FUNCTIONAL STATES OF THE 70S E. COLI RIBOSOME REVEALED BY ELECTRON CRYOMICROSCOPY
Holger Stark1, Marina Rodnina2, Richard Brimacombe3, Wolfgang Wintermeyer2, Marin van Heel1
1Imperial College of
Science, Medicine and Technologie, Department of Biochemistry,
London SW7 2AY, UK. E-mail: h.stark@ic.ac.uk
2Institut für
Molekularbiologie, Universität Witten/Herdecke, D-58448 Witten,
Germany
3Max-Planck-Institut für
Molekulare Genetik, D-14195 Berlin, Germany
Keywords: ribosome, translation, tRNA
binding sites, elongation factor, electron cryomicroscopy,
kirromycin.
The ribosome is essential for protein synthesis in practically all organisms and more than 100 macromolecules contribute to the process of translation. By antibiotic interaction, the ribosome can be fixed in different functional states of the elongation cycle. Those functional states were embedded in amorphous ice by rapidly freezing the sample in its natural environment on a holey carbon grid and imaged either at liquid helium or liquid nitrogen temperature [1,2]. For example, tRNAs or elongation factors (EF-Tu, EF-G) can be bound to their respective binding sites on the ribosome. Many functional states of the ribosome can thus be trapped, which are only milliseconds apart from each other within the functional time scale. Analysing these structures is necessary in order to gain insight into the pathway of protein synthesis, which is of immense importance for the understanding of ribosomal function at the molecular level.
Recently, the structures of the E.coli 70S ribosome in their pre- and post-translocational states have been solved at a level of ~20A and the positions of the tRNA molecules visualised and clearly identified [2]. The two tRNAs in the acceptor (A) site and peptidyl (P) site lie in close proximity at both ends of the L shaped molecules, in the intersubunit space between 50S and 30S subunits. The enclosed angle between the two tRNAs is ~50° at a distance of 38 A between the elbow regions of the tRNAs. All single particle image processing has been carried out using the IMAGIC-V software package [3]
The binding of aminoacyl-tRNAs to the ribosome is catalysed by the elongation factor Tu (EF-Tu). The release of the ternary complex (EF-Tu/tRNA/GTP) from the ribosome usually occurs after GTP hydrolysis and can be prevented by the antibiotic kirromycin. The structure of the kirromycin stalled ribosome was determined to a resolution of 18 A [4]. Domain 1 of the EF-Tu is bound to the L7/L12 stalk of the 50S subunit, whereas domain 2 is orientated towards the S12 region on the 30S subunit. The ternary complex is thus spanning the gap between the two subunits with the acceptor region of the tRNA reaching into the decoding centre.
Attempts are underway to use cryomicroscopical
3D reconstructions to boost phases for x-ray crystallographic
data of the thermophilic 70S, 50S and 30S ribosome. This project
is being carried out in collaboration with the group of Ada
Yonath.