Researchers unlock secret of RNA's versatility

December 9, 1994

By Lynn Yarris, [email protected]

Evidence is in that could help explain the variety of complex shapes that RNA--ribonucleic acid--assumes to carry out its many biological functions.

RNA is the workhorse of the genetic world, transcribing the coded instructions of DNA and assembling amino acids into proteins. It has been shown that chains of RNA can fold back on themselves and assume complex formations that enable them to perform their tasks. Until now, however, there has been little or no detailed information to explain RNA's amazing versatility.

Stephen Holbrook, a chemist in the Structural Biology Division, has found that uracil, one of the four types of nitrogenous "bases" that represent the letters of the genetic code, can pair off with any other letter, including itself. This contradicts the exclusive two-letter base-pairing pattern in DNA, which was first discovered by Nobel laureates Francis Crick and James Watson.

"Uracil can now be called the universal partner in RNA structure," says Holbrook. "That it can pair with any other base helps explain why RNA is so flexible in terms of how it interacts with itself and why, unlike DNA, it can take on so many different shapes." DNA has only one form--the double helix.

Determining the structure of biological macromolecules such as RNA requires that the material first be crystallized so that its atoms are firmly fixed in an orderly pattern. This pattern can then be identified by sending a beam of x-rays through the crystal (the x-rays are diffracted or scattered by the atoms). Whereas other researchers have used x-ray crystallography to determine protein structural shapes, Holbrook is one of the few to use it to study the structural shapes of RNA. He attributes this in part to the difficulty in synthesizing, purifying, and, ultimately, crystallizing RNA.

"Virtually all crystallizations of DNA have resulted from similar conditions of salt, buffer, precipitant, and additives, but these standard conditions are not successful for many of the different forms of RNA," says Holbrook. He and his research group have been experimenting with novel ways of crystallizing RNA, focusing on sequences of base-pairs that form structures known as "internal loops."

One of the first results of Holbrook's crystallization research was the direct observation of an RNA double helix formation that incorporated four unconventional base-pairs. Watson and Crick demonstrated that DNA's double helix is held together by chemical bonds formed between complementary pairs of bases. These complementary base-pairs are cytosine (C) and guanine (G), and adenine (A) and thymine (T). RNA has a similar structure, except that thymine is represented by uracil (U).

Watson-Crick base-pairs--C-G and A-T (or A-U in RNA)--were once thought to be the only arrangement possible in nature. Holbrook's crystals however, showed two uracil-guanine (U-G) and two uracil-cytosine (U-C) base pairs in the middle of the sequence. This mismatched pairing resulted in the formation of a stable RNA double helix in the crystal with just a slight shape distortion.

"The U-C base pairs were joined by only a single hydrogen bond (conventional base-pairs are joined by two or three bonds)," says Holbrook, "but were stabilized by the presence of numerous, tightly bound water molecules."

Holbrook subsequently determined the three-dimensional structure of an RNA molecule containing U-U base pairs. Unlike the U-G and U-C base pairs, the U-U partners formed two hydrogen bonds that were stable without the presence of tightly bound water molecules.

"Non-standard base pairs such as the U-G, U-C, and U-U partners we have observed are common in ribosomal RNA, viroids, messenger RNA, and retroviruses," says Holbrook. "Runs of these mismatched pairs in the middle of double helical RNA form internal loops."

To date, Holbrook's best x-ray crystallography images have come when using the facilities at the Stanford Synchrotron Radiation Laboratory. Although SSRL's resolution of about two angstroms provided him with "the clearest views ever obtained of RNA structures," it was still not as high as he would have liked. When the x-ray crystallography beamline opens at the Advanced Light Source, he will probably be one of its first users.

"The availability, proximity, and unique facilities for measurement of x-ray diffraction data at the ALS crystallography beam will allow us to collect data much faster and determine molecular structures which would not otherwise be feasible," Holbrook says.

With the improved resolution and other advantages of the ALS, Holbrook says he would like to tackle longer and more complex stretches of RNA. "If we know the shape of an RNA structure and can design molecules that will bind to it," he says, "we can then study and possibly control the function of that structure."


Amino acid--Any of a group of 20 molecules that combine to form proteins in living cells. A protein's structure and function is determined by the sequence of its amino acids.

Base pairs--Two nucleotides (i.e., cytosine and guanine, or adenine and thymine) that are joined by weak bonds. The bonds between base pairs hold together DNA's two strands into the shape of the double-helix.

DNA--Deoxyribonucleic acid, the double-stranded molecule in the shape of a double-helix that encodes the genetic information which determines the sequence of amino acids in protein synthesis.

Protein--A large molecule composed of chains of amino acids arranged in a specific sequence according to the instructions in the genetic code. Proteins are responsible for the structure, function, and regulation of living cells.

RNA--Ribonucleic acid, a molecule similar in structure to DNA that is found in the nucleus and cytoplasm of cells and plays an important role in protein synthesis and other vital chemical activities.

X-ray crystallography--A technique for determining the location of atoms in a crystal, based on the diffraction pattern created when a beam of x-rays is passed through the crystal.