Sharper X-Rays of Cell's Ribosome Could Lead to Better Antibiotics

Scientists at the University of California, Berkeley, and Lawrence Berkeley National Laboratory (LBNL) have announced that they have captured the high-resolution snapshots of the bacterial ribosome with the lab's Light Source, which generates intense beams of X-rays that can reveal unprecedented structural detail of such large and complex molecules.

The high-resolution data on the intact ribosome allows researchers to build detailed and realistic models of the ribosome.

While sharp images of the two main pieces of the ribosome have already provided insight into how specific antibiotics work, many antibiotics, such as the aminoglycosides, only interfere with the entire, fully assembled molecular machine.

"Many antibiotics target only the intact machine, disrupting messenger RNA decoding or movement," said lead author Jamie Cate, assistant professor of chemistry and of molecular and cell biology at UC Berkeley and a staff scientist in the Physical Biosciences Division at LBNL.

"We are now in a position to look at some of these drugs and discover things that haven't been known before."
 
Cate, a member of the California Institute for Quantitative Biomedical Research (QB3) at UC Berkeley, and his colleagues report the detailed structure of the ribosome from Escherichia coli, the common intestinal bacteria, in the Nov. 4 issue of Science.

The ribosome, about 21 to 25 nanometers across, is the original nanomachine, taking genetic information relayed by messenger RNA, decoding it and spitting out proteins.

Only four years ago, Cate was part of a team that published a picture of the ribosome with a resolution of 5.5 Angstroms, where an Angstrom, about the size of a hydrogen atom, is one-tenth of a nanometer.

The images have a resolution of 3.5 Angstroms, allowing Cate and his colleagues to see the individual nucleotides in the RNA strands of the ribosome and the amino-acid backbones of the proteins that surround the RNA core.

Both the old and new images were obtained through X-ray crystallography using Advanced Light Source beamlines, which provide extremely bright X-ray sources.

Having the light source in his backyard, Cate said, has made it easier to get the best crystallographic picture with the sharpest three-dimensional detail.

He and his laboratory colleagues grow crystals of ribosomes, check their quality in the light source, then tweak the crystals and try again.

"We've burned through thousands of crystals in the last five years," he said.

The researchers obtained two high-resolution snapshots of the intact E. coli ribosome and compared them with a wide range of conformations of other ribosomes.

These other data came from lower-resolution X-ray crystallographyic images of Thermus thermophilus and E. coli ribosomes, plus electron microscopy of E. coli, yeast and mammalian ribosomes.

Together, they yielded what Cate calls "global snapshots" and allowed him and his colleagues to deduce how individual parts of the ribosome function during the translocation process.

What the new structure shows so far is how the two large pieces of the ribosome bend, ratchet and rotate as the ribosome goes through the repetitive process of protein manufacturing.
 
The "small" subunit of the ribosome first recognizes and latches onto the messenger RNA (mRNA), which contains a copy of part of the chromosomal DNA.

Once the small subunit finds the start position, the "large" subunit moves in and latches on, clamping the mRNA between them.

The combined machine slides along the mRNA, reading each three-letter codon, matching this code to the appropriate amino acid, and then adding that amino acid - one of 20 possible building blocks - to the lengthening protein chain.

As this translation takes place, transfer RNA (tRNA) constantly brings in amino acid building blocks, while energy-supplying molecules in the form of GTP (guanosine triphosphate) cycle through.

They found that after the bond - called a peptide bond - forms between the growing chain and the newly added amino acid, the small subunit ratchets with respect to the large subunit.

Then the head of the small subunit swivels in preparation for shifting the mRNA forward by one codon.

At the same time, a groove opens that allows the mRNA to actually move and the tRNA, depleted of its amino acid, to float away.

Then, the small subunit reverses its motions, resets, and is ready to add the next amino acid. This picture of translocation - ratcheting, swiveling, opening the groove, then reversing these three steps - is repeated 10 to 20 times each second in bacteria.

Based on the researchers' analysis of the new data, Cate said that it appears, also, that the helical RNA in the ribosome acts as a spring to withstand the stress of these reversible swivels.

Also, the ribosome harbors an astounding number of positive magnesium ions - hundreds in all - that apparently neutralize the highly negative charge of the RNA.

Without these magnesium ions, Cate said, the repulsion of the RNA's negative charge would blow the ribosome apart.

Some of the magnesium ions form a salty liquid at the interface between the large and small subunits of the ribosome, perhaps lubricating the machine. These and other hypotheses need further exploration, he said.

"All the interactions we see have been seen before at lower resolution, but it was not clear how to interpret them," he said. "It took these high-resolution studies to coalesce our ideas."