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At least 1,045 records · Page 58Linked to original sources
PubMed2003

Small worlds in RNA structures.

I consider conformational spaces of tRNA(phe) defined by sets of suboptimal structures from the perspective of small-world networks. Herein, the influence of modifications on typical small-world network properties and the shape of energy landscapes is discussed. Results indicate that natural modifications influence the degree of local clustering and mean path lengths far more than random or no modifications. High frequencies in the thermodynamic ensemble coincide with high numbers of neighboring structures that one conformation can adopt by one elementary move. Conformation spaces indicate the existence of modular substructures. It can be shown that modifications leave the nature of small-world topology untouched albeit natural modifications have a reasonable enhancing and streamlining effect on the degree of clustering and therefore on the substructures of the conformational space.

Algorithms↗
PubMed1991

The effect of sinefungin and synthetic analogues on RNA and DNA methyltransferases from Streptomyces.

Sinefungin is an antibiotic structurally related to S-adenosylmethionine. It has been described as an inhibitor of RNA transmethylation reactions in viruses and eukaryotic organisms, but not in bacteria. We show here that sinefungin strongly inhibits RNA methyltransferase activity, but not the biosynthesis of these enzymes in Streptomyces. All the methylated bases found in Streptomyces RNA (1-methyladenine, N6-methyladenine, N6,N6-dimethyladenine and 7-methylguanine) are inhibited by this antibiotic. Experiments with sinefungin analogues show that specific changes in the ornithine radical of the molecule still preserve its inhibitory capability. The substitution of the adenine radical by uridine causes the loss of the inhibitory effect. These results and our former studies on Streptomyces DNA methylation, suggest that nucleic acid modification is the main target of sinefungin in Streptomyces.

Adenosine↗
PubMed1977

Study of the transfer RNAs coded by T2, T4, and T6 bacteriophages.

T2, T4, and T6 bacteriophage tRNAs coding for arginine, leucine, proline, isoleucine, and glycine were isolated under conditions of short term and long term infection of Escherichia coli B cells. The corresponding phage tRNA species were examined for sequence homology by RNA-DNA hybridization analysis and by their relative behavior on reversed phase chromatography. The results indicate that all three T-even phages code for similar tRNA species; however, some tRNA species are homologous, others are not, and not all of the same tRNA species are coded by each bacteriophage. Reversed phase chromatography showed the presence of isoacceptor tRNAs for each phage aminoacyl-tRNA species. Pulse-chase experiments for [32P]tRNAGly suggest that the multiple isoacceptor species observed derive from the intracellular modification of a single tRNAGly gene product.

Arginine↗
PubMed2006

Assembly of protein-RNA complexes using natural RNA and mutant forms of an RNA cytosine methyltransferase.

This work reveals that mutant forms of RNA methyltransferases that form 5-methylcytosine (m5C) have characteristics that may make them useful for biomacromolecular assembly. The experiments utilized bacterially expressed Trm4p, a tRNA methyltransferase cloned from Saccharomyces cerevisiae. Like DNA m5C methyltransferases, Trm4p mediates methylation using a covalent intermediate, which would allow Trm4p to be trapped as a stable protein-RNA complex when the substrate RNA contains a modified cytosine base such as 5-fluorocytosine. However, mutant forms of Trm4p are identified that fail to release RNA resulting in the formation of denaturant stable methyltransferase-RNA complexes that contain only natural nucleotides. The ability to form stable complexes with natural RNA gives these mutant forms of Trm4p greater potential versatility for biomacromolecule construction applications than the wild-type Trm4p enzyme or DNA methyltransferases for which the trapping of the covalent intermediate requires the presence of a nucleotide analogue at the site of modification.

Base Sequence↗
PubMed1980

Dissection of the active site of rabbit liver tRNA nucleotidyltransferase. Specificity and properties of subsites for donor nucleotide triphosphates.

tRNA nucleotidyltransferase incorporates both AMP and CMP into tRNA acceptors. Studies of the effects of nucleoside triphosphates, nucleotide analogues, and affinity reagents on AMP and CMP incorporation indicate that these residues are donated from different subsites. However, neither of these sites is completely specific for nucleoside triphosphate binding, and CMP can actually be incorporated from the AMP-donating site, although at a slow rate. The two donor subsites interact with each other, such that binding of a ligand to the ATP site stimulates incorporation from the CMP-donating site. This interaction accounts for the biphasic CTP saturation curve and the unusual effects of nucleoside triphosphates on CMP incorporation observed earlier. In addition to donating CMP, the CTP subsite also serves as the position of binding of the terminal C residue of tRNA-C-C and, in the absence of CTP, for binding of the terminal residue of tRNA-C. These results, together with those in the accompanying paper, have defined multiple accepting and donating subsites within the active site of tRNA nucleotidyltransferase, as predicted from our previous model for enzyme action (Deutscher, M. P. (1972) J. Biol. Chem. 247, 459-468). However, since we have been unable to obtain definitive evidence for two CMP-donating sites, we have considered a modification of this earlier model which utilizes only a single CMP-donating site. Using these models, we discuss how the specificity of the donor and acceptor subsites ensures the accurate synthesis of the -C-C-A sequence of tRNA.

Adenosine Monophosphate↗
PubMedDate not supplied

[Study of affinity modification kinetics as an approach to demonstrating cooperativity between substrate-recognition centers of bicentric enzymes].

The dependence of the initial rate of the affinity labelling of phenylalanyl-tRNA synthetase from E. coli MRE-600 by an phenylalanyl-tRNA analog--N-Br-Ac-[14C]Phe-tRNAPhe against reagent concentration was obtained. The curve runs through maximum therefore it is impossible to describe it by the traditional Kitz and Wilson scheme. The experimental results were treated in assumption of dimeric enzyme, cooperativity in the substrate analog binding and its conversion being taken into account. It was shown that such a treatment of kinetic data of the affinity labelling allows to estimate quantitatively the cooperativity arisen between substrate analog centers of the binding and conversion. The data obtained allow to assume that in the case of phenylalanyl-tRNA analog there is a slight negative cooperativity in the reagent binding (thermodynamic cooperativity) but strong negative cooperativity in reagent conversion (kinetic cooperativity). The results obtained testify the high sensitivity of the kinetic approach for elucidation of centers cooperativity.

Amino Acyl-tRNA Synthetases↗
PubMed1996

An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression.

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology.

Acetylcholine↗
PubMed2005

Transglycosylation: a mechanism for RNA modification (and editing?).

The vast majority of the ca. 100 chemically distinct modified nucleosides in RNA appear to arise via the chemical transformation of a genetically encoded nucleoside. Two notable exceptions are queuosine and pseudouridine, which are incorporated into tRNA via transglycosylation. Transglycosylation is an extremely efficient process for incorporating highly modified bases such as queuine into RNA. Transglycosylation is also a requisite process for "isomerizing" an N-nucleoside into a C-nucleoside as is the case for pseudouridine formation. Finally, transglycosylation is an attractive possibility for certain RNA editing events (e.g., pyrimidine to purine conversions) that cannot occur via the known, more straightforward enzymatic reactions (e.g., deaminations). This review discusses what is known about the mechanisms of transglycosylation for the queuine and pseudouridine RNA modifications and will speculate about a potential role for transglycosylation in certain RNA editing events.

Crystallography, X-Ray↗
PubMed1986

The nucleotide sequence of two homogeneic Drosophila melanogaster tRNATyr isoacceptors: application of a rapid tRNA anticodon sequencing method using S-1 nuclease.

The nucleotide sequence of the two major Drosophila melanogaster tRNATyr isoacceptors was determined to be pC-C-U-U-C-G-A-U-A-m2G-C-U-C-A-G-D-D-G-G-acp3 U-A-G-A-G-C-m2(2)G-G-psi-G-G-A-C-U-G/Q-psi-A-m1G-A-Um-C-C-A-U-A-G-m7 G-D-C-G-C-U-G-G-U(T)-psi-C-A-m1A-A-U-C-C-G-G-C-U-C-G-A-A-G-G-A-A-C-C-AOH . The two isoacceptors differ by the presence of a G or a Q in the wobble position. Both contain a partial modification in position 54 (U/T). Thus, these tRNAs are transcribed from a single gene (or many genes with identical sequences). A fast and sensitive postlabeling method for sequencing tRNA anticodons is described. Nuclease S-1-treated tRNA is labeled with 5[32P]-pCp using T-4 RNA ligase. The tRNA fragments are then separated on 7 M urea/20% PAA gels. After autoradiography the RNA is eluted and digested with T-2 RNase. The nature of the labeled nucleotides is determined by two-dimensional thin-layer chromatography. The same method can be used to determine the 5' sequence of a tRNA by 3' labeling 5' tRNA halves with 5[32P]-pCp and subsequent chemical sequencing.

Animals↗
PubMed1990

Regional differences in in vivo myocardial protein synthesis in the neonatal rabbit heart.

Left ventricular free wall (LV) myofibrillar proteins accumulate at a rate approximately 2.5 times faster than the rate of right ventricular free wall (RV) proteins during postnatal cardiac development of the rabbit heart. In this study, the contribution of regional differences in in vivo protein synthesis to the differential rates of growth of the RV and LV myocardium was assessed in 4-d, 3-wk, and 9-wk old rabbits. In vivo total protein fractional synthetic rates were measured by a modification of the flooding infusion method, with calculations based upon the rate of equilibration of plasma leucine and cardiac leucyl-tRNA specific radioactivities following intravenous administration of a large dose of labeled amino acid. This method was also applied to the analysis of the fractional synthetic rate of a contractile protein subunit (myosin heavy chain) in the RV and LV of 4-d and 9-wk old rabbits. Accelerated growth of the LV during the first week of postnatal cardiac development was associated with increased total protein fractional synthetic rates (35.3 +/- 3.6 vs. 45.5 +/- 4.7%/day for RV and LV total protein of 4-d old rabbits, respectively). In addition, RV and LV fractional synthetic rates for myosin heavy chain in the same 4-d old rabbits were considerably greater than those values observed for total protein (84.0 +/- 13.1 and 98.4 +/- 16.9%/day for RV and LV myosin heavy chain, respectively. However, the fractional synthetic rates of both total protein and myosin heavy chain in both the RV and LV rapidly declined during the transition from fetal to adult hemodynamics. Thus, regional and developmental differences in myocyte-specific protein synthesis both contribute to the extensive restructuring of ventricular muscle during this period of extremely rapid growth. These differences may be in response to regional and developmental changes in hemodynamic load.

Aging↗
PubMed1984

Sucrose modifies ribosomal stability and conformation.

High concentrations of sucrose have a strong protective effect on heat-induced modifications of rat liver ribosomal subunits. They prevent to a large extent subunit inactivation, measured by poly (U)-dependent [14C] Phe tRNA binding (40S subunits) and puromycin reaction (60S subunits), subunit unfolding into light forms, and the release of both free and protein-complexed 5S RNA. They also increase the temperature at which subunits start to melt. Our data indicate that sucrose affects subunit conformation.

Animals↗
PubMed1983

Competence and progression growth factors stimulate different tRNAlys modification reactions in BALB/C 3T3 cells.

Sparse cultures of Balb/C 3T3 cells were growth arrested in a medium containing 1% calf plasma. This treatment caused a decrease in tRNA4lys, a tRNA species related to cell division, and a corresponding increase in tRNA5lys. A restoration of tRNA4lys levels and an increase in cell number was obtained by the addition of either 10% calf serum or a combination of fibroblast growth factor and melanocyte stimulating activity. Neither growth factor alone evoked a proliferative response. The progression factors, insulin and multiplication stimulating activity stimulated the rapid conversion of tRNA5lys to tRNA2lys, whereas the competence factors, fibroblast growth factor and platelet-derived growth factor stimulated a slight conversion of tRNA2lys to tRNA4lys.

Animals↗
PubMed1990

Replacement of the Escherichia coli trp operon attenuation control codons alters operon expression.

To test features of the current model of transcription attenuation in amino acid biosynthetic operons, alterations were introduced into the trp operon leader region and expression of the mutated operons was examined in miaA and miaA+ Escherichia coli strains that lacked the trp repressor. The miaA mutation prevents modification of the adenosine residue immediately 3' of the anticodon of tRNAs that interact with codons beginning with uridine. The undermodified tRNA(Trp) in miaA strains is thought to increase readthrough at the trp attenuator by slowing ribosome movement over two tandem Trp codons in the 14-codon leader peptide coding region. The rate of translation of these two "control codons" is thought to be the key step in determining the extent of transcription attenuation in the trp leader region. Sequential deletion of trpL DNA specifying the leader peptide initiation region, RNA segment 1, RNA segment 2 and RNA segment 3 alternately decreased and increased trp operon expression, a result consistent with previous findings in another bacterium and the generally accepted model for transcription attenuation. Replacement of the tandem Trp control codons by AGG-UGC (Arg-Cys) codons eliminated the miaA-dependent increase in transcription readthrough. Replacement of the Trp control codons by AGG-UGA (Arg-stop) codons caused complete readthrough at the trp attenuator as well as abolishing the miaA effect. Presumably, the ribosome terminating translation at the new UGA codon mimics the effect of a stalled ribosome at the Trp control codons. This finding suggests that ribosome dissociation at some stop codons is slow relative to the time required for transcription of the trp leader region. Thus, most ribosomes translating the trp leader peptide coding region may remain attached to the natural UGA stop codon until after the attenuation decision is made. The interpretation supports models for trp operon attenuation in which the elevated basal level readthrough is determined by occasional ribosome release prior to synthesis of the 3:4 terminator hairpin.

Amino Acid Sequence↗
PubMed2002

Chemical modification of reveromycin A and its biological activities.

Various derivatives of reveromycin A, a novel inhibitor of eukaryotic cell growth, were prepared and their inhibitory effects on both isoleucyl-tRNA synthetase activity and in vitro protein synthesis, and activities on the morphological reversion of src(ts)-NRK cells were assayed. The C5 hydroxyl group and C24 carboxyl group are particularly important for these activities.

Animals↗
PubMed2000

Synthesis and biophysical characterization of tRNA(Lys,3) anticodon stem-loop RNAs containing the mcm(5)s(2)U nucleoside.

[reaction: see text] Phosphoramidite reagents of the naturally occurring modified nucleosides mcm(5)s(2)U and mcm(5)U were synthesized and along with pseudouridine were incorporated into 17-nucleotide lysine tRNA anticodon stem-loop domains. Standard RNA phosphoramidite coupling chemistry allowed us to systematically investigate the thermodynamic effects of nucleoside modification and to correlate thermodynamic trends with qualitative structure effects seen by NMR spectroscopy.

Anticodon↗
PubMed2001

Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs.

Translational stop codon readthrough provides a regulatory mechanism of gene expression that is extensively utilised by positive-sense ssRNA viruses. The misreading of termination codons is achieved by a variety of naturally occurring suppressor tRNAs whose structure and function is the subject of this survey. All of the nonsense suppressors characterised to date (with the exception of selenocysteine tRNA) are normal cellular tRNAs that are primarily needed for reading their cognate sense codons. As a consequence, recognition of stop codons by natural suppressor tRNAs necessitates unconventional base pairings in anticodon-codon interactions. A number of intrinsic features of the suppressor tRNA contributes to the ability to read non-cognate codons. Apart from anticodon-codon affinity, the extent of base modifications within or 3' of the anticodon may up- or down-regulate the efficiency of suppression. In order to out-compete the polypeptide chain release factor an absolute prerequisite for the action of natural suppressor tRNAs is a suitable nucleotide context, preferentially at the 3' side of the suppressed stop codon. Three major types of viral readthrough sites, based on similar sequences neighbouring the leaky stop codon, can be defined. It is discussed that not only RNA viruses, but also the eukaryotic host organism might gain some profit from cellular suppressor tRNAs.

Animals↗
PubMed1985

Response of a phage modification factor to enhanced production of its target molecule.

Escherichia coli cells with plasmids bearing the valS gene were constructed from the Clark-Carbon collection. Their increased valyl-tRNA synthetase (EC 6.1.1.9) activity was not matched by commensurate production of a modifying peptide during T4 infection. Thus, phage vs gene expression is set to modify the normal amount of valyl-tRNA synthetase activity.

Amino Acyl-tRNA Synthetases↗
PubMed2003

The tRNA-like domains of E coli and A.aeolicus transfer-messenger RNA: structural and functional studies.

Transfer-messenger RNA (tmRNA, 10Sa RNA or ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The understanding at molecular level of this ubiquitous quality control system in eubacteria requires structural information. Here, we describe the purification and structural analysis of a functional fragment of both Aquifex aeolicus and Escherichia coli tmRNA, recapitulating their tRNA-like domain, which were expressed in vivo from synthetic genes. Both recombinant RNA are correctly processed at both 5' and 3' ends and are produced in quantities suitable for structural analysis by NMR and/or X-ray crystallography. The sequence and solution structure of the tRNA-like domains were analysed by various methods including structural mapping with chemical and enzymatic probes and 2D NMR spectroscopy. The minimalist RNAs contain two post-transcriptional base modifications, 5-methyluridine and pseudouridine, as the full-length tmRNA. Both RNAs fold into three stems, a D-analogue, a T-loop and a GAAA tetra-loop. 2D NMR analysis of the imino proton resonances of both RNAs allowed the assignment of the three stems and of a number of tertiary interactions. It shows the existence of interactions between the TPsiC-loop and the D-analogue, exhibiting a number of similarities and also differences with the canonical tRNA fold, indicating that RNA tertiary interactions can be modulated according to the sequence and secondary structure contexts. Furthermore, the E.coli minimalist RNA is aminoacylatable with alanine with a catalytic efficiency an order of magnitude higher than that for full-length tmRNA.

Alanine↗