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

Abstract

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.

A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES

“…Initiation of protein synthesis within eukaryotic cells usually occurs by a cap-dependent mechanism in which the 59 terminal cap-structure (m 7 GpppN++) of the cytoplasmic mRNAs is recognized by the translation initiation complex eIF4F (reviewed by Merrick & Hershey, 1996)+ This complex is believed to migrate along the mRNA in association with the small ribosomal subunit until the initiation codon is reached, usually within 50-100 nt+ A second, distinct, cap-independent mechanism of translation initiation is employed by some cellular mRNAs (e+g+, BiP; Macejak & Sarnow, 1991) and the RNAs of picornaviruses (e+g+, poliovirus (PV), encephalomyocarditis virus (EMCV), and foot-and-mouth disease virus (FMDV); reviewed in Belsham & Sonenberg, 1996;Jackson & Kaminski, 1995)+ These viruses have positive-sense RNA genomes that act as mRNAs+ However their RNAs are uncapped and they are translated even when cap-dependent protein synthesis is inhibited following the cleavage of the eIF4G component of the eIF4F cap-binding complex+ A region of about 450 nt, near the 39 end of the long 59 noncoding region (600-1,300 nt in different picornaviruses), is required to achieve internal initiation of protein synthesis and this element is termed an internal ribosome entry site (IRES)+ Within the picornavirus family, there are two major classes of IRES element+ The cardioviruses (e+g+, EMCV) and aphthoviruses (FMDV) share one class of element, whereas the enteroviruses (e+g+, PV) and rhinoviruses share a second type of element+ These classes of IRES element have very different predicted secondary structures and also differ in their biology+ The cardio-/aphthovirus elements function efficiently in rabbit reticulocyte in vitro translation systems whereas the entero-/rhinovirus elements do not+ One common feature of the picornavirus IRES elements is the presence of a polypyrimidine tract near the 39 end of the element+ Kaminski et al+ (1994) also pointed out the presence of two conserved loop sequences within these IRES elements+ In each class, one loop fits the GNRA tetraloop consensus (where N is any nucleotide and R is a purine) whereas the second loop is C rich+ Within the cardio-/aphthoviruses, the GNRA tetraloop is located at the end of a stem-loop, within a hammerhead structure that constitutes part of the largest predicted domain (termed the I domain in the EMCV structure) of the secondary structure+ RNA tetraloops fitting the GNRA consensus are very highly represented within large RNAs with stable tertiary structures (Woese et al+, 1990)+ It is believed that such loops play an important role in RNA tertiary interactions (Pley et al+, 1994;Costa & Michel, 1995, 1997Cate et al+, 1996aCate et al+, , 1996b and also interactions with proteins (Glück et al+, 1992;Legault et al+, 1998)+ A single point mutation in the sequence of the EMCV IRES (nt 380-834 of the EMCV RNA) within this tetraloop (GCGA to GCGC, nt 547-550) severely reduces (.95%) the activity of the IRES element (Robe...…”

The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme

“…The recently reported crystal structure of the P4-P6 domain of the Tetrahymena group I intron has provided tremendous insight into the secondary and tertiary structures involved in RNA folding (Cate & Doudna, 1996;Cate et al+, 1996aCate et al+, , 1996bCate et al+, 1997)+ Many of the structural motifs important for domain folding involve adenosine-rich sequences+ These motifs are likely to be present in a wide variety of RNA structures+ One example is the adenosine-platform, which is observed at three different positions within the domain (Fig+ 1; FIGURE 1. Primary sequence and secondary structure of the L-21 G414 version of the Tetrahymena group I intron (Cech et al+, 1994)+ This ribozyme binds the oligonucleotide CCCUCdTAAAAA and transfers the AAAAA onto the 39 end of the intron in a reaction analogous to the reverse of the second step of splicing+ Numbering of the nucleotides discussed in the text is shown, as are the names of the helical (P1-P9) and single-stranded regions (J6/6a, J8/7, etc+) of the RNA+ The three A-platforms in P4-P6 are shown as adjacent A's with a heavy underline+ The long thin lines indicate regions known to make tertiary interactions within the three-dimensional structure+ Thick lines designate connectivity of the RNA strand+ Cate et al+, 1996b)+ This motif involves a side by side alignment of two consecutive A's to form a pseudobase pair that serves as a platform for tertiary stacking interactions+ In one of the three occurrences, the A-platform makes tertiary interactions with another example of an A-rich motif, the GAAA tetraloop frequently found at the end of RNA hairpin loops (Woese et al+, 1990)+ The A's in this and related GNRA tetraloops FIGURE 2.…”

Representation, searching and discovery of patterns of bases in complex RNA structures

“…Each adenosine of the GAAA tetraloop makes contact with part of the receptor, the specific interactions are detailed in Cate et al [12]. The interaction of the first adenosine in the tetraloop is with an A.U reverse Hoogsteen pair, with the adenosines forming an A.A N1-amino symmetric pair.…”

“…Hairpin loops, particularly tetraloops [12][13]), and internal loops [12] are common secondary structural motifs that also contribute significantly to RNA tertiary structural interactions. These elements then interact with each other through formation of base triples by hydrogen bonding and tetraloop-receptor interactions to stabilise the folding of the RNA molecule into its three-dimensional (3D) shape [12].…”

“…These elements then interact with each other through formation of base triples by hydrogen bonding and tetraloop-receptor interactions to stabilise the folding of the RNA molecule into its three-dimensional (3D) shape [12]. For a summary of structural motifs in RNA see Moore [14].…”