Synthesis of RNA containing O-beta-D-ribofuranosyl-(1'-2')-adenosine-5'-phosphate and 1-methyladenosine, minor components of tRNA

tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is well-known based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-beta-D-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5'-O-phosphorylated 2'-O-(beta-D-ribofuranosyl)-beta-D-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA(Met) will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.     

The RNA Modification Database: 1999 update.

The RNA Modification Database (http://medlib.med.utah.edu/RNAmods/) provides a comprehensive listing of naturally modified nucleosides in RNA. Each file includes: chemical structure; common name and symbol; type(s) of RNA in which found and corresponding phylogenetic distribution; Chemical s registry number and index name; and initial literature citations for structure characterization and chemical synthesis. New features include capability to search database files by name or substructural features, modifications in tmRNA, and links to related data and sites.     

A unique method utilizing antinucleotide antibodies for evaluating changes in the levels of modified nucleosides of tRNAs from crude extracts of whole cells.

The utilization of antibodies directed toward modified nucleosides in evaluating changes in the levels of certain modified nucleosides in transfer RNA is reported. Antibodies directed toward the N6-(delta 2-isopentenyl)adenosine modification were used in this model system with a mutant strain of Escherichia coli designated ipaA. The procedure is rapid, sensitive, and specific. In addition, it does not depend on the existence of an in vitro remodification system or any radiochemical labeling of the tRNA. By varying the extraction technique, the method could be applied to procaryotic or eukaryotic cell lines. The existence of antibodies specific for other nucleoside modifications makes this a system that is potentially applicable to a variety of deficiencies in the modification of both tRNA and rRNA.     

Orientation of the tRNA anticodon in the ribosomal P-site: quantitative footprinting with U33-modified, anticodon stem and loop domains.

Binding of transfer RNA (tRNA) to the ribosome involves crucial tRNA-ribosomal RNA (rRNA) interactions. To better understand these interactions, U33-substituted yeast tRNA(Phe) anticodon stem and loop domains (ASLs) were used as probes of anticodon orientation on the ribosome. Orientation of the anticodon in the ribosomal P-site was assessed with a quantitative chemical footprinting method in which protection constants (Kp) quantify protection afforded to individual 16S rRNA P-site nucleosides by tRNA or synthetic ASLs. Chemical footprints of native yeast tRNA(Phe), ASL-U33, as well as ASLs containing 3-methyluridine, cytidine, or deoxyuridine at position 33 (ASL-m3U33, ASL-C33, and ASL-dU33, respectively) were compared. Yeast tRNAPhe and the ASL-U33 protected individual 16S rRNA P-site nucleosides differentially. Ribosomal binding of yeast tRNA(Phe) enhanced protection of C1400, but the ASL-U33 and U33-substituted ASLs did not. Two residues, G926 and G1338 with KpS approximately 50-60 nM, were afforded significantly greater protection by both yeast tRNA(Phe) and the ASL-U33 than other residues, such as A532, A794, C795, and A1339 (KpS approximately 100-200 nM). In contrast, protections of G926 and G1338 were greatly and differentially reduced in quantitative footprints of U33-substituted ASLs as compared with that of the ASL-U33. ASL-m3U33 and ASL-C33 protected G530, A532, A794, C795, and A1339 as well as the ASL-U33. However, protection of G926 and G1338 (KpS between 70 and 340 nM) was significantly reduced in comparison to that of the ASL-U33 (43 and 61 nM, respectively). Though protections of all P-site nucleosides by ASL-dU33 were reduced as compared to that of the ASL-U33, a proportionally greater reduction of G926 and G1338 protections was observed (KpS = 242 and 347 nM, respectively). Thus, G926 and G1338 are important to efficient P-site binding of tRNA. More importantly, when tRNA is bound in the ribosomal P-site, G926 and G1338 of 16S rRNA and the invariant U33 of tRNA are positioned close to each other.     

Metabolism of tRNA in rats with aflatoxin B1-induced hepatomas

This study describes effects of aflatoxin B1-induced hepatomas on RNA metabolism in rats. At 4 and 24 hours after the administration of L-(14CH3)-methionine, tRNA was isolated from the livers and hydrolyzed enzymatically to nucleosides which were quantitatively measured by HPLC. Radioactivity of the nucleosides was also determined. The data indicate that although tRNA methylation may be more rapid in livers with hepatomas, catabolism of tRNA in tumorous tissue is slower than in control livers. The large increase in some radioactive methylated nucleosides and bases by the tumor-bearing rats during the 24-hour period following the administration of labeled methionine indicates increased turnover of mRNA and rRNA as well as tRNA. Since degradation of tumor tRNA appears to be delayed, the excessive amounts of the urinary methylated nucleosides must be derived from RNA in nonneoplastic tissue.     

An embarrassment of riches: the enzymology of RNA modification

The maturation of transfer RNA (tRNA) involves extensive chemical modification of the constituent nucleosides and results in the introduction of significant chemical diversity to tRNA. Many of the pathways to these modified nucleosides are characterized by chemically complex transformations, some of which are unprecedented in other areas of biology. To illustrate the scope of the field, recent progress in understanding the enzymology leading to the formation of two distinct classes of modified nucleosides, the thiouridines and queuosine, a 7-deazaguanosine, is reviewed. In particular, recent data validating the involvement of several proposed intermediates in the formation of thiouridines are discussed, including two key enzyme intermediates and the activated tRNA intermediate. The discovery and mechanistic characterization of a new enzyme activity in the queuosine pathway is discussed.     

Role of modified nucleosides of yeast tRNAPhe in ribosomal binding

Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.     

Chemotactic responses of human polymorphonuclear leukocytes to cyclic GMP and other compounds

Some metabolic properties of small molecular weight nuclear RNA (snRNA) components have been studied in human lymphocytes cultured with PHA. Pulse-labelling experiments with ³H-uridine in 3 h-intervals around the onset of DNA synthesis showed no qualitative or quantitative differences in the snRNA labelling pattern. Long labelling experiment with ³H-methionine demonstrated the following relative degrees of methylation: tRNA (1.0), 5S RNA (0), D (0.3), 5.5S RNA (0.2), C (0.6), A (0.2), L (0) and rRNA (0.2). Chase-experiments with ³H-methionine showed that the snRNA components D, C and A are metabolically stable with half-lives of not less than 30 h. Actinomycin D (0.05 μg/ml) reduced markedly the synthesis of rRNA and 5 S RNA whereas the synthesis of D, C, A and L was unaffected or only slightly affected. Actinomycin D at a concentration of 0.25 μg/ml inhibited the synthesis of D, C and A. Cycloheximide (0.19 μg/ml) reduced the synthesis of D, C and rRNA to about 50% of control whereas 5S RNA synthesis was only slightly inhibited and tRNA synthesis was unaffected.     

Recombinant photoreactive tRNA molecules as probes for cross-linking studies

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.     

植物核小RNA研究进展

核小RNA (small nuclear RNA,snRA)是一类长度为60～300 nt的非编码RNA,是真核生物RNA剪接体的主要成分.snRNA在各种生物中具有较高的保守性和同源性,主要参与了mRNA和rRNA前体的加工过程.本文对当前植物中snRNA领域的研究成果进行简要概述,介绍了植物snRNA的分类、基因结...     

Complete analysis of tRNA-modified nucleosides by high-performance liquid chromatography: the 29 modified nucleosides of Salmonella typhimurium and Escherichia coli tRNA

A high-performance liquid chromatography (HPLC) method has been developed to quantify the major and modified nucleoside composition of total, unfractionated transfer RNA. The method is rapid and sensitive and offers a high degree of chromatographic resolution suitable for quantifying both stable and unstable modified nucleosides. It is nondestructive and allows the recovery of nucleosides for further characterization. We apply the method in the analysis of the 29 modified nucleosides in tRNA from Salmonella typhimurium (and Escherichia coli) and show it to be useful in examining changes in the modified nucleoside content of tRNA. Such changes may be important in regulation.     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.     

General screening procedure for RNA modificationless mutants: isolation of Escherichia coli strains with specific defects in RNA methylation.

A general method for the isolation of mutants of Escherichia coli that are defective in RNA modification is described. The method is based on the fact that RNA with specific undermodifications accumulates under nonpermissive growth conditions and that such a defect can be detected by remodification either in vivo at permissive conditions or in vitro. The method provides a means by which to study mutations affecting essential modification reactions. The usefulness of the method was demonstrated by the isolation of two rRNA and two tRNA methylation defective mutants. Both rRNA mutants accept methyl groups into their 23S rRNA in vitro. Analyses of in vitro methylated 23S rRNA from one of the mutants revealed the presence of several methylated nucleosides, of which 6-methyladenosine was the most abundant (40% of recovered radioactivity). In 23S rRNA from the other mutant, the only product formed in vitro was 5-methylcytidine. The tRNA mutants are characterized in the accompanying paper.