News

Three papers by the Frank Lab have just been accepted by Nature, Nature Communications, and Journal of Structural Biology.

NATURE
High-resolution cryo-EM structure of Trypanosoma brucei eukaryotic ribosome
Yaser Hashem, Amedee des Georges, Jie Fu, Sarah N. Buss, Fabrice Jossinet, Amy Jobe, Qin Zhang, Hstau Y. Liao, Robert A. Grassucci, Chandrajit Bajaj, Eric Westhof, Susan Madison-Antenucci, and Joachim Frank

Abstract:
Ribosomes, constituting protein factories in living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome^1-6 reveal its increased complexity compared to its prokaryotic counterpart^7-8, manifested mainly in the presence of eukaryotic-specific ribosomal proteins and additional rRNA insertions, called expansion segments (ES)⁹. These structures also point out some differences among species, in part represented by the size and arrangement of these ESs. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosomefrom Trypanosoma brucei, the parasite transmitted by the Tse-tse fly and causing African sleeping sickness. Our atomic model reveals the unique features of this ribosome, characterized mainly by the presence of extraordinarily large ESs and ribosomal proteins extensions leading to the formation of four additional intersubunit bridges, and additional rRNA insertions including one large rRNA domain nonexistent in other eukaryotes. The kinetoplastid large ribosomal subunit (LSU) rRNA chain is uniquely cleaved into six pieces^10-12; our structure reveals the five cleavage sites and suggests the importance of the cleavage for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA ESs for the translation regulation process. Our structure could serve as a basis for future experiments aiming at understanding the functional importance of these kinetoplastid-specific ribosomal features in the protein translation regulation, an essential step toward finding effective and safe kinetoplastid-specific drugs.

NATURE COMMUNICATIONS
Mechanism of tetracycline resistance by ribosomal protection protein Tet(O)
Wen Li, Gemma C. Atkinson, Nehal S. Thakor, Ülar Allas, Chuao-chao Lu, Kwok-Yan Chan, Tanel Tenson, Klaus Schulten, Kevin S. Wilson, Vasili Hauryliuk, and Joachim Frank

Abstract:
Tetracycline resistance protein Tet(O), which protects the bacterial ribosome from binding with the antibiotic tetracycline, is a translational GTPase with significant similarity in both sequence and structure to elongation factor EF-G. Here, we present an atomic model of the Tet(O)-bound 70S ribosome based on our cryo-electron microscopic reconstruction at 9.6 Å resolution. This atomic model allowed us to identify the Tet(O)-ribosome binding sites, which involve three characteristic loops in domain 4 of Tet(O). Replacements of the three-amino acid tips of these loops by a single glycine residue resulted in loss of Tet(O)-mediated tetracycline resistance. On the basis of these findings, the mechanism of Tet(O)-mediated  tetracycline resistance can be explained in molecular detail.

JOURNAL OF STRUCTURAL BIOLOGY
Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome
Gyanesh Sharma, Jesper Pallesen, Sanchaita Das, Robert Grassucci, Robert Langlois, Cheri Hampton, Deborah F. Kelly, Amedee des Georges, and Joachim Frank

Abstract:
Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC βII binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map that shows PKC βII interacts with RACK1, a seven-bladed β-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1. This study is a first step in understanding the molecular framework of PKC βII/RACK1 interaction and its role in translation.