Ribosomes are RNA-based protein factories found in all living cells, responsible for translating the genetic information encoded in messenger RNA (mRNA) into proteins. The first x-ray structures of the complete 70S ribosome were determined in 1999 at 7.8 Å and in 2001 at 5.5 Å, using diffraction data collected at ALS Beamline 5.0.2. These structures showed how the ribosomal RNA and the more than 50 ribosomal proteins are organized to form the structure of the complete ribosome, and the positions of the mRNA and transfer RNAs (tRNAs) in the ribosome. Now, using data collected at ALS Beamline 12.3.1, researchers from the University of California, Santa Cruz, have solved the structure of a Thermus thermophilus 70S ribosome functional complex at 3.7 Å resolution. Due to the large cell dimensions of ribosome crystals, they give weak diffraction, and spots are crowded close together in the diffraction patterns. Consequently, the high-flux beams, sensitive large-area detectors, and well-focused, compact beam cross sections available at the ALS all played a crucial role in this work. Research in this area may lead to novel antibiotics targeting bacterial ribosomes that have developed resistance to current drugs.

X-ray crystal structure of a 70S ribosome functional complex at 3.7Å resolution. The complex contains a defined mRNA (green) and two tRNAs, bound to the P and E sites (orange and red) at the subunit interface. (a) The 30S subunit is shown on the left (16S rRNA in cyan; 30S proteins in dark blue) and the 50S subunit is on the right (23S rRNA in grey, 5S rRNA in grey-blue, and 50S proteins in magenta); (b) The 30S subunit; (c) The 50S subunit.

The improved structure resolution allows construction of the first all-atom model of a ribosome functional complex containing its mRNA and tRNA substrates. This provided two kinds of information crucial to the understanding of the protein synthesis mechanism: (1) details of molecular interactions between the ribosome and its substrate RNAs, and (2) ways in which the structures of both the ribosome and the tRNAs are altered by their functional interactions.

During protein synthesis, the genetic code is used to translate the sequence of nucleotides in a mRNA into the sequence of amino acids in the protein. Groups of three nucleotides (codons) in the mRNA are read by base pairing with a complementary three-nucleotide sequence of nucleotides (anticodon) in the tRNAs, which carry the growing protein chain during synthesis. There are three binding sites for tRNA in the ribosome, called the A (aminoacyl), P (peptidyl) and E (exit) sites. In the crystals that were used for the structure determination, tRNAPhe was bound to the P site and noncognate endogenous tRNA to the E site of the ribosome, in addition to a 10-nucleotide mRNA fragment. The tRNA is bound most tightly to the ribosomal P site, which is responsible for maintaining the translational reading frame of the mRNA and for holding the growing protein chain in the ribosome via the peptidyl-tRNA. The structure reveals a high density of contacts between the tRNA and ribosomal RNA bases and backbone, as well as ribosomal proteins, explaining the high affinity of the P site for tRNA. Catalysis of peptide bond formation takes place on the 50S subunit by a ribosomal activity called peptidyl transferase. In the new structure, an intriguing structural rearrangement is observed in the peptidyl transferase center.

Interactions of the anticodon stem-loop (ASL) of elongator tRNAPhe (orange) and mRNA (green) with 16S rRNA (cyan) and small-subunit proteins (blue) in the 30S subunit P site.

In the course of protein synthesis, tRNAs move through the ribosome, coupled to movement of mRNA like an assembly line, in a process called translocation. Translocation of tRNA from the P to the E site is crucial for the energetics of this process, and requires that the terminal nucleotide A76 of tRNA is deacylated—i.e., no longer contains a bound protein chain, which is its chemical state following peptide bond formation. The new structure explains this requirement, showing that binding of tRNA to the E site requires hydrogen bonding between the ribose moiety of A76 and C2394 of 23S rRNA; the presence of a peptidyl, aminoacyl, or even a methyl group bound to the terminal ribose would thus prevent these interactions.

Structural changes in the peptidyl transferase center. View of the T. thermophilus 70S ribosomal complex containing deacylated tRNA bound to the P site (blue) compared with the H. marismortui 50S subunit model complex (magenta).

Future ribosome structure studies that include functional states may eventually lead to an atomic-resolution 3D "movie"—the ultimate description of the molecular mechanism of protein synthesis.

E-site tRNA interactions. (a) Interaction of the elbow of E-site tRNA (red) with 23S rRNA (blue) in the L1 stalk region, showing the large-scale displacement of the stalk relative to its position in the vacant ribosome, induced by tRNA binding. The blue arrow indicates the extreme compression of the major groove of helix 76 of 23S rRNA that accompanies this movement. (b) Interactions of the CCA tail of E-site tRNA with the large subunit.

Research conducted by A. Korostelev, S. Trakhanov, M. Laurberg, and H.F. Noller (University of California, Santa Cruz).

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

Publication about this research: A. Korostelev, S. Trakhanov, M. Laurberg and H. Noller, "Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements," Cell 126, 1065–1077 (2006).