BERKELEY, CA -- Chemists with the Lawrence Berkeley Laboratory and the University of California at Berkeley 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. It may be possible, the researchers say, to design drugs that could fight retroviruses by binding to the pseudoknot at this site and blocking these interactions.
This research was reported in the April 14 issue of the Journal of Molecular Biology in a paper co-authored by Ignacio Tinoco, Jr., with LBL's Structural Biology Division and the UCB Chemistry Department, and his student, Ling X. Shen.
A retrovirus is a protein-coated packet of RNA (ribonucleic acid) that requires the chemicals of a host cell to make viral DNA and proteins 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 frameshifting" and involves a shift in the order in which the virus' RNA genetic code is read. Retroviruses use a "minus-one" frameshift, which means the reading of the code starts one nucleotide from where it should.
"The efficiency of frameshifting is modulated by messenger RNA structures such as a pseudoknot downstream of the frameshift sight," says Tinoco. "The minus-one frameshifting translational mechanism allows controlled synthesis of viral enzymes and structural proteins."
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 is one of the few researchers to use NMR to study RNA which is the workhorse of the genetic world, transcribing the coded instructions of DNA and assembling amino acids into proteins. In 1992, Tinoco led a research team that 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 loop of a stem-loop hairpin pairs with a complementary sequence outside the loop to form a second stem, the resulting structure becomes a pseudoknot," says Tinoco. The structure is only partially twisted, otherwise it would form a knot.
From their NMR images, Tinoco and Shen discovered that the presence of the nucleotide adenine at the junction of the two stems of the pseudoknot derived from the mouse mammary tumor virus creates a bend in the shape of the pseudoknot. Subsequently, the Tinoco and Varmus research groups experimented with modifying the pseudoknot's nucleotide sequences. The idea was to find out which sequences resulted in frameshifting and which did not.
They found that with the bend in its shape, the pseudoknot promotes high-efficiency (up to 20-percent) frameshifting. If the intervening adenine nucleotide is removed, a pseudoknot is formed without a bend and no frameshifting occurs. The next step will be to find which ribosomal proteins recognize this bend and interact with it in order to frameshift.
"Our NMR data indicate that there are internal dynamics associated with the pseudoknot," says Tinoco. "The unique, compact structure and conformational flexibility of the pseudoknot may be required for recognition and favorable interaction with the translating ribosome, or with the translation factors associated with the ribosome."
LBL is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California.