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Biomedical subjects

M Yarus

Publications and source records attributed to M Yarus.

At least 19 recordsLinked to original sources
PubMed2001

Binding and disruption of phospholipid bilayers by supramolecular RNA complexes.

In an RNA world, RNAs would have regulated traffic through normally impermeable bilayer membranes. Using selection-amplification we previously found RNAs that bind stably and increase the ionic conductance of phospholipid membranes at high Mg(2+) and Ca(2+) concentrations. Now selection in reduced divalents yields RNAs that bind phosphatidylcholine liposomes under conditions closer to physiological. Such affinity for phospholipid membranes requires interactions between RNAs. In fact, we detected no functional monomeric membrane-binding RNAs. A membrane-active end-to-end heterotrimer consisting of 2 RNA 9 and 1 RNA 10 is defined by nucleotide protection, oligonucleotide competition, and mutant analysis. Oligomers of the heterotrimer bind stably, cause release of liposome-encapsulated solutes, and disrupt model black membranes. Individual RNA molecules do not show any of these activities. This novel mechanism of RNA binding to lipid membranes may not only regulate membrane permeability, but suggests that arrays of catalytic or structural RNAs on membranes are plausible. Finally, a selection met only by RNA complexes evokes new possibilities for selection-amplification itself.

Base Sequence↗
PubMed2001

RNA-catalyzed amino acid activation.

We have selected RNAs that perform a new reaction that chemically activates amino acids, paralleling mixed phosphate anhydride synthesis by protein aminoacyl-transfer RNA synthetases. Care with recovery of the unstable reaction product was apparently essential to this selection. The best characterized RNA, KK13, requires only Ca2+ for reaction and is optimally active at low pH with KM = 50 mM and kcat = 1.1 min(-1) for activation of leucine. In conjunction with previous RNA-catalyzed aminoacyl-RNA synthesis, peptide bond formation, and RNA-based coding, these amino acid-activating RNAs complete an experimental demonstration that the four fundamental reactions of protein biosynthesis can be RNA-mediated. The appearance of translation in an RNA world is therefore supported.

Amino Acids↗
PubMed2001

Translation termination: a ghost ballet?

In the October 5th issue of Cell, clarify the end of translation on a eubacterial mRNA. By elucidating the molecular choreography of class I and class II release factors within the ribosome, they show how the steps around the release of nascent protein are ordered.

Escherichia coli↗
PubMed2000

RNA-Catalyzed CoA, NAD, and FAD synthesis from phosphopantetheine, NMN, and FMN.

A novel in vitro selection method was developed to isolate RNA sequences with coenzyme-synthesizing activities. We used size-heterogeneous libraries containing randomized ribonucleotide sequences of four different lengths (30N, 60N, 100N, and 140N), all with 5'-ATP initiation. Two RNAs, CoES7 (30N) and CoES21 (60N), are able to catalyze the synthesis of three common coenzymes, CoA, NAD, and FAD, from their precursors, 4'-phosphopantetheine, NMN, and FMN, respectively. Both ribozymes require divalent manganese for activities. The results support the availability of these coenzymes in an RNA world, and point to a chemical explanation for the complex bipartite structures of many coenzymes.

Coenzyme A↗
PubMed2000

Peptidyl transferase: ancient and exiguous.

The finding that the universal ribosomal peptidyl transferase is an RNA enzyme casts new light on its ancient origins, on the use of transition state analogues for ribozymes, and on the role of selection-amplification in studies of molecular evolution.

Base Sequence↗
PubMed2000

Suppression of eukaryotic translation termination by selected RNAs.

Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1.eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1.eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.

Animals↗
PubMed2000

RNA-ligand chemistry: a testable source for the genetic code.

In the genetic code, triplet codons and amino acids can be shown to be related by chemical principles. Such chemical regularities could be created either during the code's origin or during later evolution. One such chemical principle can now be shown experimentally. Natural or particularly selected RNA binding sites for at least three disparate amino acids (arginine, isoleucine, and tyrosine) are enriched in codons for the cognate amino acid. Currently, in 517 total nucleotides, binding sites contain 2.4-fold more codon sequences than surrounding nucleotides. The aggregate probability of this enrichment is 10(-7) to 10(-8), had codons and binding site sequences been independent. Thus, at least some primordial coding assignments appear to have exploited triplets from amino acid binding sites as codons.

Amino Acids↗
PubMed1999

RNAs that bind and change the permeability of phospholipid membranes.

The RNA world hypothesis presumes that RNA will be competent for varied essential cellular functions. One such indispensable cell function is regulation of membrane permeability. Though this was not a known RNA activity, selection-amplification yielded RNAs that bound phosphatidylcholine:cholesterol liposomes. At least eight distinct, approximately 95-mer sequences bind well to the outside of the lipid bilayer, though randomized sequences had no such activity. No distinct sequence motif for lipid binding was found. However, truncation of one such RNA shows that a smaller, 44-nucleotide irregular RNA hairpin is an active membrane binding domain. Bound RNA increases the permeability of liposomes to (22)Na(+). In addition, using voltage clamp technique, four individual RNAs increase the ion permeability of the plasma membrane of cultured human cells. The existence of multiple sequences that bind membranes and provoke permeability changes suggests that these may be elementary RNA functions that could be selected in vivo.

Base Sequence↗
PubMed1999

Specific, rapid synthesis of Phe-RNA by RNA.

RNA 77, derived by selection amplification, accelerates its own conversion to Phe-RNA (relative to randomized RNA) more than 6 x 10(7)-fold, by using amino acid adenylates as substrate. A modified assay system allows measurement of slow rates of aa-RNA formation, which for disfavored amino acid substrates can be more than 10(4)-fold slower than phenylalanine. Thus unlike previously characterized self-aminoacylators, RNA 77 catalysis is highly amino acid selective. Remarkably, both rates of aminoacyl transfer and amino acid specificities are greater for RNA 77 than measured for protein PheRS. These data experimentally support the possible existence of an ancestral amino acid-specific translation system relying entirely on RNA catalysis. RNA 77 itself embodies a possible transitional evolutionary state, in which side-chain-specific aa-RNA formation and anticodon-codon pairing were invested in the same molecule.

Acylation↗
PubMed1999

Boundaries for an RNA world.

Before large, specific peptides could be made, in an RNA world our ancestors may have used an RNA-like molecule for both catalysis and information storage. Taken together, RNA activities suggest RNA could have performed most of the essential informational functions of an ancient organism (i.e. those functions most similar to RNA's role today). In contrast, though compellingly reasoned optimism about RNA-catalyzed small-molecule metabolism is plentiful, fundamental problems remain to be solved.

Animals↗
PubMed1999

A tiny RNA that catalyzes both aminoacyl-RNA and peptidyl-RNA synthesis.

A 29-nt RNA catalyst successively forms the aminoacyl ester phe-RNA, and then peptidyl-RNA (phe-phe-RNA), given phenylalanine adenylate (phe-AMP) as substrate. Catalysis of two related reactions at similar rates supports the argument that RNA catalysts would evolve as groups with similar mechanisms. In particular, successive aminoacyl- and peptidyl-RNA synthesis by one RNA suggests that uncoded but RNA-catalyzed peptide synthesis would evolve before the synthesis of coded peptides.

Acylation↗
PubMed1998

Kinetics at a multifunctional RNA active site.

Kinetics of a self-capping RNA, Iso6, have been investigated to constrain the catalytic mechanism. The role of phosphates has been examined by varying the number of phosphates on the nucleophilic attacking group or on the RNA. While the number of phosphates in the nucleophile affects capping kinetics, only KM but not kcat is altered. The KM values for GMP, GDP, GTP and ppppG are 200, 11, 13 and 31 microM, respectively. A reaction product, pyrophosphate, is also found to strongly inhibit RNA activities through a competitive exchange mechanism with an apparent Ki of 200 nM. Uniquely strong binding of pyrophosphate supports the idea that capping originated by utilization of the initial pyrophosphate leaving group site for capping nucleophiles. In contrast to the nucleophile phosphate, change of 5' RNA terminus from triphosphate to tetraphosphate enhances the overall rate and kcat by 40%, with little effect on KM. Thus, only the leaving group appears to affect the rate of the chemical transformation. We propose two possible mechanisms that explain this apparent rate-limiting chemical step, either dissociation of pyrophosphate to form a metaphosphate monoester intermediate or formation of a circular phosphoramidate intermediate, using an internal RNA nitrogenous group. A single essential Ca ion is required for all activities.

Calcium↗
PubMed1998

Amino acids as RNA ligands: a direct-RNA-template theory for the code's origin.

Numerous RNA binding sites for specific amino acids are now known, coming predominantly from selection-amplification experiments. These sites are chemically discriminating despite being predominantly small, simple RNA structures: internal and bulge loops. Recent studies of sites for hydrophobic side chains suggest that there are other generalizable structural features which recur in hydrophobic RNA sites. Further, sites for hydrophobic side chains can contain codons for the bound amino acid, as has also long been known for the polar amino acid arginine. Such findings are comprehensively reviewed, and the implications for the origin of coded peptide synthesis are considered. An origins hypothesis which accommodates all the data, DRT (direct RNA templating), is formulated.

Amino Acids↗
PubMed1998

RNA enzymes with two small-molecule substrates.

BACKGROUND: The 'RNA world' hypothesis posits ancient organisms employing versatile catalysis by RNAs. In particular, such a metabolism would have required RNA catalysts that join small molecules. Such anabolic reactions now occur very widely, for example in phospholipid, terpene, amino acid and nucleotide synthetic pathways in modern organisms. Present RNA systems, however, do not perform such reactions using substrates that do not base pair. Here we ask whether this lack is a methodological artifact due to the practice of selection-amplification, or a fundamental property of active sites reconstructed within RNA structures. RESULTS: Three rationally modified RNA enzymes, Iso6-G, Iso6-2G and Iso63G, catalyze the formation of (5'-->5') polyphosphate-linked oligonucleotides in trans. One of these, Iso6-G RNA, has a specific substrate site for a guanosine triphosphate, GTP, dGTP or ddGTP, and one nonspecific substrate site for a terminal-phosphate-containing small molecule. This ribozyme catalyzes multiple turnovers, proceeding at a constant rate. Guanosine specificity is probably not attributable to Watson-Crick base pairing. CONCLUSIONS: Ribozymes can readily bind multiple small-molecule substrates simultaneously and catalyze reactions that build up larger products, apparently independent of substrate-RNA Watson-Crick base pairing. RNA enzymes therefore parallel proteins, which often overcome the entropic difficulties of positioning multiple small substrates for catalysis of anabolic reactions. These results support the idea of a complex ancestral metabolism based on RNA catalysis.

Algorithms↗
PubMed1998

Isoleucine:RNA sites with associated coding sequences.

An RNA family that binds isoleucine with Kd = 200-500 microM was repetitively isolated from a ribonucleotide transcript pool containing 50 randomized positions. The RNA site is specific, discriminating against branched side chains of different size (valine--one methylene smaller than isoleucine) by at least 1.3 kcal/mol and against the shape (linear) of norleucine by 0.6 kcal/mol. The binding site was localized by sequence comparison, by synthesis of mutant and truncated active derivatives, and by chemical modification-interference. The binding site is small, no more than 10-12 nt, containing an asymmetric internal loop (6 over 2 nt) that includes the isoleucine codon AUU and a sequence of four G's, two of which are involved in G-U and G-C base pairs. Areas of U/G concentration like these may signal a hydrophobic RNA site.

Amides↗