Opposing Translations

Two structures of the ribosome ignite debate and discovery in structural biology.

Andrea Gawrylewski
May 1, 2008
<figcaption>The 70S ribosome at 2.8 Å Credit: Courtesy Venki Ramakrishnan / Medical Research Council, Cambridge</figcaption>
The 70S ribosome at 2.8 Å Credit: Courtesy Venki Ramakrishnan / Medical Research Council, Cambridge

The ribosome is the cellular machine that translates DNA into proteins. Its two subunits, 30S and 50S (which together make up the 70S ribosome), scan over the messenger RNA and spit out polypeptides using amino acids delivered by transfer RNA (tRNA).

The first glimpse of the much anticipated structure came in 2001 when Harry Noller's group at the University of California, Santa Cruz, published the complete bacterial ribosome structure at the modest resolution of 5.5 Å. Several subsequent efforts were published, and then in September 2006, two groups provided high resolution views of the whole ribosome. These two Hot Papers reported the 70S bacterial ribosome structure bound with messenger and transfer RNAs.

"There was no high resolution structure of the whole ribosome with tRNA and mRNA until this work came along," says Venki Ramakrishnan of...

Structural discrepancies

While the two structures showed that the tRNA was distorted and bent when bound, one structure at 2.8 Å, from Ramakrishnan's group, confirmed the structure of the peptidyl-transferase center shown in previous work.1 But Harry Noller's structure, at 3.7 Å, showed that several nucleotides on the large ribosomal subunit seemed to have shifted.2 This shift appeared to occur within the peptidyl-transferase center of the P site, the location where amino acids are bound together to form polypeptide chains.

Noller's structure suggests that these nucleotides are perhaps involved in the process of building the polypeptide chain. "For many people that didn't make that much sense," says Marina Rodnina, from Witten/Herdecke University. Her group and others have conducted mutation experiments, discovering each nucleotide's role in the translation process, and showing that translation occurs well, even without the very nucleotides that Noller's group sees slipping into the transferase center.

Perhaps more importantly, Noller's structure undermines a proton shuttle mechanism that enables peptides to bond in the peptadyl-transferase center; by this mechanism a hydroxyl group carries a proton from the tRNA to the bond of the extending peptide chain, enabling amino acids to attach to each other. Thomas Steitz's lab at Yale University performed a cross-crystal average of the two data sets and found that the two structures actually conformed.3 Especially at the peptadyl-transferase center, they suggested that the Noller structure had been incorrectly modeled. "Noller had the orientation of these groups different and their interactions with the ribosome were different," Steitz says.

Andrei Korostelev, first author of the Noller paper, says that their group is checking out Steitz's calculations, but at first blush he accuses Steitz's analysis of being inherently biased toward Ramakrishnan's higher resolution structure.

The biochemical side

"Since we now know where the tRNAs bind and we also know that they are somewhat distorted from the structure you see of a free tRNA, that's led to lots of experiments which seek to trace the movement of the tRNA as it goes through the ribosome in great detail," says University of Pennsylvania's Barry Cooperman. His lab uses fluorescence resonance energy transfer (FRET) between single molecules to measure the strength of interactions among various parts of the ribosome. Last year they reported that the ribosome has some 10-12 intermediate states through which it moves during translation.4

Many groups are trying to manipulate the ribosome's "ground state" - its conformation of lowest energy - into one of the several conformations that it might normally move through quickly. Scott Blanchard at Cornell University showed that the ribosome is a dynamic, moving machine that requires no energy or other factors to change conformation several times through the process of translation.5 Soon after, another paper from Harry Noller's group showed that a twisting movement between the ribosome's two subunits is essential for translation to occur.6

Jamie Cate, at the University of California, Berkeley, and collaborators have also shown how movement between conformations is essential to the ribosome's function. They published last year on how an antibiotic inhibits translation by sending one helix of the subunit swinging away from where the two subunits meet.7 Conversely, Ramakrishnan showed last year that at the final stage of ribosome action, known as recycling, requires no conformational changes.

Meanwhile, Noller's group continues to probe the ribosome structure. Using their 2006 model as a starting point, they are exploring how a promoter region upstream of the start codon, called the Shine-Dalgarno sequence, influences the position of the 30S subunit at the beginning of translation.

But the most exciting work on the ribosome has yet to happen, says Cate. "It takes a while for structures like this to really sink in."

Data derived from the Science Watch/Hot Papers database and the Web of Science (Thomson ISI) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. M. Selmer et al., "Structure of the 70S ribosome complexed with mRNA and tRNA," Science, 313:1935-42, 2006. (Cited in 111 papers) A. Korostelev et al., "Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements," Cell, 126:1065-77, 2006. (Cited in 58 papers)

References

1. M. Selmer et al., "Structure of the 70S ribosome complexed with mRNA and tRNA," Science, 313:1935-42, 2006. (Cited in 111 papers) 2. A. Korostelev et al., "Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements," Cell, 126:1065-77, 2006. (Cited in 58 papers) 3. M. Simonovic et al., "Cross-crystal averaging reveals that the structure of the peptidyl-tranferase center is the same in the 70S ribosome and 50S subunit," Proc Natl Acad Sci, 105:500-5, 2008. 4. Y. Wang et al., "Single-molecule structural dynamics of EF-G-rbosome interaction during translocation," Biochem, 46:10767-75, 2007. 5. J.B. Munro et al., "Identification of two distinct hybrid state intermediates on the ribosome," Mol Cell, 25:505-17, 2007. 6. L.H. Horan et al., "Intersubunit movement is required for ribosomal translocation," Proc Natl Acad Sci, 104:4881-5, 2007. 7. M.A. Borovinskaya et al., "Structural basis for aminoglycoside inhibition of bacterial ribosome recycling," Nat Struct Mol Biol, 14:727-32, 2007.