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M Yarus

Publications and source records attributed to M Yarus.

At least 37 records · Page 2Linked to original sources
PubMed1997

Essential structures of a self-aminoacylating RNA.

Comparison of six independent self-aminoacylating RNAs derived from selection-amplification, as well as deletion, addition, substitution, fragmentation of one particular RNA, are used to analyze the requirements for the RNA-catalyzed aminoacylation. All elements required for catalysis by one RNA family: sequence at the 3' acceptor end, calcium and magnesium sites, as well as the Phe-AMP substrate site and the essential 5' triphosphate terminus, are closely grouped near a bihelix junction in the parental molecule. All elements of the active center for aminoacyl transfer can therefore be captured by a peripherally-deleted helix junction RNA, defining a much smaller 43 nucleotide ribozyme, of which only 17 nucleotides were initially randomized. It appears that a complex RNA active center can be assembled by specifying unexpectedly few nucleotides, perhaps with a critical contribution from an essential calcium ion.

Acylation↗
PubMed1997

A calcium-metalloribozyme with autodecapping and pyrophosphatase activities.

A previously-isolated ribozyme with capping activity has self-decapping activity, here characterized alongside its additional, somewhat parallel, pyrophosphatase reaction. Decapping is 10-50 times slower than the pyrophosphatase activity, depending on pH. The RNA accelerates pyrophosphate release 170 000 times over a control composed of randomized pppRNA, and 5' capped RNA accelerates decapping 1000-fold over random capped RNA. Triphosphate-linked G(5')pppRNA also supports an unusual cap-exchange reaction, exchanging its cap with guanosine 5'-tetraphosphate to form pentaphosphate-linked G(5')pppppRNA. GDP, a capping reactant for the RNA, appears to suppress both decapping and pyrophosphatase activities. Autodecapping and pyrophosphatase activities have in common an unusual divalent metal ion requirement for Ca2+ or less effectively Mn2+, and both are active over a broad pH range of 4.5-9. 0. These characteristics resemble the capping activity of the same RNA. Kinetic analysis reveals a well-defined Ca2+-RNA complex, and Mg2+ and Sr2+ act as competitive inhibitors of Ca2+. A strong Ca2+-binding site is suggested by a low KM of 40-60 microM at pH >/= 7.0. The role of Ca2+ in these reactions can be surmized from literature data on reactivity of nucleotide phosphates. Pyrophosphatase, capping, and decapping activities of isolate 6 RNA are apparently carried out by a single reaction center, whose rate of reaction with all nucleophiles sums to a constant total rate. This suggests a universal rate-limiting step. Versatile activation of alpha-phosphate by this reaction center raises the possibility of combinatorial ribozymes.

Base Sequence↗
PubMed1997

Versatile 5' phosphoryl coupling of small and large molecules to an RNA.

A Ca2+-requiring catalytic RNA is shown to create 5' phosphate-phosphate linkages with all nucleotides and coenzymes including CoA, nicotinamide adenine dinucleotide phosphate, thiamine phosphate, thiamine pyrophosphate, and flavin mononucleotide. In addition to these small molecules, macromolecules such as RNAs with 5'-diphosphates, and nonnucleotide molecules like Nepsilon-phosphate arginine and 6-phosphate gluconic acid also react. That is, the self-capping RNA isolate 6 is an apparently universal 5' phosphate-linker, reacting with any nucleophile containing an unblocked phosphate. These RNA reactions demonstrate a unique RNA catalytic capability and imply versatile and specific posttranscriptional RNA modification by RNA catalysis.

Calcium↗
PubMed1997

23S rRNA similarity from selection for peptidyl transferase mimicry.

RNAs from a randomized pool were selected by affinity elution for binding to the molecule CCdApPuro, a high-affinity ligand of ribosomal peptidyl transferase designed as a transition-state analogue of peptide formation. The selected RNAs show affinity for CCdApPuro comparable to that of the peptidyl transferase center itself (Kd approximately 10 nM). Chemical modification/protection experiments implicate bases completely conserved among the selected RNAs in CCdApPuro interaction, which appears to involve both CCdA and puromycin moieties, that is, both A- and P-site homologues. The apparent selected binding site shows up to 17 nucleotides with similarity to conserved nucleotides of the peptidyl transferase loop domain of 23S rRNA and is conserved when reselected under mutagenesis. Thus, these nucleotides of 23S rRNA likely provide elements of the peptidyl transferase active center that bind the reactants near the site of peptide bond formation. Binding of CCdApPuro by a peptidyl transferase-like motif in the absence of protein strengthens the hypothesis that peptidyl transfer originated in an RNA world.

Base Sequence↗
PubMed1997

5'-RNA self-capping from guanosine diphosphate.

A selected RNA (isolate 6) efficiently catalyzes a self-capping reaction with free GDP, yielding the same 5'-capped structure as is formed by protein GTP:RNA guanylyltransferase. This unexplored RNA-catalyzed reaction type involving nucleophilic attack on phosphate by phosphate adds to the variety of possible postsynthetic RNA-catalyzed RNA modifications. The selected RNA requires only Ca2+ for activation and has a broad active pH range of 4.5-9.0. The RNA also has a 5'-pyrophosphatase activity.

Base Sequence↗
PubMed1997

Small-molecule-substrate interactions with a self-aminoacylating ribozyme.

A self-aminoacylating RNA catalyst is shown to carry out the chemistry required for turnover, being reacylated several times from aminoacyl-AMP with an unaltered rate, thereby meeting one definition of an enzyme. Furthermore, a newly applied gel electrophoresis assay suggests first order kinetics in RNA and saturation kinetics in the substrate aminoacyl-adenylate, implying a Michaelis complex. AMP is a competitive inhibitor, though phenylalanine is not detectably inhibitory, consistent with a Michaelis complex through the AMP moiety of phenylalanyl-adenylate substrate. This idea is supported by measurement of elevated acylation velocities with seryl and alanyl-adenylates. The rate of aminoacylation increases with pH, consistent with attack of a terminal ribose oxyanion on the carbonyl carbon atom of the adenylate.

Acylation↗
PubMed1996

On malleability in the genetic code.

To explain now-numerous cases of codon reassignment (departure from the "universal" code), we suggest a pathway in which the transformed codon is temporarily ambiguous. All the unusual tRNA activities required have been demonstrated. In addition, the repetitive use of certain reassignments, the phylogenetic distribution of reassignments, and the properties of present-day reassinged tRNAs are each consistent with evolution of the code via an ambiguous translational intermediate.

Codon↗
PubMed1996

Small RNA-divalent domains.

From a potentially completely sampled set of randomized 23-mer sequences, we selected RNAs that bind a Zn-column and also show KD approximately 100-400 microM for free Zn2+, probably relying on one or two direct ion coordinations. Comparison of selected sequences with previously known divalent sites suggests three or four small RNA motifs repeatedly found to interact with divalent ions. We suggest that the GC cluster, the augmented GC cluster, and the E element may be useful generalized ion-binding structures. Such structures may help identify similar divalent sites in sequenced RNAs and serve as substructures for design of functional RNA metallodomains.

Base Sequence↗
PubMed1995

Aminoacyl-RNA synthesis catalyzed by an RNA.

An RNA has been selected that rapidly aminoacylates its 2'(3') terminus when provided with phenylalanyl-adenosine monophosphate. That is, the RNA accelerates the same aminoacyl group transfer catalyzed by protein aminoacyl-transfer RNA synthetases. The best characterized RNA reaction requires both Mg2+ and Ca2+. These results confirm a necessary prediction of the RNA world hypothesis and represent efficient RNA reaction (> or = 10(5) times accelerated) at a carbonyl carbon, exemplifying a little explored type of RNA catalysis.

Acylation↗
PubMed1995

An inhibitor of ribosomal peptidyl transferase using transition-state analogy.

The phosphoramidate of CCdAp and puromycin (CCdApPuro) is a potent inhibitor of ribosomal peptidyl transferase, as assayed by the fragment reaction. Inhibition is competitive at the ribosomal A-site. CCdApPuro protects P-site-associated bases in the peptidyl transferase loop region of 23S rRNA from carbodiimide modification. The Ki's of structural homologues of CCdApPuro suggest that both the CCdA and puromycin moieties participate in binding. Thus, CCdApPuro appears to bridge the A- and P-sites of the ribosome, implying that substrates are juxtaposed with a geometry suitable for direct reaction during peptidyl transfer.

Escherichia coli↗
PubMed1995

Diversity of oligonucleotide functions.

SELEX is a technology for the identification of high affinity oligonucleotide ligands. Large libraries of random sequence single-stranded oligonucleotides, whether RNA or DNA, can be thought of conformationally not as short strings but rather as sequence dependent folded structures with high degrees of molecular rigidity in solution. This conformational complexity means that such a library is a source of high affinity ligands for a surprising variety of molecular targets, including nucleic acid binding proteins such as polymerases and transcription factors, non-nucleic acid binding proteins such as cytokins and growth factors, as well as small organic molecules such as ATP and theophylline. The range of applications of this technology for new discovery extends from basic research reagents to the identification of novel diagnostic and therapeutic reagents. Examples of these applications are described along with a discussion of underlying principles and future developments expected to further the utility of SELEX.

Amino Acid Sequence↗
PubMed1995

Selection of an RNA domain that binds Zn2+.

We have selected an RNA that depends on zinc for affinity to a column, starting from a pool of ribooligonucleotides with 50 randomized positions. This RNA's chemical sensitivities, calculated folding thermodynamics, and activity when fragmented suggest that an ion binding site lies within a complex 21-nt hairpin loop, near the junction with an imperfect helical stem. This RNA site has an unselected selectivity among divalents, preferring nickel, cobalt, and cadmium to calcium, magnesium, and manganese, as expected for a simple site of chelation. A moderate zinc-dependent change in loop structure accompanies divalent binding and can be detected by chemical probing and zinc-dependent UV-induced crosslinking. The latter also demonstrates the apposition of loop sequences to make a structure that may be related to the E-loop motif found in a number of other RNA molecules; the E-loop motif, accordingly, may be a divalent site.

Base Sequence↗
PubMed1995

An RNA pocket for the planar aromatic side chains of phenylalanine and tryptophane.

This is a preliminary report of RNA's with affinity for the aromatic side chains of the amino acids phenylalanine and tryptophane that have been isolated utilizing in vitro selection to phenylalanine-affinity matrix. Cloning and sequencing of a binding pool identified a frequently occurring 6-9 base motif (-CUCGUGU-) common to most of the RNA's; other potential binding motifs were revealed but occurred less frequently. Four of five clones analyzed bind free phenylalanine and tryptophane with dissociation constants in the low millimolar range, the fifth binds resin only. Isolate F7, the most common sequence in the binding pool, demonstrated moderate specificity for aromatic rings, no stereoselectivity was observed. Preliminary Pb cutting structural analysis agrees with the most stable predicted secondary structure. A striking similarity between the sequence and predicted structure of F7 and the bridged-biphenylisomerase RNA (J.R. Prudent, T. Uno, and P.G. Schultz, Science 264,1924 (1994)) suggests a 19 base loop contains the important aromatic binding elements.

Amino Acid Sequence↗
PubMed1994

RNAs with dual specificity and dual RNAs with similar specificity.

The biological role of RNA is delimited by its possible reactions, which can be explored by selection. A comparison of selected RNAs that bind one ligand with those that bind two related ligands suggests that a single nucleotide substitution can expand binding specificity. An RNA site with dual (joint) specificity has adenine and cytosine bases whose pKa's appear shifted upward, thereby mimicking an efficient general acid-base catalyst. The joint site also contains two conserved, looped arginine-coding triplets implicated in arginine site formation. Two selected joint RNAs are identical in some regions and distinct in others. The distinct regions, like some peptides, seem to function similarly without being similar in primary structure.

Arginine↗
PubMed1994

Transfer RNA mutation and the malleability of the genetic code.

We propose that evolutionary reassignment of codons is facilitated by a translationally ambiguous intermediate. For example, recently discovered tRNA mutations that allow relatively efficient simultaneous cognate and near-cognate coding (sharing 2 contiguous nt) in vivo may speed reassignment of the near-cognate codon. As predicted by this notion, characterized codon reassignments are strikingly non-random, and half can be immediately explained by unusual tRNA activities already demonstrated. In addition, sequences of reassigned tRNAs contain sequences that promote ambiguity. tRNA structural change may provide a transitional pathway that allows rapid selection of a new specificity, rather than slow mutation toward a new codon-amino acid association.

Biological Evolution↗
PubMed1994

tRNA structure and ribosomal function. I. tRNA nucleotide 27-43 mutations enhance first position wobble.

Transfer RNA su7 G36 is a derivative of tRNA(Trp) with a 3'GUC anticodon complementary to the glutamine codon CAG. This tRNA requires a normally forbidden G-U wobble at the first codon position to suppress a UAG (amber) termination codon. Measurement of amber suppression by mutated su7 G36 tRNAs and correction for tRNA levels and aminoacylation allowed calculation of KUAG, a linearized index of in vivo ribosomal function. Following saturating mutagenesis of the anticodon arm of su7 G36, screening for UAG suppression using a lacZ reporter yielded tRNAs with up to 40-fold increased first position G-U wobble, judged from KUAG. The parental anticodon helix has minimized this type of miscoding, and virtually all changes in the top base-pair of the anticodon helix, nucleotides (nt) 27-43, increased the error. Thus, misincorporation of amino acids due to aberrant first position wobble is apparently prevented by normal tRNA structure, which is specifically altered by substitution at nt 27-43, the top base-pair of the anticodon helix. All 16 permutations of nt 27-43, the hotspot for increased wobble, were subsequently constructed and compared. Comparison of values for tRNA coding function, tRNA level, and aminoacylation for the 16 suggest that a tRNA conformational change, specifically involving both nt 27-43, differentially affects all these tRNA functions. This conformational alteration, which presumably occurs normally on the ribosome, appears more complex than simple breakage of the normal 27-43 base-pair. We suggest that the change is in the angle and/or flexibility of the tRNA L-shape. Among these 16 tRNAs, efficient wobble is strongly and inversely correlated with good aminoacylation and high tRNA levels; this quality may have been selected. Constraints on the sequences of natural tRNAs suggest that nt 27-43 have effects on function in many tRNAs.

Anticodon↗