Home Faculty and Research Faculty by Name Jennifer A. Doudna

Jennifer A. Doudna

Howard Hughes Investigator and Professor of Biochemistry and Molecular Biology

Lab Homepage: http://rna.berkeley.edu/

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Research Interests

RNA molecules are uniquely capable of encoding and controlling the expression of genetic information, often as a consequence of their three-dimensional structures. We are interested in understanding RNA-mediated initiation of protein synthesis, and RNA-protein complexes involved in targeting proteins for export out of cells. We are also investigating the early steps in gene regulation by RNA interference.

Current Projects

Internal Ribosome Entry Site (IRES) RNAs

Most eukaryotic and viral messages initiate translation by a mechanism involving recognition of a 7-methylguanosine cap at the 5' end of the mRNA. In a few cases, however, translation occurs via a cap-independent mechanism in which an Internal Ribosome Entry Site (IRES) in the 5' untranslated region of the mRNA recruits the ribosome. In Hepatitis C virus (HCV), the ~400 nucleotide IRES folds into a magnesium-dependent structure in which loops thought to interact with the ribosome are exposed on the surface of the RNA. Point mutations that destroy IRES activity disrupt the folded structure of the RNA. The IRES is formed from two independently folding structural domains. One of these, the "core", binds specifically to the 40S subunit of eukaryotic ribosomes, while the other domain interacts with initiation factor eIF3. Structures of the IRES-40S subunit complex, determined by cryo-electron microscopy, revealed that the IRES induces a significant conformational change in the 40S subunit upon binding. This conformational change helps lock the start of the viral mRNA protein coding sequence into the correct site on the 40S subunit.

We determined structures of the HCV IRES in complex with the human translational machinery, showing how the IRES can functionally replace proteins that help position most cellular mRNAs on the ribosome. Using affinity purified samples, mass spectrometry has revealed the full composition and post-translational modification states of IRES-bound complexes that assemble in human cell extracts. We are analyzing intact IRES-ribosome complexes by mass spectrometry to determine how the HCV IRES induces assembly of active human 80S ribosomes.

In related experiments, we have discovered a class of IRES RNA sequences in yeast that activate translation of genes in response to starvation, enabling protein expression under conditions where most mRNAs are translationally silenced. Unlike viral IRESs, the yeast IRESs do not require three-dimensional RNA structures to recruit the ribosome, although recent data hint that RNA structure might regulate IRES activity. Exciting experimental data show that yeast IRES function is essential for invasive growth, the developmental pathway that haploid yeast enter in response to starvation. Current work focuses on elucidating the mechanism of these cellular IRES RNAs, which appears to be distinct from that of viral IRES elements, and determining whether this kind of RNA-mediated translational control is conserved in higher eukaryotes.

Structure and Mechanism of the Signal Recognition Particle (SRP)

The signal recognition particle (SRP) is a highly conserved ribonucleoprotein responsible for transport of nascent polypeptides targeted for secretion or membrane insertion. In prokaryotes, the SRP consists of one protein (Ffh) and one RNA molecule (4.5S RNA), and both are required for SRP activity. The RNA sequence corresponding to the Ffh binding site has been maintained through evolution, and is virtually identical in organisms from the three kingdoms of life - bacteria, archaea and eukaryotes. The RNA plays a key, yet undetermined, role in the protein targeting pathway. In 2000 we determined the crystal structure of the complex at 1.5 Å resolution, revealing a fascinating network of contacts at the RNA-protein interface that explain the observed evolutionary conservation. Using site-directed hydroxyl radical probing, we discovered that the association of the SRP with its receptor triggers a dramatic conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide binding site at the SRP-receptor heterodimer interface. The orientation of the RNA explains how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation. Recent experiments show that the position of the SRP RNA within the SRP-receptor complex enhances the rate of GTP hydrolysis in the complex above a critical threshold required in vivo. Work towards a crystal structure of the SRP-receptor complex (a ~130 kD assembly) has been aided by the selection and purification of multiple antibody proteins using phage display technology.

RNA Recognition by Dicer Enzymes

Double-stranded RNA induces potent and specific gene silencing in a broad range of eukaryotic organisms. This mode of gene silencing, called RNA interference (RNAi), acts at the transcriptional level through formation of heterochromatin and at the post-transcriptional level through mRNA degradation and translational suppression. In all cases, RNAi begins with the processing of endogenous or introduced precursor RNA into micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) 21-25 nucleotides in length by the enzyme Dicer. We recently solved the crystal structure of an intact Dicer enzyme, revealing how Dicer functions as a molecular ruler to measure and cleave duplex RNAs of a specific length. The structure has now been refined to higher resolution, and a series of mutant forms of Dicer have been used to delineate the roles of various domains and interactions both in vitro and in vivo. Ongoing work focuses on determining how Dicer interacts with other components of the RNAi pathway and how diced RNAs are targeted to specific mRNAs.

Selected Publications

Doudna, J.A. and Sarnow, P. (2007) Translation initiation by viral internal ribosome entry sites. In Translational Control in Biology and Medicine (Mathews, M.B., Sonenberg, N. and Hershey, J.W.B., Eds.), Cold Spring Harbor Monograph 48, pp. 129-153.

Kiessling, L.L., Doudna, J.A., Johnsson, K., Mapp, A.K., Marletta, M.A., Seeberger, P.H., Williamson, J.R. and Wedde S.G. (2007) A higher degree of difficulty. ACS Chem Biol. 2, 197-199.

Ke, A., Ding, F., Batchelor, J.D. and Doudna, J.A. (2007) Structural roles of monovalent cations in the HDV ribozyme. Structure. 15, 281-287.

Macrae, I.J. and Doudna, J.A. (2007) Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Curr. Opin. Struct. Biol. 17, 1-8.

Fraser, C.S. and Doudna, J.A. (2007) Structural and mechanistic insights into hepatitis C viral translation initiation. Nat. Rev. Microbiol. 5, 29-38.

Macrae, I.J., Li, F., Zhou, K., Cande, W.Z. and Doudna, J.A. (2006) Structure of dicer and mechanistic implications for RNAi. Cold Spring Harb. Symp. Quant. Biol. 71, 73-80.

Siu, F.Y., Spanggord, R.J. and Doudna, J.A. (2006) SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting. RNA 13, 240-250.

Green, R. and Doudna, J.A. (2006) RNAs regulate biology. ACS Chem. Biol. 1, 335-338.

Wu, S., Ke, A. and Doudna, J.A. (2006) A fast and efficient procedure to produce scFvs specific for large macromolecular complexes. J. Immunol. Methods 318, 95-101.

Chmiel, N.H., Rio, D.C., and Doudna, J.A. (2006) Distinct contributions of KH domains to substrate binding affinity of Drosophila P-element somatic inhibitor protein. RNA 12, 283-291.

MacRae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.Z., Adams, P.D., and Doudna, J.A. (2006) Structural basis for double-stranded RNA processing by Dicer. Science 311, 195-198.

Karbstein, K. and Doudna, J.A. (2006) GTP-dependent Formation of a Ribonucleoprotein Subcomplex Required for Ribosome Biogenesis. J. Mol. Biol. 356, 432-443.

Doudna, J.A. (2005) Chemical biology at the crossroads of molecular structure and mechanism. Nat. Chem. Biol. 1, 300-303.

Siridechadilok, B., Fraser, C.S., Hall, R.J., Doudna, J.A. and Nogales, E. (2005) Structural roles for human translation factor eIF3 in initiation of protein synthesis. Science 310, 1513-1515.

Karbstein, K., Jonas, S. and Doudna, J.A. (2005) An Essential GTPase Promotes Assembly of Preribosomal RNA Processing Complexes. Mol. Cell 20, 633-643.

Spanggord, R.J., Siu, F., Ke, A. and Doudna, J.A. (2005) RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle. Nat. Struct. Mol. Biol. 12, 1116-1122.

Chen, L., Lullo, D.J., Ma, E., Celniker, S.E., Rio, D.C. and Doudna, J.A. (2005) Identification and analysis of U5 snRNA variants in Drosophila. RNA 11, 1473-7.

Yu, Y., Ji, H., Doudna, J.A. and Leary, J.A. (2005) Mass spectrometric analysis of the human 40S ribosomal subunit: Native and HCV IRES-bound complexes. Protein Sci. 14, 1438-1446.

Ji, H., Fraser, C.S., Yu, Y., Leary, J. and Doudna, J.A. (2004) Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA. PNAS 101, 16990-16995.

Ke, A. and Doudna, J.A. (2004) Crystallization of RNA and RNA-protein complexes. Methods, 34, 408-414.

Rambo, R.P. and Doudna, J.A. (2004) Assembly of an active group II intron-maturase complex by protein dimerization. Biochemistry, 43, 6486-6497.

Luptak, A. and Doudna, J.A. (2004) Distinct sites of phosphorothioate substitution interfere with folding and splicing of the Anabaena group I intron. Nucleic Acid Res., 32, 2272-2280.

Sagar, M.B., Lucast, L. and Doudna, J.A. (2004) Conserved but nonessential interaction of SRP RNA with translation factor EF-G. RNA, 10, 772-778.

Ke, A., Zhou, K., Ding, F., Cate, J.H.D. and Doudna, J.A. (2004) A conformational switch controls hepatitis delta virus ribozyme catalysis. Nature, 429, 201-205.

Fraser, C.S., Lee, J.Y., Mayeur, G.L., Bushell, M., Doudna, J.A. and Hershey, J.W. (2003) The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40S ribosomal subunits in vitro. J. Biol. Chem. 279, 8946-8956.

Last Updated 2007-07-28