which the TC is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near‐cognate aa‐tRNA with respect to elongation factor Tu (EF‐Tu) and the ribosome, as well as the domain movements of the ribosome. One of the interesting findings is that near‐cognate tRNAs acceptor stem region is flexible and CCA end becomes disordered. The data bring direct structural insights into the induced‐fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa‐tRNA and EF‐Tu and comparison with the cognate case (UGG codon) offers clues on how the conformational signals conveyed to the GTPase differ in the two cases.

Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase

Kinetics and cryo-electronmicroscopy data provide insights into GTPase ObgE’s role as a ribosome anti-association factor that is modulated by nutrient availability, coupling growth control to ribosome biosynthesis and protein translation.

Cryo-EM structure of the mammalian eukaryotic release factor eRF1-eRF3-associated termination complex

Eukaryotic translation termination results from the complex functional interplay between two eukaryotic release factors, eRF1 and eRF3, and the ribosome, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, using cryo-electron microscopy (cryo-EM) and flexible fitting, we determined the structure of eRF1–eRF3–guanosine 5′-[β,γ-imido]triphosphate (GMPPNP)-bound ribosomal pretermination complex (pre-TC), which corresponds to the initial, pre-GTP hydrolysis stage of factor attachment. Our results show that eukaryotic translation termination involves a network of interactions between the two release factors and the ribosome. Our structure provides mechanistic insight into the coordination between GTP hydrolysis by eRF3 and subsequent peptide release by eRF1.

Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM.

The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF‐Tu, which forms a ternary complex with aminoacyl(aa)‐tRNA. To study the binding modes of different aa‐tRNAs, we compared cryo‐EM maps of the kirromycin‐stalled ribosome bound with ternary complexes containing Phe‐tRNAPhe, Trp‐tRNATrp, or Leu‐tRNALeuI. The three maps suggest a common binding manner of cognate aa‐tRNAs in their specific binding with both the ribosome and EF‐Tu. All three aa‐tRNAs have the same ‘loaded spring’ conformation with a kink and twist between the D‐stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF‐Tu‐binding CCA end of aa‐tRNA, proposed to signal cognate codon–anticodon interaction to the GTPase centre and tune the accuracy of aa‐tRNA selection.

Structural basis for the function of a small GTPase RsgA on the 30S ribosomal subunit maturation revealed by cryoelectron microscopy

The bacterial RsgA, a circularly permutated GTPase, whose GTPase activity is dependent on the 30S ribosomal subunit, is a late-stage ribosome biogenesis factor involved in the 30S subunit maturation. The role of RsgA is to release another 30S biogenesis factor, RbfA, from the mature 30S subunit in a GTP-dependent manner. Using cryoelectron microscopy, we have determined the structure of the 30S subunit bound with RsgA in the presence of GMPPNP at subnanometer resolution. In the structure, RsgA binds to the central part of the 30S subunit, close to the decoding center, in a position that is incompatible with multiple biogenesis factors, all three translation initiation factors, as well as A-, P-site tRNAs and the 50S subunit. Further structural analysis not only provides a structural model for the RsgA-dependent release of RbfA from the nascent 30S subunit, but also indicates RsgA’s role in the ribosomal protein assembly, to promote some tertiary binding protein incorporation. Moreover, together with available biochemical and genetic data, our results suggest that RsgA might be a general checkpoint protein in the late stage of the 30S subunit biogenesis, whose function is not only to release biogenesis factors (e.g., RbfA) from the nascent 30S subunit, but also to block the association of initiation factors to the premature 30S subunit.

Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process.

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3′-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3′-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3′-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.