Large RNA Molecules
Three-dimensional structural studies of large RNAs (>50 nucleotides) are a particularly difficult task, notes Michael Summers, Ph.D., Howard Hughes professor at the University of Maryland, Baltimore. “Historically, x-ray crystallography has been the approach of choice for investigating the 3-D structure of biomolecules, but RNAs do not yield easily to this technology. Largve RNAs are negatively charged on their exterior and tend to be heterogeneous in terms of their conformation, all of which means they don’t form good crystals.”
RNAs are quite a different target as compared to protein molecules, which are much more easily analyzed through crystallographic techniques. In national data banks there are many more protein structures available than large RNA structures. To expand this slim catalog, one could employ nuclear magnetic resonance for the analysis, but this is also a problematic approach for the large RNAs.
While proteins contain approximately 20 amino acids, there are only four basic nucleotide building blocks in RNA. This means that while the spectral NMR signals are well defined for protein molecules, they crowd together for the RNA molecules, with much overlapping, making the data difficult to interpret.
In nucleic acid NMR investigations, one must rely on aromatic signals for information since the repeated ribose molecule is too homogeneous. Because of the reliance on these ring structures, the use of C13 isotopes is not effective. For this reason, Dr. Summers’ group turned to another strategy.
“We rely on an older technique, that is, using 2-D spectra with duterated nucleotides,” continues Dr. Summers. “We can duterate specific nucleotides to improve our resolution. NMR is very good for obtaining local structural information regarding the location of hydrogen atoms. This works well with proteins, but with nucleic acids it is more difficult, since the hydrogen atoms are sequestered in the middle of the helices and not easily resolvable. So we combine the high-resolution NMR with low-resolution global structural data from cryoelectron tomography.”
Dr. Summers is particularly concerned with how viral genomes assemble in the cell, and for addressing the replication cycle of HIV, a knowledge of the 3-D relationships of the RNA and protein molecules is essential. Dr. Summers argues that the regulation of the assembly process is guided by a specific, highly conserved region within the RNA genome. Antiretroviral drug design depends on a detailed understanding of the 3-D structure of the HIV RNA and protein molecules.
“At present, we don’t know how the assembly process works at the atomic level. However, we are aware that all of the regulation of replication is guided by genes in the 5´ untranslated portion of the genome,” Dr. Summers adds.
It is known that during the process of retrovirus assembly, RNA plays many different roles, as it must be spliced and packaged in a variety of ways in order to produce functional viral particles. Dr. Summers’ investigations are focused on basic science at this point, but it is clear that in the future these finding will contribute to new therapeutics for the control of RNA viral-based diseases.