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 one explanation for the abundance of adenosine residues in internal loops of many RNAs.

The sarcin/ricin loop, a modular RNA.

The conformation of a 29 base oligonucleotide called E73 has been determined in solution by NMR. E73 includes a 23 nucleotide sequence that is identical with that of a the alpha-sarcin and ricin-sensitive loop (SRL) from rat 28 S rRNA, and like the SRL in intact ribosomes, E73 is a substrate for both toxins. The SRL includes a long, conserved sequence found in the RNA of all large ribosomal subunits, which plays a critical role in the factor-dependent steps of protein synthesis. The spectroscopic observations and analysis that led to the determination of the conformation of E73 are presented. The SRL in E73 has a highly structured conformation, which is stabilized by several non-Watson-Crick base-pairs, and many properties of the SRL in the ribosome can be understood assuming that the conformation of E73 and that of the SRL in the ribosome are the same. The role of the SRL in protein synthesis is discussed in light of the conformation of E73, as is the modular relationship that exists between the conformation of the SRL and other smaller RNAs.

Assignment of NH resonances in nucleic acids using natural abundance 15N-1H correlation spectroscopy with spin-echo and gradient pulses.

It is well known that 2D 15N-1H correlation spectra can resolve overlapped imino proton resonances in the downfield NMR spectra of nucleic acids according to their 15N chemical shifts, and that these resonances can be assigned by base type on that basis, independent of conformation. This information can be extremely important in determining the solution structure of a nucleic acid by NMR, but previously could only be obtained using 15N-labeled, or very concentrated samples. Here we report the design of a gradient-enhanced, jump-return spin echo version of an 15N-1H HMQC experiment (GE-JRSE HMQC) that is sensitive enough to work on unlabeled nucleic acid samples at normal NMR concentrations. This experiment has led to the assignment of imino proton resonances with non-Watson Crick chemical shifts in the spectrum of a 29 residue oligoribonucleotide that models the sarcin/ricin loop from 28S rRNA.

On the conformation of the alpha sarcin stem-loop of 28S rRNA.

A synthetic RNA that is a substrate for the cytotoxin alpha sarcin has been examined by NMR. The molecule in question includes the entire sequence of the so-called alpha sarcin loop from rat 28S rRNA (U4316-C4332), and it is cleaved at the residue that corresponds to G4325, the site of alpha sarcin cleavage in 28S rRNA. The data show that the terminal stem designed into the molecule's sequence exists, as expected, and that its loop has a definite structure, which is stable to at least 40 degrees C under ionic conditions compatible with its cleavage by alpha sarcin.

An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis.

RNA molecules commonly consist of helical regions separated by internal loops, and in many cases these internal loops have been found to assume stable structures. We have examined the function and dynamics of an internal loop, J5/5a, that joins the two halves of the P4-P6 domain of the Tetrahymena self-splicing group I intron. P4-P6 RNAs with mutations in the J5/5a region showed nondenaturing gel electrophoretic mobilities and levels of Fe(II)-EDTA cleavage protection intermediate between those of wild-type RNA and a mutant incapable of folding into the native P4-P6 tertiary structure. Mutants with the least structured J5/5a loops behaved the most like wild-type P4-P6, and required smaller amounts of Mg2+ to rescue folding. The activity of reconstituted introns containing mutant P4-P6 RNAs correlated similarly with the nature of the J5/5a mutation. Our results suggest that, in solution, the P4-P6 RNA is in a two-state equilibrium between folded and unfolded states. We conclude that this internal loop mainly acts as a flexible hinge, allowing the coaxially stacked helical regions on either side of it to interact via specific tertiary contacts. To a lesser extent, the specific bases within the loop contribute to folding. Furthermore, it is crucial that the junction remain unstructured in the unfolded state. These conclusions cannot be derived from a simple examination of the P4-P6 crystal structure (Cate JH et al., 1996, Science 273:1678-1685), showing once again that structure determination must be supplemented with mutational and thermodynamic analysis to provide a complete picture of a folded macromolecule.

A minor groove RNA triple helix within the catalytic core of a group I intron.

Close packing of several double helical and single stranded RNA elements is required for the Tetrahymena group I ribozyme to achieve catalysis. The chemical basis of these packing interactions is largely unknown. Using nucleotide analog interference suppression (NAIS), we demonstrate that the P1 substrate helix and J8/7 single stranded segment form an extended minor groove triple helix within the catalytic core of the ribozyme. Because each triple in the complex is mediated by at least one 2'-OH group, this substrate recognition triplex is unique to RNA and is fundamentally different from major groove homopurine-homopyrimidine triplexes. We have incorporated these biochemical data into a structural model of the ribozyme core that explains how the J8/7 strand organizes several helices within this complex RNA tertiary structure.

On the use of T7 RNA polymerase transcripts for physical investigation.

A few years ago we made some observations which raised questions about the accuracy with which T7 RNA polymerase transcribes templates in vitro, and the suitability of its in vitro products for biophysical study (1). The experiments described below demonstrate that there is no reason for concern; the products of T7 RNA polymerase transcription in vitro are as suitable for biophysical characterization as RNAs synthesized in vivo. It is likely that aggregation involving the transcribed portions of the T7 RNA polymerase promoter caused our initial observations.

An NMR characterization of the regA protein-binding site of bacteriophage T4 gene 44 mRNA.

The conformations of two RNA dodecamers that differ markedly in affinity for the regA protein from bacteriophage T4 have been examined by NMR to see if the ability of that protein to discriminate between mRNAs is based on pre-existing differences in their three-dimensional structures. One of the RNAs examined has the same sequence as the site where regA protein binds when it inhibits the expression of gene 44's mRNA. The second RNA differs from the first in having a U instead of a G at position -9; it binds regA protein 100 times less tightly. The NMR data indicate that both RNAs have similar single-stranded conformations and that they each resemble an isolated strand of a double helix. They also show that most, if not all of the ribose rings in both molecules have appreciable 2'-endo puckering. It is unlikely that regA protein distinguishes between these two molecules on the basis of differences in their global conformations in solution.

The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme.

Adenosines are present at a disproportionately high frequency within several RNA structural motifs. To explore the importance of individual adenosine functional groups for group I intron activity, we performed Nucleotide Analog Interference Mapping (NAIM) with a collection of adenosine analogues. This paper reports the synthesis, transcriptional incorporation, and the observed interference pattern throughout the Tetrahymena group I intron for eight adenosine derivatives tagged with an alpha-phosphorothioate linkage for use in NAIM. All of the analogues were accurately incorporated into the transcript as an A. The sites that interfere with the 3'-exon ligation reaction of the Tetrahymena intron are coincident with the sites of phylogenetic conservation, yet the interference patterns for each analogue are different. These interference data provide several biochemical constraints that improve our understanding of the Tetrahymena ribozyme structure. For example, the data support an essential A-platform within the J6/6a region, major groove packing of the P3 and P7 helices, minor groove packing of the P3 and J4/5 helices, and an axial model for binding of the guanosine cofactor. The data also identify several essential functional groups within a highly conserved single-stranded region in the core of the intron (J8/7). At four sites in the intron, interference was observed with 2'-fluoro A, but not with 2'-deoxy A. Based upon comparison with the P4-P6 crystal structure, this may provide a biochemical signature for nucleotide positions where the ribose sugar adopts an essential C2'-endo conformation. In other cases where there is interference with 2'-deoxy A, the presence or absence of 2'-fluoro A interference helps to establish whether the 2'-OH acts as a hydrogen bond donor or acceptor. Mapping of the Tetrahymena intron establishes a basis set of information that will allow these reagents to be used with confidence in systems that are less well understood.

Thermodynamic stability of the P4-P6 domain RNA tertiary structure measured by temperature gradient gel electrophoresis.

The P4-P6 domain RNA from the Tetrahymena self-splicing group I intron is an independent unit of tertiary structure that, in the kinetic folding pathway, folds before the rest of the intron and then stabilizes the remainder of the intron's tertiary structure. We have employed temperature gradient gel electrophoresis (TGGE) to examine the unfolding of the tertiary structure of P4-P6. In 0.9 mM Mg2+, the global tertiary fold of the molecule has a melting temperature of approximately 40 degreesC and is completely unfolded by 60 degreesC. Calculated thermodynamic parameters for folding of P4-P6 are DeltaH degrees' = -28 +/- 3 kcal/mol and DeltaS degrees' = -91 +/- 8 eu under these conditions. Chemical probing of the P4-P6 tertiary structure using dimethyl sulfate and CMCT confirms that these TGGE experiments monitor the unfolding of the global tertiary fold of the domain and that the secondary structure is largely unaffected over this temperature range. Thus, unlike the entropically driven P1 docking and guanosine binding steps of Tetrahymenagroup I intron self-splicing, which have positive or zero DeltaH terms, P4-P6 tertiary structure formation is stabilized by a negative DeltaH term. This implies that enthalpically favorable hydrogen bond formation, nucleotide base stacking, and/or binding of Mg2+ within the folded structure are responsible for stabilizing the P4-P6 domain.

The conformation of the sarcin/ricin loop from 28S ribosomal RNA.

The sarcin/ricin loop is a highly conserved sequence found in the RNA of all large ribosomal subunits. The cytotoxins alpha-sarcin and ricin both inactivate ribosomes by cleaving a single bond in that loop. Once it has been attacked, ribosomes no longer interact with elongation factors properly, and translation stops. We have determined the conformation of the sarcin/ricin loop by multinuclear NMR spectroscopy using E73, a 29-nucleotide RNA that has the sarcin/ricin loop sequence and that is sensitive to both toxins in vitro. The sarcin/ricin loop has a compact structure that contains several purine.purine base pairs, a GAGA tetraloop, and a bulged guanosine adjacent to a reverse Hoogsteen A.U pair. It is stabilized by an unusual set of cross-strand base-stacking interactions and imino proton to phosphate oxygen hydrogen bonds. In addition to having interesting structural features, this model explains many of the biochemical observations made about the loop's structure and its reactivity with cytotoxins, and it sheds light on the loop's interactions with elongation factors.

An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.