Jeffrey S. Kieft Assistant Professor Ph.D., University of California, Berkeley, 1997 Phone: (303) 724-3257 jeffrey.kieft@uchsc.edu

RNA is arguably the most versatile of the biological macromolecules, participating in processes as diverse as catalyzing formation of the peptide bond (rRNA), sensing intracellular metabolite concentrations (riboswitches), controlling developmental decisions (miRNAs), packaging DNA (pRNA), serving as the template for translation (mRNA and viral RNAs), and many others. These functions depend on the three-dimensional structure of the RNA, placing RNA structure at the center of biological processes and many diseases. Efforts in the Kieft Lab focus on understanding the structure and function of complex biologically important RNAs, especially those involved in viral disease. Our research involves collaborations with several other groups on this campus as well as at other universities.

Some of the projects in the Kieft Lab:

1. Structure and function of viral IRES RNAs. Viral internal Ribosome entry sites (IRESes) are structured RNAs that initiate protein synthesis by a mechanism that is radically different from the mechanism used by the major of mRNAs in the host cell. IRESes are essential for infection in many medically and economically important viruses such as hepatitis C, hepatitis A, polio, foot-and-mouth disease, rhinovirus, coxsackievirus-B3, and HIV-1. Some cellular mRNAs also use IRESes, including mRNAs implicated in cancer and other diseases. IRESes are important in biology, but the molecular rules and interactions underlying this RNA structure-driven mechanism remain mysterious. How do IRESes work? What is the structure basis for their function? Could they be good drug targets?

We are using a variety of biochemical, biophysical, and structural methods to decipher the folded architecture of these IRES RNAs and relate that architecture to function. Among the IRESes we are interested in are those from hepatitis C virus and the Dicsitroviridae. Of particular interest to the lab is that fact that IRES RNAs seem to be dynamic - undergoing change in conformation as they interact with and manipulate the cell's protein making machinery. Recently, we solved the first high-resolution structure of a complete ribosome binding domain of an IRES RNA, giving us tremendous insight into how IRESes might operate and laying the foundation for continued study. (see movie).

2. tRNA-like structures from viral RNAs. Certain RNA viruses have evolved sequences on their 3' ends that resemble transfer RNA (tRNAs). These viral RNAs are aminoacylated by the host cell's enzymes. Despite the fact that these tRNA-like sequences (TLS) were discovered long ago, we know very little about them. Some of them have sequences and topologies that are very different from authentic tRNAs, yet they seem to mimic tRNA. How are these TLS RNAs recognized by the aminoacylation and translation machinery? What are the structures of these RNAs and how do they differ from authentic tRNAs? What can the mechanism of action tell us about "normal" translation? To address these goals, we are pursing biochemical, biophysical, and structural studies on TLS sequences obtained from various viral sources. We now know that in three dimensional space, the TLS folds in a manner similar to tRNAs, but with some differences. We have obtained crystals of a TLS RNA and therefore we will compare the high-resolution structure of this RNA to an authentic tRNA in the near future and we hope to also crystallize this RNA in complex with purified recombinant valyl-synthetase to understand how this viral RNA mimics authentic cellular tRNAs.


3. Development of RNA purification and RNA crystallography methods. An important component to our research is the development of new methods for RNA structural studies. Recently, we developed a new method to purify structural quantities of RNAs using an affinity-tag based system (in collaboration with R. Batey, UC Boulder). We are now in the process of improving the method and developing a second-generation system that we anticipate will make gel purification of RNAs obsolete and will allow RNA sequences to be screened for crystallization at a rate that was previously impossible. We are also developing a phasing module that will provide a means to incorporate heavy atoms into RNA structures, allowing "first-time, every-time" phasing of RNA crystal diffraction data. These techniques have the potential to change the way RNA structural studies are conducted.


4. Other RNAs. The lab is also pursuing the atomic-resolution structure and mechanistic understanding of several other interesting RNAs, including sequences critical for poliovirus replication and packing of DNA in phage. Again our approach is to combine x-ray crystallography with a mix of biochemical and biophysical experiments in order to not only obtain the structure of the RNA, but truly understand its function in the context of complex biological systems and its roles in the pathogenesis of the virus. In one case, this work is part of a close collaboration linking in vivo studies with structural studies, giving us the change to study how atomic-level structure affects viral pathogenesis in animals. In the future, we hope to explore other RNAs from other pathogenic viruses including Dengue, Yellow Fever, and West Nile.

Pfingsten, J.S., Costantino, D.A., & Kieft, J.S. Structural basis for cap-independent ribosome recruitment and manipulation by a viral IRES, submitted.

Keel, A. , Batey, R.T., & Kieft, J.S. Understanding and exploiting the rules of cation binding in the RNA major groove, in preparation.

Costantino, D.A., Pfingsten J.S., Keel, A. & Kieft, J.S. A conserved IRES RNA fold at the heart of ribosome recruitment and translation initiation, in preparation.

Nelson, B & Kieft, J.S., 80S ribosome formation by the hepatitis C virus internal ribosome entry site RNA, in preparation.

Pfingsten, J.A., & Kieft, J.S., Three-dimensional architecture of the class II Dicitroviridae IRESes: sequence diversity, a conserved fold, in preparation.

Kieft, J.S. & Pfingsten, J. (2005) Weapons in the molecular arms race (News and Views). Nat. Struct. Mol. Biol. 12, 938-939.

Costantino, D. & Kieft, J.S. (2005) A pre-formed compact ribosome-binding domain in the cricket paralysis-like virus IRES RNAs, RNA 11, 332-343.

*Kieft, J.S. & *Batey, R.T. (2004) A general method for rapid and nondenaturing purification of RNAs. RNA, 10, 995-998. *These authors contributed equally to this work

Kieft, J.S., Zhou, K., Grech, A., Jubin, R., & Doudna, J.A. (2002) Crystal structure of a four-way RNA junction from the HCV IRES, Nat. Struct. Biol. 9, 370-374.

Kieft, J.S., Grech, A., Adams, P. & Doudna, J.A. (2001). Mechanisms of internal ribosome entry in translation initiation. Cold Spring Harb Symp Quant Biol. 66, 277-283.

*Spahn, C.M.T., *Kieft, J.S., Grassucci, R.A., Pencek, P., Zhou, K., Doudna, J.A. & Frank, J. (2001). Hepatitis C virus IRES RNA induced changes in the conformation of the 40S subunit. Science 291, 1959-1962. *These authors contributed equally to this work

Rijnbrand, R., Bredenbeck, P.J., Haasnoot, P.C., Kieft, J.S., Spaan, W.J.M. & Lemon, S.M. (2001). The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs. RNA 7, 585-597.

Kieft, J.S. Zhou, K. Jubin, R. & Doudna, J.S. (2001). Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 7, 194-206.

Jubin, R., Vantuno, N.E., Kieft, J.S., Murray, M.G., Doudna, J.A., Lau, J.Y.N. & Baroudy, B.M. (2000). Hepatitis C virus internal ribosome entry site loop IIId contains a phylogenetically conserved GGG triplet essential for translation initiation and IRES folding. J. Virol. 74, 10430-10437.

Kieft, J.S., Zhou, K., Jubin, R., Murray, M.G., Lau, J.Y.N. & Doudna, J.A. (1999). The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold. J. Mol. Biol. 292, 513-529.

Kieft, J.S., & Tinoco, I. Jr. (1997). Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine. Structure 5, 713-721.