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Highest-Resolution Ribosome Structure Print

The last step in converting the genetic information stored in DNA into the major functional parts of cells is protein biosynthesis. Protein synthesis occurs on the ribosome, a cellular factory found in all forms of life. In contrast to most cellular machines, the ribosome contains a functional core of RNA that is enhanced by ribosomal proteins and accessory factors. Two structures of the intact ribosome from the common bacterium Escherichia coli, determined by a Berkeley–Berlin collaboration to a resolution of 3.5 Å, the highest yet achieved, provide many new insights into how the ribosome factory works.

Ribosomes Ready for Extreme Close-Up

In 1999, the first structure of the intact ribosome—a very large, asymmetric protein that is difficult to crystallize—was solved by x-ray crystallography at the ALS (see "Solving the Ribosome Puzzle"). Since then, scientists have developed quite an extensive photo gallery of ribosomes from various organisms and in various configurations. More importantly, they have sharpened the focus significantly, going from a resolution of 7.8 Å in 1999, to 5.5 Å in 2001 (see "Zooming in on Ribosomes"), to an amazing 3.5 Å in this latest work. What was initially seen as fuzzy "fingers" of electron density can now be resolved into individual nucleotides in the RNA strands. Serendipitously, the crystals used in this particular study contained two versions of the ribosome, each one in a different "pose," allowing the researchers to compare the positions of the various parts and deduce how they work. With these sharper images, scientists are now better able to interpret previous data, test models, and develop new theories, both practical (how do antibiotics that target the ribosome work?) and theoretical (how much has the ribosome evolved from bacteria to human?). Stay tuned.

Schematic of protein synthesis. The intact ribosome (lower left) reads messenger RNA (long strand at bottom) to synthesize a protein (the strand growing from the top of the ribosome) out of amino acids brought by transfer RNAs (upper right).

The ribosome is a multicomponent machine that, in bacteria, is 21 nm in diameter. Given the large size of the ribosome, it has been difficult to determine how it works at a molecular level. To provide a complete picture of the protein synthesis cycle, many groups have been striving to make an atomic-resolution "movie" of the intact ribosome synthesizing a protein. Cate et al. have determined the first "frames" of this movie by solving the atomic-resolution structures of two intact ribosomes from the model organism, Escherichia coli. To determine the ribosome structures, they used x-ray crystallography at ALS Beamlines 8.3.1 and 12.3.1. The researchers' close proximity to the ALS was critical for optimizing x-ray diffraction from the fragile ribosomes that they had crystallized.

Swiveling of the head of the small ribosomal subunit within the intact ribosome. Left: View of the intact ribosome showing the small subunit (dark and light blue) and large subunit (grey and magenta). Right: Swiveling of the head of the small ribosomal subunit (blue difference vectors) determined from the two ribosome structures.

The ribosome crystals happened to contain a repeating unit of two ribosomes, which turned out to be an unexpected boon. Each of the two ribosomes adopted a different conformation, providing clues as to how the ribosome moves along messenger RNA (mRNA), the genetic template for protein synthesis. Comparison of the two ribosome structures revealed movement of the "head" of the small ribosomal subunit that the researchers think helps to complete one step along the mRNA, a process called "translocation." Using models and lower-resolution structures of the ribosome, the group proposed a sequence of steps in translocation that finishes with the swiveling of the small subunit head to allow the mRNA and the transfer RNAs to move by one step. Swiveling of the head may be driven by a protein enzyme called Elongation Factor G (EF-G), which burns one guanosine triphosphate (GTP) molecule to catalyze the stepping along the mRNA.

A second intriguing finding from comparison of the two structures is the mode of long-range movements within the ribosome. The researchers were able to identify three long-range motions of 15 Å or more that are concentrated in standard RNA helices within the ribosomal RNA. The researchers think that these may be molecular versions of torsion springs that absorb the strains induced by the rotation of the head domain. The fact that similar neck-helix dynamics have also been identified by biochemical means in human ribosomes suggests that the role of these helices is conserved in all forms of life.

Twisting of the neck helix within the small subunit allows swiveling of the head domain. One ribosome is colored red, while the other is blue.

Finally, the researchers found that the interface between the two ribosomal subunits, the 30S and 50S subunits in bacteria, to a large extent consists of salt water. The number of direct interactions between the two subunits, which are primarily RNA, was much lower than expected. This may explain why ribosome function is incredibly sensitive to the salts in the solution around it.

Many commonly used antibiotics target the function of the intact ribosome. Now that intact ribosome crystals that diffract x rays to high resolution have been obtained, the researchers are now working to probe the structural basis for how these antibiotics work. In particular, they are probing how different classes of antibiotic affect three steps of translation. One class of antibiotic blocks translation initiation, whereas a second class degrades accurate mRNA decoding or slows translocation. Yet a third specifically targets translocation by a different mechanism. This future work may potentially open new avenues for antibiotic design.

Research conducted by B.S. Schuwirth (University of California, Berkeley, and Free University of Berlin, Germany); M.A. Borovinskaya, A. Vila-Sanjurjo, and J.M. Holton (Berkeley Lab); C.W. Hau and Wen Zhang (University of California,Berkeley); and Jamie H. Doudna Cate (University of California, Berkeley, and Berkeley Lab).

Research funding: National Institutes of Health and U.S. Department of Energy, Office of Basic Energy Sciences (BES). Operation of the ALS is supported by BES.

Publication about this research: B.S. Schuwirth, M.A. Borovinskaya, C.W. Hau, W. Zhang, A. Vila-Sanjurjo, J.M. Holton, and J.H.D. Cate, "Structures of the bacterial ribosome at 3.5 Å resolution," Science 310, 827 (2005).