"Frozen Pictures" Allow Far-Reaching Glimpse Into The Cell’s Protein Factory

Ribosomes, the universal cellular organelles responsible for protein production, are essential to life. Receiving genetically encoded instructions from the cell nucleus, the ribosomal factory churns out proteins - the body's primary component and the basis of all enzymatic reactions. Understanding protein biosynthesis is therefore the gateway to grasping life itself, and it's darker side - the emergence of disease when production goes haywire. This explains why ribosomes have been the target of numerous biochemical, biophysical, and genetic studies. However, throughout nearly four decades of research, these pivotal biological units have stubbornly "resisted" scientific attempts to reveal their detailed functional design.

In order to examine microscopic structures scientists expose crystals of the material in question to high intensity x-ray beams - a method known as x-ray crystallography. The beam diffracted from crystals of natural and slightly modified materials creates a pattern that coupled with sophisticated computational analysis demonstrates the crystal's components and the nature of their spatial interactions. However, the ribosome, a notoriously unstable giant nucleoprotein complex, represents a daunting crystallographic challenge. To further complicate matters, it also lacks the internal symmetry and repetitions that eased the way to understanding the structure of other biological entities, such as viruses.

Nevertheless, using novel crystallographic techniques, Professor Ada Yonath of Weizmann's Department of Structural Biology and the Max Planck Research Units for Ribosomal Structure in Hamburg, has now overcome this obstacle. Her study, due to appear in the 7 December issue of the Proceedings of the National Academy of Sciences (PNAS), introduces an electron density map of the small ribosomal subunit from bacterium Thermus thermophilus, constructed at 4.5 angstroms (1A = 10 -10 meter).

In contrast to other published studies, which are subject to considerable ambiguity due to their reliance on indirect, non-crystallographic models, according to Yonath, the new map provides an unbiased positioning of ribosomal components.

The uniqueness of her approach lies in phasing - designing heavy atoms as markers that stand out like flares in the ribosomal map due to their high electron density. These markers significantly enhance the ability to pinpoint functional units within the ribosome.

"In order to examine the spatial structure of ribosomes at high resolution, we prepare "derivative crystals", consisting of the original material to which heavy atoms were bound in key sites," explains Yonath.

And the resultant image, constructed from twice the diffraction data than collected elsewhere, provides a far-reaching glimpse into the microscopic ribosomal world, revealing certain outstanding, heretofore unobserved features. These include the site where protein production begins, as well as exceptionally clear RNA double helixes and various ribosomal protein locations.

Opening the Ribosomal Gate

Ribosomes consist of two independent subunits of unequal size. It is on 30S - the smaller subunit critical to initiating protein biosynthesis and decoding genetic information, that Yonath set her sites. More specifically, she wanted to capture "snapshots" of 30S in its "active form" - during the precise moment in time when protein biosynthesis begins. To do so, her team had to first activate the ribosomal particles within the crystal lattice as well as trigger the initiation step. Both tasks called for a creative stretch of scientific imagination.

Conformational changes are not routinely induced within crystals due to the limitation of motion imposed by the crystal network. However, since controlled heating had previously been shown to enhance in-vitro ribosomal activation, Yonath decided to take a chance at heating up the crystals despite concerns that this would cause their disintegration.

Next, the team introduced a messenger RNA analogue designed to trigger protein biosynthesis, which is essentially kept under lock and key. "The mRNA has to attach itself to a specific site in order to open the gate to protein production," explains Yonath. Once activated and bound it was time to record the 30S subunit "in the act" - a feat accomplished by flash freezing the crystals through sudden exposure to cryo-temperature (-185 Celsius).

Yonath's findings are the result of almost twenty years of determined experimentation into largely uncharted territories. In the course of her pursuit she became the first scientist to successfully create ribosomal crystals that diffract to high resolution, around 3A.

Her quest for durable crystals also gave rise to two novel approaches: the use of ribosomal material from hardy bacterial strains isolated from the Dead Sea and cryo-cystallography. This method, which later became standard research procedure in structural biology, is based on exposing (previously formed) crystals to cryo-temperature during x-ray measurements in order to minimize their disintegration.

Future Prospects

The achievements generated by Yonath‘s team indicate that the road to near-atomic resolution of ribosomal structures is closer than ever before. Their approach and procedures have recently been repeated by a growing contingent of international researchers - all racing to elucidate the mystery of ribosomal functioning (Science, Vol. 285, p. 2084, 2095 and 2133;Nature, Vol. 400, p. 811, 833 und 841). According to Yonath, this understanding should pave the way to improved antibiotics, targeting bacterial agents at the ribosomal level. Additionally, the enhanced understanding of normal protein biosynthesis may one day improve the ability to fight the pathogenic, uncontrolled protein production characteristic of cancer cells.

Prof. Ada Yonath holds the Martin S. Kimmel Professorial Chair. This research was funded by the Max Planck Society, the US National Institute of Health, the German Federal Ministry for Education and Research and the Kimmelman Center for Macromolecular Assembly at the Weizmann Institute.