l6 ribozymes

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Self-splicing and Enzymatic Activity of an Intervening Sequence RNA From Tetrahymena (Nobel Lecture, T.R. Cech ) (1990) Angew. Chem. Int. Ed. Eng. 29, 759-768. Ribozymes.

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Schematic diagram of the Tetrahymena thermophila group I ribozyme secondary structure. The P, L, and J nomenclature is as described for Figure 4. The universally conserved catalytic core is inside the gray box, and the P5abc, P2-P2.1, and P9.1-P9.2 peripheral elements are outside the box. Lines with arrowheads show connectivities between different secondary structural elements. The site of 5' splicing is marked with an arrow.

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Figure 8. Active site of the Tetrahymena group I intron, showing the close positioning of several secondary structural elements. (Adapted from Reference 122.) Figure 7. The Tetrahymena ribozyme reaction. The 3' oxygen of an exogenous guanosine bound to the P7 helix attacks the phosphate adjacent to a universally conserved base pair with G-U wobble in P1. Magnesium ions inferred from sulfur or nitrogen derivatization to be directly involved in catalysis are shown. (Adapted from References 50 and 112.)

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The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in green. The substrate or exon portion is printed in black. Arrows indicate sites of cleavage by ribozymes. (A) Left, the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis. Right, the -shaped structure of the hammerhead ribozyme–substrate complex. (B–F) The two-dimensional structures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from Saccharomyces cerevisiae (ai 5) and escherichiathe ribozyme of RNase P from E.coli, respectively.

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Figure 1. The P4-P6 domain is a central component of the self-splicing group I intron from Tetrahymena thermophila. A schematic representation of the intron is shown above, with the phylogenetically conserved catalytic core shaded in gray.

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Figure 2. The P4-P6 crystal structure. (A) The molecule viewed in the same orientation as the secondary structure shown in Fig. 1. A bend of 150° at one end of the molecule allows helices of the conserved core (in light blue and in red) to pack against helices of the P5abc extension. Specific contacts occur between the P4 helix (light blue) and the A-rich bulge (orange), and between the tetraloop receptor (green) and the GAAA tetraloop (gold). (B) View of the structure from the side, facing the core. The domain is about one helix thick in this dimension (25 Å), except for the P5c helix and loop (gray). (C) Stereo representation of the structure from the opposite side (180° from Fig. 2A).

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Figure 4. The GAAA tetraloop-receptor interaction.

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Figure 1. The P4-P6 domain is a central component of the self-splicing group I intron from Tetrahymena thermophila. A schematic representation of the intron is shown above, with the phylogenetically conserved catalytic core shaded in gray.

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Figure 2. (A) View of an adenosine platform looking down the helix axis. N3 of the 5 A and N6 of the 3 A are within hydrogen bonding distance (2.8 to 3.4 Å). Closed arrow, N1 position of the 5 A, which is protected from dimethyl sulfate when the long-range contact is formed; open arrow, N1 position of the 3 A which shows variable dimethyl sulfate protection (see 18). (B) The adenosine platform in the tetraloop receptor; color scheme as in Fig. 1. (C) Stereo view from underneath the platform, looking up the helix axis.

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Figure 3. The different kinds of long-range interactions that occur near the adenosine platforms. At left and in the center, reciprocal interactions occur between L5c and J6/6a in the two molecules in the asymmetric unit of the crystal; at right, the tetraloop docks above the platform in the tetraloop receptor. The docking partner for each platform is shaded. Blue adenosines (circles) are accessible to dimethyl sulfate in the presence and absence of the docking partner associated with the platform. Green adenosines (squares) are protected from dimethyl sulfate modification when the docking partner is present. The noncanonical base pair below each platform is highlighted in red. RNA Tertiary Structure Mediation by Adenosine Platforms Jamie H. Cate, Anne R. Gooding, Elaine Podell, Kaihong Zhou, Barbara L. Golden, Alexander A. Szewczak, Craig E. Kundrot, Thomas R. Cech, Jennifer A. Doudna * Science (1996) 273, 1696-1699

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Figure 3. Possible catalytic functions of metal ions in the cleavage of a phosphodiester bond. Metal ions can act as (a) a general acid catalyst, (b) a general base catalyst, (c) a Lewis acid that stabilizes the leaving group, (d) a Lewis acid that enhances the deprotonation of the attacking nucleophile and (e) an electrophilic catalyst that increases the electrophilicity of the phosphorus atom. Figure 1. RNA cleavage by acid-base catalysis and two-metal ion catalysis. :B is a general base and H-A is a general acid. Thick dashed lines show direct inner-sphere coordination between divalent cations MA and MB and oxygen atoms. N represents a purine or pyrimidine base and R denotes chain continuation. (From Doherty and Doudna)

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The mechanism of cleavage by ribonuclease A. Two imidazole residues function as general acid–base catalysts. (B) The single-metal-ion mechanism proposed for cleavage by the hammerhead ribozyme. One metal ion binds directly to the pro-Rp oxygen and functions as a general base catalyst. (C) The double-metal-ion mechanism proposed for cleavage by the hammerhead ribozyme. Two metal ions bind directly to the 2'- and 5'-oxygens.

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The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in green. The substrate or exon portion is printed in black. Arrows indicate sites of cleavage by ribozymes. (A) Left, the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis. Right, the -shaped structure of the hammerhead ribozyme–substrate complex. (B–F) The two-dimensional structures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from Saccharomyces cerevisiae (ai 5) and escherichiathe ribozyme of RNase P from E.coli, respectively.

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Figure 2. Secondary and tertiary structure of a hammerhead ribozyme. Universally conserved bases are shown as letters and those referred to in the text are numbered. Dashed lines and dots show non–Watson-Crick interactions between bases; solid lines represent Watson-Crick base pairs. The site of cleavage is marked with an arrow. (Adapted from Reference 58.) Famous “U-turn” The role of metal ions in catalysis for the hammerhead is still controversial, but the answer appears to be “structural”.

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Figure 1 Sequence and reactions catalysed by the hairpin ribozyme. a, Reversible transesterification reaction catalysed by the ribozyme. Hypothetical acid, base and positive charge catalytic groups are indicated. b, Sequence and secondary structure of the crystallization construct. The RNA consists of a 92-nucleotide ribozyme and a 21-nucleotide substrate strand. In the latter, the 2'-OH adjacent to the scissile bond was substituted with a methoxy to inhibit cleavage. To facilitate crystallization27, a cognate site for the RNA-binding protein U1A was grafted at the distal end of stem B. This construct was decided on after screening 21 different RNA–U1A complexes. The RNA is numbered according to convention1. In the substrate strand, numbers of nucleotides in the 5' and 3' direction of the cleavage site (yellow arrow) are preceded by minus and plus signs, respectively. Ribozyme-strand nucleotides are numbered starting at the 5' terminus, skipping the grafted U1A-binding site. Lines with embedded arrows indicate the direction of the chains at the crossovers. Solid letters, wild-type sequence; outline letters, nucleotides added for crystallization. Thin lines indicate Watson–Crick pairs; black circles indicate non-canonical pairs involving at least one hydrogen bond between co-planar bases. Nucleotides that participate in a ribose zipper25 are connected by dotted lines. Nucleotides with C2'-endo ribose puckers (asterisk), those in the syn conformation (double asterisk), and the A-1 residue that carries the 2'-methoxy modification (triple asterisk) are indicated. The same nucleotide conformations are present in both molecules in the crystallographic asymmetric unit. Secondary structure of a minimal hairpin ribozyme construct. Conserved nucleotides within domains A and B are lettered. The cleavage site is at the arrow. The dashed line at the junction of the two domains can vary in sequence and length. Tertiary folding causes thetwo helical domains to pack side by side, stabilized by tertiary interactions between the conserved nucleotides in the internal loops. The cleavage site is proposed to pack against portions of the domain B internal loop, rendering it inaccessible to solvent. (Adapted from Reference 66.) From Doherty and Doudna (2000) Ann. Rev. Biochem. Rupert, PB and Ferre-D’Amare, AR (2001) Nature 410, 780-786.

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Figure 2 Architecture of the hairpin ribozyme. The backbone of the two RNA strands is depicted as ribbons; the nucleotide bases as sticks (colour code as in Fig. 1). Two tightly bound calcium ions are represented as green spheres. a, Stereoview of the docked conformation of the hairpin ribozyme. The scissile bond lies between the two yellow nucleotides, which are splayed apart. The U1A protein is shown as a grey ribbon. b, c, Views orthogonal to that in a, showing the path of the RNA chains in the four-helix junction, the crossing angle between stems A and B, and the approximately 30° bend at the site of the bulged nucleotides in stem B.

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Figure 1: Type I and Type II base triples. a, Adenosines that form Type I and Type II base triple interactions in the P4–P6 domain of the Tetrahymena group I intron2 are labeled in cyan and green, respectively. b, Examples of base triple Type I from P4–P (A184C109–G212) and H. marismortui 23S rRNA (A306U325–340) (ref. 11) and a Type I-like interaction involving protein and RNA components from the signal recognition particle (A39Ser 397–Arg 401–C62–A63) (ref. 22). The adenosine N1, C2, N3 and 2' hydroxyl (O2') groups interact with the entire minor groove surface of the base pairs, including both riboses. The strands C109 and U325. c, Examples of Type II base triples between adenosines and Watson-Crick C–G and Hoogsteen A–U base pairs in the P4–P6 domain. The adenosine N1, C2, N3 and 2' hydroxyl groups interact with one-half of the minor groove surface of the base pair. The adenosine is oriented locally antiparallel to the strand (containing G110 or U224) with which it primarily interacts. From: Doherty, Batey, Masquida, Doudna (2001) Nature Struct. Biol. 8, 339-343 Type I and Type II base triples. This is a big deal!

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Figure 3: Comparison of Type I/II base triples from different RNAs. a, Superposition of consecutive Type I/II base triples from the 23S rRNA from H. marismortui [PDB entry code 1FFK]11. The ribose-phosphate backbone of the helices (gold) from 10 individual examples of this interaction with nonidentical sequences were superimposed using LSQMAN33, with a r.m.s. deviation of 1.40 Å. Six individual structures from this family are shown in stereo, with the coloring scheme of the helix and adenosines consistent with Fig. 1. The adenosines from one structure have been colored red to emphasize that the two adenosines remain stacked, irrespective of their orientation relative to the helix minor groove. b, Surface representations of adenosines within Type I/II base triples docked into the minor groove from (left to right) the P4–P6 domain (the A183 and A184 mediated interaction)2, the hepatitis delta virus ribozyme4 and the L11 protein–RNA complex6. Nucleotides that stack upon the adenosines are included within the molecular surface of the P4–P6 and hepatitis delta virus figures. This figure was created with GRASP34. From: Doherty, Batey, Masquida, Doudna (2001) Nature Struct. Biol. 8, 339-343