Unraveling
Pseudoknots

LBL chemists in the Structural Biology Division, Ignacio Tinoco, Jr. and Ling Shen, have produced the first three-dimensional image of an RNA structure that plays a vital role in enabling retroviruses to replicate within cells. The structure, a double looped strand of RNA that forms what is called a "pseudoknot," was revealed to contain a bend in its shape that may serve as the site where key host proteins interact.

A retrovirus is a protein-coated packet of RNA that requires the chemicals of a host cell to make DNA from its RNA genome. When a retrovirus -- the most notorious of which is HIV -- invades a cell it synthesizes three enzymes (integrase, protease, and reverse transcriptase) that enable it to transform the host into a virus replication factory. The mechanism by which this enzyme synthesis is carried out is called "ribosomal frame-shifting" and involves a shift in the order in which the virus' RNA genetic code is read. Retroviruses all use a "minus-one" frameshift, which means the reading of the code starts one nucleotide from where it should. This controlled frameshifting enables the retrovirus to pack extra information into its genome.

The presence of adenosine (red) between the two stems of a pseudoknot creates a bend in the pseudoknot's shape that helps retroviruses to reproduce. Without the adenosine there is no bend, and the retrovirus cannot reproduce.

To understand how pseudoknots promote frameshifting, scientists need detailed structural information. Tinoco and Shen, working in collaboration with the UC San Francisco group of current NIH head Harold Varmus, used nuclear magnetic resonance (NMR) spectroscopy to produce a three-dimensional high resolution image of a 34-nucleotide pseudoknot that is known to cause high-efficiency frameshifting in the mouse mammary tumor virus. In NMR spectroscopy, atomic nuclei are identified and spatially located by their characteristic absorbance of radiowaves in a magnetic field. Tinoco has been using this technique to study RNA and in 1992 produced the first 3-D image of a stem-loop "hairpin," a common and highly stable RNA structural element with critical folding and protein-recognition properties. When the loops of a pair of hairpins are joined by base-pairing the combined structure becomes a pseudoknot. (The structure is only partially twisted, otherwise it would form a knot.)

In producing their NMR images, the Tinoco and Varmus research groups experimented with modifying the nucleotide sequences of their pseudoknots to determine which resulted in frameshifting and which did not. They discovered that the presence of the nucleotide adenosine at the junction where the two stems of the pseudoknot are connected creates a bend in the shape of the pseudoknot. In this form, the pseudoknot promotes high-efficiency (up to 20-percent) sequence frameshifting. If the adenosine is removed, a pseudoknot is still formed but no frameshifting occurs. The next step will be to find which ribosomal proteins recognize this bend and interact with it in order to frameshift. It may be possible, the researchers say, to design drugs that could fight retroviruses by binding to the pseudoknot and blocking frameshifting.

-- Lynn Yarris