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RNA Structure and RNA-Protein Recognition

J.R. Williamson, S. Agalarov,* I. Baxter, R. Burris, C.D. Cilley, K.T. Dayie, P. Funke, H. Mao, J.W. Orr, P.K. Radha, M.S. Rook, L.G. Scott, D.K. Treiber, S.A. White**

* Institute of Protein Research, Pushchino, Russia
** Bryn Mawr College, Bryn Mawr, PA

At many levels, regulation of gene expression involves the formation of RNA-protein complexes. Transcription of DNA to give premessenger RNA, processing of premessenger RNA to messenger RNA, and translation of the messenger RNA into protein all involve RNA-protein complexes. These complexes often increase or decrease the rate of a particular step and so regulate expression of the gene. In addition, the large molecular assemblies that carry out processing and translation are themselves RNA-protein complexes. The RNA in these complexes can have a structural or a catalytic role, or possibly both. Despite the fundamental importance of these complexes, little is understood about their structures. We are applying a broadly based biochemical and biophysical approach to better understand RNA structure and RNA-protein interactions in several key systems.

INTERACTION OF RIBOSOMAL PROTEIN S15 WITH RIBOSOMAL RNA

The bacterial ribosome is composed of a large 50S subunit and a small 30S subunit. The small subunit is composed of 21 small proteins and a 1500-nucleotide 16S rRNA. Four of the 21 proteins can bind independently to the rRNA, and these 4 proteins initiate the cascade of protein binding for ribosomal assembly. The protein S15 is one of these key primary binding proteins.

S15 is 88 residues long, and the minimal binding site in 16S rRNA is a 3-helix junction (Fig. 1). Using transient electric birefringence methods, we showed that the 3 arms of this RNA undergo a dramatic conformational change upon binding of the protein and that this change in the structure of the rRNA allows the second wave of ribosomal proteins to bind. We are using nuclear magnetic resonance (NMR) spectroscopy and x-ray crystallography to investigate the 3-dimensional structure of this RNA-protein complex.

INTERACTION OF RIBOSOMAL PROTEIN L32 WITH MESSENGER RNA

The yeast ribosomal protein L32 can regulate its own expression by binding to RNA sequences at the 5´ end of its messenger RNA. Binding to these sequences blocks access to the site necessary for splicing of the messenger RNA and thus inhibits expression. Using NMR spectroscopy, we determined the 3-dimensional structure of L32 bound to a 30-nucleotide fragment from the messenger RNA site (Fig. 2).

L32 is a sandwich composed of a 4-stranded ß-sheet between 2 sets of -helices. The RNA-binding face of L32 is composed of 3 loops at 1 end of the protein that interact with bases from the RNA. One loop inserts into the major groove of the RNA, and 2 other loops form binding sites for 2 of the RNA bases. The RNA structure involves formation of unusual nonstandard base pairs that bend the RNA and present 3 nucleotides to the protein for specific recognition. One of these 3 nucleotides is inserted into a hydrophobic pocket on the protein; the other 2 are involved in extensive hydrogen-bonding interactions with the protein. This complex is the first structure of a ribosomal protein--RNA complex and reveals new principles of RNA-protein recognition.

ISOTOPE LABELING METHODS FOR NMR OF RNA

The use of stable isotopic labels for NMR studies has revolutionized NMR of proteins. However, until recently, these same methods could not be applied to RNA molecules because molecules with the appropriate isotopic labels were not available. We developed the first biosynthetic methods for uniform labeling of RNAs, and we are continuing to develop new methods. Recently, we developed methods that enable us to synthesize labeled nucleotides completely in vitro by using enzymes of the metabolic pathways. With these methods, we can efficiently prepare a wide variety of labeling patterns that were not previously available, and we are developing new NMR experimental methods that take advantage of these labeling strategies. These methods are crucial to enable us to determine the structure of larger RNAs and RNA-protein complexes of biological importance.

RNA-PROTEIN INTERACTIONS IN HIV

Two key regulatory proteins in HIV, Tat and Rev, bind to RNA structural elements to regulate the expression of HIV genes. Previously, using NMR spectroscopy, we determined the structure of the complex formed by Rev and its RNA-binding site. Recently, we determined the high-resolution structure of the binding site for the Tat protein, which is an RNA element called TAR. The TAR RNA binds to the small molecule argininamide, which is a Tat analog that occupies the binding site on TAR for one of the arginine side chains from Tat. The RNA folds up into an arginine binding pocket, where the arginine is sandwiched between 2 bases, and the edge of the arginine is involved in hydrogen-bonding contacts to the RNA. This binding is facilitated by formation of a base triple interaction between a bulged base and a base pair on TAR. Studies of mutant TAR RNAs indicate that the structure of the base triple rather than the sequence of bases is important for argininamide and Tat binding.

PUBLICATIONS

Batey, R.T., Williamson, J.R. Effects of polyvalent cations on the folding of an rRNA three-way junction and binding of ribosomal protein S15. Nucleic Acids Res., in press.

Brodsky, A.S., Erlacher, H.A., Williamson, J.R. NMR evidence for a base triple in the HIV-2 CGC-mutant TAR argininamide complex. Nucleic Acids Res. 26:1991, 1998.

Brodsky, A.S., Williamson, J.R. Solution structure of the HIV-2 TAR-argininamide complex. J. Mol. Biol. 267:624, 1997.

Dayie, K.T., Tolbert, T.J., Williamson, J.R. 3D C(CC)H TOCSY experiment of assigning protons and carbons in uniformly ¹³C and selectively ²H labeled RNA. J. Mag. Reson. 130:97, 1997.

Orr, J.W., Hagerman, P.J., Williamson, J.R. Protein and Mg²⁺-induced conformational changes in the S15 binding site of 16S ribosomal RNA. J. Mol. Biol. 275:453, 1998.

Tolbert, T.J., Williamson, J.R. Preparation of specifically deuterated and ¹³C-labeled RNA for NMR studies using enzymatic synthesis J. Am. Chem. Soc. 119:12100, 1997.

Treiber, D.K., Rook, M.S., Zarrinkar, P.P., Williamson, J.R. Kinetic intermediates trapped by native interactions in RNA folding. Science 279:1943, 1998.

Zamore, P.D., Williamson, J.R., Lehmann, R. The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins. RNA 3:1421, 1997.