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August 28, 2009

Watching the Birth of a Ribosome: Assembly Quicker, Squishier Than Thought

Using powerful x-rays at the NSLS, a group of researchers from Johns Hopkins University has shown how the intricate process of ribosome assembly happens on a very quick time scale. Their unexpected results challenge common thought about the early stages of this vital molecular complex.

Sarah Woodson

A ribosome is a large particle made of protein and RNA that manufactures the numerous proteins required for living cells. Rapidly growing cells produce thousands of new ribosomes in mere minutes through a series of complicated, and little understood, steps. The challenge for researchers is to unveil how this assembly is completed correctly in such a short amount of time.

To do this, the researchers took "snapshots" of the speedy interactions between ribosomal RNA from the bacteria E. coli and the 20 proteins that make up the smaller of the ribosome's two subunits.

"We wanted to know not only how RNA folds but how RNA from part of the ribosome comes together with proteins to form a subunit," said Johns Hopkins University researcher Sarah Woodson. "We knew the structure of the ribosome. We didn't know what the intermediates look like along the way."

In this study, the researchers mixed together the E. coli RNA and proteins in a motorized syringe and then pushed the fluid stream through a flow cell at NSLS beamline X28C while performing a technique called x-ray footprinting. In this setup, the x-rays create a very reactive chemical (hydroxyl radical) that causes the RNA strand to break when it's exposed. However, the RNA is protected when it's tucked inside of the structure.

"It's a little like shooting a BB gun at the complex and seeing what parts get nicked off," Woodson said about the technique, which was fine-tuned for exploring ribosomes by Johns Hopkins researcher and lead author Adilakshmi Tadepalli. "If the protein covers up part of the RNA, it leaves a "footprint" where the RNA is not nicked. We varied the amount of time that we allowed the RNA and proteins to come together, which gave us a series of snapshots, or footprints, throughout the reaction."

The team learned two major things. First, the different parts of the subunit start to assemble all at the same time, rather than wait for one part of the structure to finish before proceeding. This process is extremely rapid.

"Even though we started observing the assembly just 20 milliseconds after the protein and RNA were mixed, we already missed a lot," Woodson said.

X-ray footprinting of 30S ribosome assembly. 16S rRNA was mixed with all 20 30S proteins to begin assembly. Exposure to x-rays (X28C, NSLS) creates hydroxyl radical (•OH) that nicks the RNA. The absence of nicks at certain locations tells which parts of the rRNA were folded and covered by proteins. Structure of E. coli 30 S ribosome (pdb 2avy) with proteins in yellow and rRNA in gray. RNA regions protected in the first 100 ms are in red.

The researchers also were very surprised to find that the "lock-and-key" model – a popular biological assembly mechanism that describes how one piece perfectly clicks into the other in just one step – is not followed in this process. Instead, in a process referred to as "induced fit," the proteins and RNA continuously adjust their structures, squishing like Play-Doh until they're in place.

"This was extremely unexpected and very interesting," Woodson said, adding that they'll continue to look for the induced fit model in other protein assemblies. "It goes against the common school of thought."

These RNA footprinting investigations started through a collaboration with Michael Brenowitz (Albert Einstein College of Medicine) and Mark Chance (Case Western Reserve University), and have been underway at the NSLS since the late 90s. This latest study is one of the most complicated to date, Woodson said.

"What's unique is being able to look at this assembly with such short times – about 1,000 times shorter than what's been done before," Woodson said.

In the future, the researchers hope to use x-ray footprinting to study ribosomes in a whole cell, rather than in a test tube.

Their results were published in the October 30, 2008 edition of Nature. In addition to Woodson and Tadepalli, Johns Hopkins researcher Deepti Bellur also contributed to the work.

Funding was provided by the National Institutes of Health (NIH). NSLS beamline X28C is operated by the Case Center for Synchrotron Biosciences, of Case Western Reserve University, and is supported by NIH.