Structural analysis of key intermediary between genetic code and protein synthesis required years of patient effort
The three-dimensional structure of transfer RNA (tRNA) was determined by Alexander Rich and coworkers at Massachusetts Institute of Technology in 1973, but it didn’t come easy. “The story of how you get there, from knowing nothing to knowing everything, is a lot of hard work,” says Rich, now a spry 89-year-old living in Woods Hole, Mass.
tRNA mediates translation of the genetic code into the amino acid sequence of a protein. It picks up amino acids inside cells and delivers them to the protein-assembling ribosome factory, where messenger RNA gene transcripts guide their incorporation into growing protein chains.
“We used to think amino acids bound directly to mRNA” before getting strung into proteins, Rich says. Then Francis H. C. Crick “got the idea that maybe there’s a small amino acid-bound molecule that binds to mRNA,” and that turned out to be tRNA. “Then the question was, How does this molecule carry out its function?”
Robert W. Holley of Cornell University and others predicted that the sequence of tRNA could be folded into a three-leaf cloverleaf pattern. But proving it by X-ray analysis was challenging.
Indeed, it took Rich and coworkers about three years to grow suitable crystals. The key turned out to be a polyamine called spermine, which they used to stabilize the tRNA molecule’s fold, making it possible to form a stable crystal lattice.
In the simple 3-D diffraction pattern of tRNA that Rich’s group obtained in 1971, they found evidence for the presence of a short helix consistent with Holley’s cloverleaf formulation.
To proceed further, Rich and coworkers needed to find an end-terminus of tRNA to properly “phase” the diffraction pattern, a process required to obtain detailed high-resolution structures. Analyzing an osmium salt of tRNA did the trick.
But solving the tRNA structure remained difficult. For example, “We had an antiquated data collection system we had developed ourselves, where you collect data point by point, one at a time,” Rich says. Using those data to trace the molecule’s folding pattern “was very slow and laborious.”
They finally succeeded in 1973 (Science, DOI: 10.1126/science.179.4070.285).
“We found the chain had unusual folding in which the cloverleaf shape was preserved but was folded over to form an L shape,” Rich says. “No one had anticipated that the molecule would be organized in this fashion.”
Later that year, Aaron Klug of the University of Cambridge and coworkers reported substantially the same structure, based on a similar years-long effort.
“We in the end could explain in great detail the chemistry of the tRNA molecule and the way its folding facilitated its biological behavior,” Rich says. But it took years of work to get to that point.—Stu Borman