Minor groove recognition of the conserved G.U pair at the Tetrahymena ribozyme reaction site.

The guanine-uracil (G.U) base pair that helps to define the 5'-splice site of group I introns is phylogenetically highly conserved. In such a wobble base pair, G makes two hydrogen bonds with U in a geometry shifted from that of a canonical Watson-Crick pair. The contribution made by individual functional groups of the G.U pair in the context of the Tetrahymena ribozyme was examined by replacement of the G.U pair with synthetic base pairs that maintain a wobble configuration, but that systematically alter functional groups in the major and minor grooves of the duplex. The substitutions demonstrate that the exocyclic amine of G, when presented on the minor groove surface by the wobble base pair conformation, contributes substantially (2 kilocalories.mole-1) to binding by making a tertiary interaction with the ribozyme active site. It contributes additionally to transition state stabilization. The ribozyme active site also makes tertiary contacts with a tripod of 2'-hydroxyls on the minor groove surface of the splice site helix. This suggests that the ribozyme binds the duplex primarily in the minor groove. The alanyl aminoacyl transfer RNA (tRNA) synthetase recognizes the exocyclic amine of an invariant G.U pair and contacts a similar array of 2'-hydroxyls when binding the tRNA(Ala) acceptor stem, providing an unanticipated parallel between protein-RNA and RNA-RNA interactions.

Site-specific cleavage of a yeast chromosome by oligonucleotide-directed triple-helix formation.

Oligonucleotides equipped with EDTA-Fe can bind specifically to duplex DNA by triple-helix formation and produce double-strand cleavage at binding sites greater than 12 base pairs in size. To demonstrate that oligonucleotide-directed triple-helix formation is a viable chemical approach for the site-specific cleavage of large genomic DNA, an oligonucleotide with EDTA-Fe at the 5' and 3' ends was targeted to a 20-base pair sequence in the 340-kilobase pair chromosome III of Saccharomyces cerevisiae. Double-strand cleavage products of the correct size and location were observed, indicating that the oligonucleotide bound and cleaved the target site among almost 14 megabase pairs of DNA. Because oligonucleotide-directed triple-helix formation has the potential to be a general solution for DNA recognition, this result has implications for physical mapping of chromosomes.

A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center.

Biochemical and crystallographic evidence suggests that 23S ribosomal RNA (rRNA) is the catalyst of peptide bond formation. To explore the mechanism of this reaction, we screened for nucleotides in Escherichia coli 23S rRNA that may have a perturbed pKa (where Ka is the acid constant) based on the pH dependence of dimethylsulfate modification. A single universally conserved A (number 2451) within the central loop of domain V has a near neutral pKa of 7.6 +/- 0.2, which is about the same as that reported for the peptidyl transferase reaction. In vivo mutational analysis of this nucleotide indicates that it has an essential role in ribosomal function. These results are consistent with a mechanism wherein the nucleotide base of A2451 serves as a general acid base during peptide bond formation.

Site-specific cleavage of human chromosome 4 mediated by triple-helix formation.

Direct physical isolation of specific DNA segments from the human genome is a necessary goal in human genetics. For testing whether triple-helix mediated enzymatic cleavage can liberate a specific segment of a human chromosome, the tip of human chromosome 4, which contains the entire candidate region for the Huntington's disease gene, was chosen as a target. A 16-base pyrimidine oligodeoxyribonucleotide was able to locate a 16-base pair purine target site within more than 10 gigabase pairs of genomic DNA and mediate the exact enzymatic cleavage at that site in more than 80 percent yield. The recognition motif is sufficiently generalizable that most cosmids should contain a sequence targetable by triple-helix formation. This method may facilitate the orchestrated dissection of human chromosomes from normal and affected individuals into megabase sized fragments and facilitate the isolation of candidate gene loci.

RNA seeing double: close-packing of helices in RNA tertiary structure.

Structured RNA molecules play essential roles in RNA processing, chromosome maintenance and protein biosynthesis. RNA necessarily uses different strategies than proteins for folding and assembly of complex architectures. The RNA-folding problem is largely an issue of helical packing: how does RNA organize and pack short, double-helical segments to produce active sites and recognition motifs for proteins? Noncanonical base pairs, metal ions and 2'-hydroxyl groups are key elements in RNA higher-order structure formation.

Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis.

The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and import.

A chemogenetic approach to RNA function/structure analysis.

Almost two dozen nucleotide analogs have been synthesized with alpha-phosphorothioate-tagged triphosphates and utilized in an interference modification approach termed Nucleotide Analog Interference Mapping. This method has made it possible to determine the chemical basis of RNA function and structure, including the identification of new rules for RNA helix packing, the functional analysis of a binding site for monovalent metal ions within RNA and the characterization of the catalytic mechanism of RNA enzymes.

An efficient ligation reaction promoted by a Varkud Satellite ribozyme with extended 5'- and 3'-termini.

The Neurospora Varkud Satellite (VS) RNA is capable of promoting a reversible self-cleavage reaction important for its replication pathway. In vivo the VS RNA performs a cis-cleavage reaction to generate monomeric length transcripts that are subsequently ligated to produce circular VS RNA. The predominant form of VS RNA observed in vivo is the closed circular form, though minimal VS ribozyme self-cleavage constructs lack detectable ligation activity. MFOLD analysis of the entire VS RNA sequence revealed an extended region 5' and 3' of the minimal self-cleaving region that could anneal to form a complementary helix, which we have termed helix 7. In full-length VS RNA, this helix appears to span over 40 bp of sequence and brings the 5'- and 3'-ends of the RNA into proximity for the ligation reaction. Here we report a variant of the VS ribozyme with an extended 5'- and 3'-terminus capable of forming a truncated helix 7 that promotes the ligation reaction in vitro. Through mutation and selection of this RNA we have identified a ribozyme containing two point mutations in the truncated helix 7 that ligates with >70% efficiency. These results show that an additional helical element absent in current VS ribozyme constructs is likely to be important for the ligation activity of VS RNA.

Nucleotide analog interference mapping of the hairpin ribozyme: implications for secondary and tertiary structure formation.

The hairpin ribozyme is a small, naturally occurring RNA capable of folding into a distinct three-dimensional structure and catalyzing a specific phosphodiester transfer reaction. We have adapted a high throughput screening procedure entitled nucleotide analog interference mapping (NAIM) to identify functional groups important for proper folding and catalysis of this ribozyme. A total of 18 phosphorothioate-tagged nucleotide analogs were used to determine the contribution made by individual ribose 2'-OH and purine functional groups to the hairpin ribozyme ligation reaction. Substitution with 2'-deoxy-nucleotide analogs disrupted activity at six sites within the ribozyme, and a unique interference pattern was observed at each of the 11 conserved purine nucleotides. In most cases where such information is available, the NAIM data agree with the previously reported single-site substitution results. The interference patterns are interpreted in comparison to the isolated loop A and loop B NMR structures and a model of the intact ribozyme. These data provide biochemical evidence in support of many, but not all, of the non-canonical base-pairs observed by NMR in each loop, and identify the functional groups most likely to participate in the tertiary interface between loop A and loop B. These groups include the 2'-OH groups of A10, G11, U12, C25, and A38, the exocyclic amine of G11, and the minor groove edge of A9 and A24. The data also predict non-A form sugar pucker geometry at U39 and U41. Based upon these results, a revised model for the loop A tertiary interaction with loop B is proposed. This work defines the chemical basis of purine nucleotide conservation in the hairpin ribozyme, and provides a basis for the design and interpretation of interference suppression experiments.

A chemical phylogeny of group I introns based upon interference mapping of a bacterial ribozyme.

Despite its small size, the 205 nt group I intron from Azoarcus tRNA(Ile) is an exceptionally stable self-splicing RNA. This IC3 class intron retains the conserved secondary structural elements common to group I ribozymes, but lacks several peripheral helices. These features make it an ideal system to establish the conserved chemical basis of group I intron activity. We collected nucleotide analog interference mapping (NAIM) data of the Azoarcus intron using 14 analogs that modified the phosphate backbone, the ribose sugar, or the purine base functional groups. In conjunction with a complete interference set collected on the Tetrahymena group I intron (IC1 class), these data define a "chemical phylogeny" of functional groups that are important for the activity of both introns and that may be common chemical features of group I intron catalysts. The data identify the functional moieties most likely to play a conserved role as ligands for catalytic metal ions, the substrate helix, and the guanosine cofactor. These include backbone functional groups whose nucleotide identity is not conserved, and hence are difficult to identify by standard phylogenetic sequence comparisons. The data suggest that both introns utilize an equivalent set of long range tertiary interactions for 5'-splice site selection between the P1 substrate helix and its receptor in the J4/5 asymmetric bulge, as well as an equivalent set of 2'-OH groups for P1 helix docking into most of the single stranded segment J8/7. However, the Azoarcus intron appears to make an alternative set of interactions at the base of the P1 helix and at the 5'-end of the J8/7. Extensive differences were observed within the intron peripheral domains, particularly in P2 and P8 where the Azoarcus data strongly support the proposed formation of a tetraloop-tetraloop receptor interaction. This chemical phylogeny for group I intron catalysis helps to refine structural models of the RNA active site and identifies functional groups that should be carefully investigated for their role in transition state stabilization.

A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme.

BACKGROUND: The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. RESULTS: Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by approximately 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chemical step. CONCLUSIONS: These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.

The human amylin analog, pramlintide, corrects postprandial hyperglucagonemia in patients with type 1 diabetes.

Mealtime amylin replacement with the human amylin analog pramlintide as an adjunct to insulin therapy improves postprandial glycemia and long-term glycemic control in type 1 diabetes. Preclinical animal studies indicate that these complementary effects may result from at least 2 independent mechanisms: a slowing of nutrient delivery to the small intestine and a suppression of nutrient-stimulated glucagon secretion. The former effect of pramlintide has previously been demonstrated in patients with type 1 diabetes. The present studies characterize the effect of pramlintide on postprandial glucagon secretion in this patient population. Plasma glucagon and glucose concentrations were measured before and after a standardized liquid meal in 2 separate randomized, double-blind, placebo-controlled studies of pramlintide administration to patients with type 1 diabetes. In a 2-day crossover study, 18 patients received a 5-hour intravenous infusion of pramlintide (25 microg/h or 50 microg/h) or placebo in addition to subcutaneous (SC) insulin injections. In a 14-day parallel-group study, 84 patients received SC injections of 30, 100, or 300 microg of pramlintide or placebo 3 times daily in addition to SC injections of insulin. In both studies plasma glucagon concentrations increased in response to the meal in the placebo-plus-insulin group but not in any of the pramlintide-treated groups (all pramlintide treatment arms v placebo, P <.05). We conclude that mealtime amylin replacement with pramlintide prevents the abnormal meal-related rise in glucagonemia in insulin-treated patients with type 1 diabetes, an effect that likely contributes to its ability to improve postprandial glucose homeostasis and long-term glycemic control.

Site specific incorporation of 6-azauridine into the genomic HDV ribozyme active site.

The HDV ribozyme is proposed to catalyze its self cleavage reaction by a proton transfer mechanism wherein the N3 of its C75 acts as a general acid. The C75 to U mutation, which raises the N3 pKa from about 4 to almost 10. abolishes all enzymatic activity. To test if a U analogue with a neutral pKa can restore ribozyme function we incorporated 6-azauridine (n6U), a uridine analogue with histidine-like N3 pKa. into the genomic HDV ribozyme active site by 2'-O-ACE oligoribonucleotide protection chemistry. The resulting ribozymes were analyzed for their ability to undergo the HDV ribozyme cis-cleavage reaction. Incorporation of n6U at nucleotide position 75 did not restore ribozyme function compared to the U75 mutant. This suggests that the HDV ribozyme reaction mechanism involves more than positioning of a neutral nucleobase at the active site and implies that the exocyclic amino group of C75 participates in establishing the proper active site fold.

Linguistic analysis of project ownership for undergraduate research experiences.

We used computational linguistic and content analyses to explore the concept of project ownership for undergraduate research. We used linguistic analysis of student interview data to develop a quantitative methodology for assessing project ownership and applied this method to measure degrees of project ownership expressed by students in relation to different types of educational research experiences. The results of the study suggest that the design of a research experience significantly influences the degree of project ownership expressed by students when they describe those experiences. The analysis identified both positive and negative aspects of project ownership and provided a working definition for how a student experiences his or her research opportunity. These elements suggest several features that could be incorporated into an undergraduate research experience to foster a student's sense of project ownership.

Ribozyme chemogenetics.

In this review I will outline several chemogenetic approaches used to determine the chemical basis of large ribozyme function and structure. The term chemogenetics was first used to describe site-specific functional group modification experiments in the analysis of DNA-protein interactions. Within the past few years equivalent experiments have been performed on large catalytic RNAs using both single-site substitution and interference mapping techniques with nucleotide analogues. While functional group mutagenesis is an important aspect of a chemogenetic approach, chemical correlates to genetic revertants and suppressors must also be realized for the genetic analogy to be intellectually valid and experimentally useful. Several examples of functional group revertants and suppressors have now been obtained within the Tetrahymena group I ribozyme. These experiments define an ensemble of tertiary hydrogen bonds that have made it possible to construct a detailed model of the ribozyme catalytic core. The model includes a functionally important monovalent metal ion binding site, a wobble-wobble receptor motif for helix-helix packing interactions, and a minor groove triple helix.

Biochemical identification of A-minor motifs within RNA tertiary structure by interference analysis.

A-minor motifs are the most common tertiary structural elements in RNA helix packing. Biochemical identification of these interactions is now feasible using interference mapping analysis with the adenosine analogues 2'-deoxyadenosine and 3-deaza-adenosine. This approach was used to demonstrate that A-minor motifs mediate helix packing interactions that are important for 5'-splice site selection in the group I intron. By analysing the interference pattern of several analogues it is possible to identify and distinguish the four variants of the A-minor motif.

Translocation of an RNA duplex on a ribozyme.

RNA cleavage by the Tetrahymena ribozyme requires recognition of the reaction-site helix by the catalytic apparatus. This binding can occur in several registers, each of which results in reaction at a different nucleotide in the helix. We now identify commensurate sets of 2'-hydroxyl interactions on both strands of the reaction-site helix that account for its translocation into alternative binding registers. These results indicate that the ribozyme has a relatively rigid substrate-binding pocket into which the helix can bind in different alignments. A similar mechanism of reaction site recognition is proposed to occur during intron circularization and ribozyme polymerase activity. Translocation of the reaction site duplex provides an example of structural heterogeneity in packing of helices during the tertiary folding of RNA.