Mechanisms of protein synthesis by the ribosome

The translation of genetic information into proteins is essential for life. At the core of this process lies the ribosome, a quintessential large (2.5-4.5 MDa) molecular machine responsible for translating genetic material into functional proteins. In a growing cell, ribosomes comprise up to half of the net dry weight. Because of its fundamental role in the cell, 50% of all efforts to develop antibiotics target bacterial ribosomes, taking advantage of the structural diferences between bacterial and human ribosomes.

The structure and function of the ribosome are fascinatingly complex. Two-thirds of the ribosome consist of ribosomal RNA (rRNA), while over 50 ribosomal proteins make up the rest. The genetic information is delivered to the ribosome by a messenger RNA (mRNA). Transfer RNAs (tRNAs) are adapter molecules, each equipped with an anticodon to matchs the codons in the mRNA, and charged with an amino acid that corresponds to the anticodon as dictated by the genetic code. The ribosome contains three tRNA-binding sites: A, P, and E (see elongation cycle box, or watch a movie). In addition to mRNA and tRNAs, the ribosome interacts with protein factors such as the elongation factors Tu (EF-Tu) and G (EF-G), that are important players in the so-called elongation cycle. The elongation cycle results in the addition of an amino acid to the nascent peptide chain, and consists of three main steps. In the decoding step, a ternary complex comprised of an aminoacyl-tRNA (aa-tRNA), EF-Tu, and GTP binds to the ribosome, leading to the recognition of the codon by the anticodon. The following step is the peptidyl transfer. Here the peptide chain bound to the P-site tRNA is covalently linked to the amino acid bound to the A-site tRNA. In the translocation step, the position of the mRNA/tRNA complex shifts by one codon, accompanied by a ratchet-like motion of the ribosomal subunits.

Elongation Cycle Fig 1. Elongation cycle of protein synthesis. The ribosome is shown in top view, with the small subunit (transparent yellow) below the large subunit (transparent blue). (i) The ribosome in the pre-translocational state with tRNAs in the A (magenta) and P (green) sites. After spontaneous peptidyl transfer, the nascent peptide is covalently attached to the A-site tRNA. (ii) The elongation factor EF-G in complex with GTP (blue) has bound to the ribosome to facilitate the translocation of tRNAs to the P (green) and E (yellow) sites. The translocation is induced by GTP hydrolysis accompanied by large transient conformational changes in the EF-G and the ribosome. (iii) The release of the EF-G after GTP hydrolysis leaves the ribosome in the post-translocational state. It is now ready to accept a new aminoacyl-tRNA (white) presented to the ribosome by the ternary complex, comprising, in addition to the new aminoacyl-tRNA, the the elongation factor EF-Tu and a GTP (red). (iv) The ribosome with a bound ternary complex (magenta and red) in place. It has been suggested that when the ternary complex binds to the ribosome, the E-site tRNA moves further away from the P site to the so-called E2 site (orange). The snapshot shown here is part of the decoding step, where the aminoacyl-tRNA whose anticodon matches the next codon in the mRNA is selected to enter the A site, accompanied by GTP hydrolysis and conformational changes. In case of a match, EF-Tu with the hydrolyzed GTP and the E-site tRNA leave the ribosome, leaving it in the pre-translocational state (i). This figure is adapted from Frank, J. in Conformational Proteomics of Macromolecular Architechtures. World Scientific Publishing Comp. Singapore, 2004. Watch a movie of this process.


Due to great advances in the structural resolution of the ribosome, an impressive feat given its large size, the system is considered one of the hottest focal areas in molecular cell biology today. During the process of translation, the ribosome undergoes several conformational changes and binds to different factors that catalyze specific reactions. As detailed below, techniques to determine structure of the ribosome can only image snapshots of the ribosome, often at medium to low resolution. Atomic details of the interactions between the factors and the ribosome, along with a dynamic description of the conformational changes of the ribosome itself, are crucial to understanding its function.

Our goal is to provide a complete description of the structural dynamics of translation using computational biology. Eventually, we would like to be able to show you an animation much like this beautiful illustrative movie but with every frame based on physically realistic simulations that incorporate experimental data.

Obtaining atomic models of the ribosome at functional states

Detail of MDFF of EF-Tu bound to the ribosome

Fig. 2. Detail of a fitted atomic model into the EM density using MDFF.

Due to its sheer size and complexity, the ribosome presents an outstanding challenge for traditional methods for high-resolution structure determination such as X-ray crystallography and nuclear magnetic resonance spectroscopy. X-ray crystallographers have conquered this challenge: today, the Protein Data Bank has several structures of entire ribosomes from different laboratories. However, these structures remain difficult to obtain for factor-bound ribosomes, which are key to understand the dynamics of translation.

Cryo-electron microscopy (cryo-EM) provides an alternative to obtain structural information of the ribosome in many different functional states. Cryo-EM produces three-dimensional density maps of ribosomes at medium resolution (7-12Å) without the need to purify large amounts of sample or to form highly ordered crystals, both required in X-ray crystallography. This technique thus can capture the ribosome at different conformational states, as shown in Fig. 1. However, these maps don't reach atomic resolution, needed to understand the function of the ribosome in detail along its functional cycle.

In order to reach atomic resolution, we have developed a technique called molecular dynamics flexible fitting (MDFF) to morph atomic-resolution structures emerging from X-ray crystallography into cryo-EM maps. Details of the MDFF method can be found here. Fig. 2 shows a detail of an atomic model flexibly fitted into a cryo-EM map of the ribosome.

We have teamed with the laboratory of Joachim Frank, a leader in cryo-EM, to study the dynamics of the ribosome.

How does the ribosome induce the GTPase activity of EF-Tu?

Fig. 3. Atomic model of the ribosome bound to factors derived from a 6.7-Å EM map. The EM data is shown in transparent surface, with atomic model generated with MDFF showed in cartoon.

During elongation, the mRNA-programmed ribosome is presented with an amino acid by a tRNA in complex with elongation factor Tu (EF-Tu) and GTP. The tRNA assumes the bent A/T position, storing elastic energy. If the ribosome recognizes the right codon-anticodon interaction, GTPase activity in EF-Tu is stimulated over 75Å away. How the ribosome induces GTP hydrolysis has been subject of intensive study. We now know that GTP hydrolysis takes places through an in-line attack on the gamma phosphate by a water molecule, and that His84 is the catalytic residue.

Atomic model of EF-Tu from cryo-EM

Fig. 4. Atomic model of a ribosome-boung EF-Tu. The switch regions are highlighted: Switch I (Sw1) in blue, Switch II (Sw2) in orange, and P-loop in green.

In order to understand GTP hydrolysis in EF-Tu, we studied the ribosome-induced EF-Tu conformational changes that trigger GTP hydrolysis on the factor as revealed by cryo-electron microscopy (cryo-EM) studies. We applied MDFF to obtain an atomic model of a 6.7-Å cryo-EM map of the pre-accommodated E. coli 70S ribosome bound to the Phe-tRNAPhe:EF-Tu:GDP ternary complex stalled by the antibiotic kirromycin (kir), showed in Fig. 3.

EF-Tu has three important regions that play a prominent role in its GTPase activity: Switch I (EF-Tu residues 40--62; E. coli numbering) Switch II (80--100) and P-loop (18--23). All of these regions undergo characteristic conformational changes in EF-Tu. Crystal structures of EF-Tu in the GTP form display a "hydrophobic gate" formed by residues Val20 (in the P-loop) and Ile60 (In Switch I) that controls access to the GTP binding pocket. A schematic representation of this gate is shown in Fig 5A, the crystal structure shown in Fig. 5B. His84 (in Switch II) needs to enter through this gate to perform its catalytic role. Thus, this gate must be opened when the right codon-anticodon interaction is recognized by the ribosome. A crystal structure of EF-Tu outside the ribosome, but bound to the antibiotic aurodox that is thought to simulate interaction with the ribosome, displays an open gate (Fig. 5D). This antibiotic binds to EF-Tu and stimulates GTPase activity, preventing EF-Tu from binding the ribosome, and therefore preventing translation and killing the cell. The fact that an open gate is found in the case of enhanced GTPase activity hints that this mechanism is used by the ribosome as well.

Hydrophobic gate of EF-Tu

Fig. 5. Hydrophobic gate of EF-Tu. (A) Schematic representation of the gate. (B) The closed gate revealed by an X-ray structure of EF-Tu free from the ribosome (PDB 1OB2). (C) The open gate revaled by MDFF (PDB 3FIH). (D) The open gate present in an antibiotic-bound X-ray structure (PDB 1HA3).



But how does the ribosome open this hydrophobic gate? Let's look at each wing of the gate:

Interaction between the P-loop and SRL

Interaction between the P-loop and SRL

Fig. 6. Interaction between the P-loop (green) and the SRL in the 50S (blue).
One wing of the hydrophobic gate is formed by residue Val20. The MDFF model reveals that the P-loop interacts with the sarcin-ricin loop (SRL; nucleotides 2646--2674 of the 23S rRNA). Specifically, the interaction occurs between His19 of EF-Tu and A2660 in the tetraloop of SRL. This contact with SRL stabilizes the P-loop in its position, acting as an anchor point for this wing of the gate, while the other wing opens as described below.

Interaction between Switch I and the 16S rRNA

The other wing of the hydrophobic gate (Ile60) is part of Switch I, which due to its flexibility sometimes is difficult to observe in structural studies. Our data and analysis reveal that Switch I interacts with the junction formed by helices h8 and h14 of the 16S rRNA (Fig. 7A,B). This interaction involves an opening of the hydrophobic gate, as shown in Fig. 5C. The contact between Switch I and the 16S rRNA h8/h14 junction could serve as a pathway for signal transduction to/from the decoding center through helix h44 of the 16S rRNA; this helix interacts with the decoding center at one end and with the h8/h14 junction at the other (Fig. 7C).

Interaction between the Switch I and the 16S rRNA

Fig. 7. Interaction between the Switch I of EF-Tu and the 16S rRNA. (A) Stereo view of the different conformations of Switch I when bound (blue) and unbound (lime) to the ribosome. The EM map is shown in gray mesh. (B) Detail of the interaction. Elements of the ribosome-bound EF-Tu (red and blue) and the 30S (yellow) and 50S (cyan) subunits are shown along with the crystal structure of the Switch I in the ribosome-unbound EF-Tu (lime; PDB 1OB2). (C) Overview of the ternary complex (EF-Tu in red, tRNA in purple) bound to the 30S subunit (yellow).

Open Sesame

Once the gate is opened, the side chain of His84 can reach the GTP binding pocket and activate hydrolysis, as shown in Fig. 5C; note the similarity to the conformation seen in the aurodox-bound structure of EF-Tu outside of the ribosome, shown in Fig. 5D.

Publications

Publications Database

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Elizabeth Villa, Jayati Sengupta, Leonardo G. Trabuco, Jamie LeBarron, William T. Baxter, Tanvir R. Shaikh, Robert A. Grassucci, Poul Nissen, Måns Ehrenberg, Klaus Schulten, and Joachim Frank. Proceedings of the National Academy of Sciences, USA, 106:1063-1068, 2009.

EM Map and Atomic Models

The atomic coordinates have been deposited in the Protein Data Bank, with PDB ID codes 3FIH (30S and factors) and 3FIK (50S). The cryo-EM density map has been deposited to the 3D-EM database , with code EMD-5036.

Get all the files and a VMD saved state to visualize them together here (107 Mb).

Investigators

Page created and maintained by Elizabeth Villa and Leonardo Trabuco.

The contents of this website, including figures 3 to 7, were reproduced in part with permission from Publications Database

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis. Elizabeth Villa, Jayati Sengupta, Leonardo G. Trabuco, Jamie LeBarron, William T. Baxter, Tanvir R. Shaikh, Robert A. Grassucci, Poul Nissen, Måns Ehrenberg, Klaus Schulten, and Joachim Frank. Proceedings of the National Academy of Sciences, USA, 106:1063-1068, 2009.

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