Jennifer A. Doudna

Crystal Structure of a Group I Ribozyme Domain: Principles of RNA Packing

Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction. The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these, the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved 11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome, and the ribosome.

Crystal structure of a hepatitis delta virus ribozyme

The self-cleaving ribozyme of the hepatitis delta virus (HDV) is the only catalytic RNA known to be required for the viability of a human pathogen. We obtained crystals of a 72-nucleotide, self-cleaved form of the genomic HDV ribozyme that diffract X-rays to 2.3u2009Å resolution by engineering the RNA to bind a small, basic protein without affecting ribozyme activity. The co-crystal structure shows that the compact catalytic core comprises five helical segments connected as an intricate nested double pseudoknot. The 5′-hydroxyl leaving group resulting from the self-scission reaction is buried deep within an active-site cleft produced by juxtaposition of the helices and five strand-crossovers, and is surrounded by biochemically important backbone and base functional groups in a manner reminiscent of protein enzymes.

The chemical repertoire of natural ribozymes

Although RNA is generally thought to be a passive genetic blueprint, some RNA molecules, called ribozymes, have intrinsic enzyme-like activity — they can catalyse chemical reactions in the complete absence of protein cofactors. In addition to the well-known small ribozymes that cleave phosphodiester bonds, we now know that RNA catalysts probably effect a number of key cellular reactions. This versatility has lent credence to the idea that RNA molecules may have been central to the early stages of life on Earth.

RNA Tertiary Structure Mediation by Adenosine Platforms

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides oneexplanation for the abundance of adenosine residues in internal loops of many RNAs.

Tertiary Motifs in RNA Structure and Folding

Specific tertiary structural motifs determine the complete architecture of RNA molecules (see picture for examples). Within the last few years a number of high-resolution crystal structures of complex RNAs have led to new insights into the mechanisms by which these complex folds are attained. In this review the structures of these tertiary motifs and how they influence the folding pathway of biological RNAs are discussed, as well as new developments in modeling RNA structure based upon these findings.

Mechanism of ribosome recruitment by hepatitis C IRES RNA

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5 untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

A universal mode of helix packing in RNA.

RNA molecules fold into specific three-dimensional shapes to perform structural and catalytic functions. Large RNAs can form compact globular structures, but the chemical basis for close helical packing within these molecules has been unclear. Analysis of transfer, catalysis, in vitro-selected and ribosomal RNAs reveal that helical packing predominantly involves the interaction of single-stranded adenosines with a helix minor groove. Using the Tetrahymena thermophila group I ribozyme, we show here that the near-perfect shape complementarity between the adenine base and the minor groove allows for optimal van der Waals contacts, extensive hydrogen bonding and hydrophobic surface burial, creating a highly energetically favorable interaction. Adenosine is recognized in a chemically similar fashion by a combination of protein and RNA components in the ribonucleoprotein core of the signal recognition particle. These results provide a thermodynamic explanation for the noted abundance of conserved adenosines within the unpaired regions of RNA secondary structures.

Metal-binding sites in the major groove of a large ribozyme domain

BACKGROUNDnGroup I self-splicing introns catalyze sequential transesterification reactions within an RNA transcript to produce the correctly spliced product. Often several hundred nucleotides in size, these ribozymes fold into specific three-dimensional structures that confer activity. The 2.8 A crystal structure of a central component of the Tetrahymena thermophila group I intron, the 160-nucleotide P4-P6 domain, provides the first detailed view of metal binding in an RNA large enough to exhibit side-by-side helical packing. The long-range contacts and bound ligands that stabilize this fold can now be examined in detail.nnnRESULTSnHeavy-atom derivatives used for the structure determination reveal characteristics of some of the metal-binding sites in the P4-P6 domain. Although long-range RNA-RNA contacts within the molecule primarily involve the minor groove, osmium hexammine binds at three locations in the major groove. All three sites involve G and U nucleotides exclusively; two are formed by G.U wobble base pairs. In the native RNA, two of the sites are occupied by fully-hydrated magnesium ions. Samarium binds specifically to the RNA by displacing a magnesium ion in a region critical to the folding of the entire domain.nnnCONCLUSIONSnBound at specific sites in the P4-P6 domain RNA, osmium (III) hexammine produced the high-quality heavy-atom derivative used for structure determination. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivatives with hexammine compounds. The features of the observed metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G.U base pairs in ribosomal RNAs are magnesium-binding sites.

A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor

Metal ions are essential for the folding and activity of large catalytic RNAs. While divalent metal ions have been directly implicated in RNA tertiary structure formation, the role of monovalent ions has been largely unexplored. Here we report the first specific monovalent metal ion binding site within a catalytic RNA. As seen crystallographically, a potassium ion is coordinated immediately below AA platforms of the Tetrahymena ribozyme P4-P6 domain, including that within the tetraloop receptor. Interference and kinetic experiments demonstrate that potassium ion binding within the tetraloop receptor stabilizes the folding of the P4-P6 domain and enhances the activity of the Azoarcus group I intron. Since a monovalent ion binding site is integral to the tetraloop receptor, a tertiary structural motif that occurs frequently in RNA, monovalent metal ions are likely to participate in the folding and activity of a wide diversity of RNAs.

CRYSTAL STRUCTURE OF AN RNA TERTIARY DOMAIN ESSENTIAL TO HCV IRES-MEDIATED TRANSLATION INITIATION

The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives internal initiation of viral protein synthesis during host cell infection. In the tertiary structure of the IRES RNA, two helical junctions create recognition sites for direct binding of the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). The 2.8 Å resolution structure of the IIIabc four-way junction, which is critical for binding eIF3, reveals how junction nucleotides interact with an adjacent helix to position regions directly involved in eIF3 recognition. Two of the emergent helices stack to form a nearly continuous A-form duplex, while stacking of the other two helices is interrupted by the insertion of junction residues into the helix minor groove. This distorted stack probably serves as an important recognition surface for the translational machinery.