Presence of phosphorylatedO-ribosyl-adenosine In T-ψ-stem of yeast methlonine initiator tRNA

We report in this paper on isolation and characterization of two unknown nucleosides G* and [A*] located in the T-psi-stem of yeast methionine initiator tRNA, using the combined means of HPLC protocols, real time UV-absorption spectrum, and post-run mass spectrometry by electron impact or fast atom bombardment. The G* nucleoside in position 65 was identified as unmodified guanosine. The structure of the unknown [A*] in position 64 was characterized as an isomeric form of O-ribosyl-adenosine by comparison of its chromatographic, UV-spectral and mass spectrometric properties with those of authentic O-alpha-ribofuranosyl-(1"----2')-adenosine isolated from biosynthetic poly(adenosine diphosphat…

Sequence of a new tRNALeu(U∗AA) from brewer's yeast

The nucleotide sequence of a new tRNA(Leu)(anticodon U*AA) from Saccharomyces cerevisiae which could recognize exclusively the UUA codon has been determined. Its primary structure is: pGGAGGGUUGm2GCac4CGAGDGmGDCDAAGGCm2(2)GGCAGACmUU*AAm1GA++ + psi CUGUUGGACGGUUGUCCGm5CGCGAGT psi CGm1A(orA)ACCUCGCAUCCUUCACCA. This tRNA has a large extraloop and contains 15 modified nucleotides. So far it is the third isoacceptor tRNA for leucine in yeast. It has 61% homology with tRNA(Leu)(anticodon m5CAA) and 63% homology with tRNA(Leu)(anticodon UAG), the two other known yeast tRNAs(Leu).

Use of two-dimensional thin-layer chromatography for the components study of poly(adenosine diphosphate ribose)

Two-dimensional thin-layer chromatography on cellulose plates has been used for separating and quantifying the three adenosine derivatives: AMP, phosphoribosyl AMP (PRAMP), and (PR)2AMP obtained by venom phosphodiesterase digestion of poly(ADP-ribose). In vitro synthesized polymer, up to 300 derivatives in length were studied. Some parameters of the complexity of poly(ADP-ribose) could be deduced from our results: (i) The first branching point appears in fragments of approximately 21 derivatives in length. (ii) The branching points are located at regular distances of approximately 41 derivatives from each other.

Determination of queuosine derivatives by reverse-phase liquid chromatography for the hypomodification study of Q-bearing tRNAs from various mammal liver cells

Three queuosine derivatives (Q-derivatives) have been found at position 34 of four mammalian so-called Q-tRNAs: queuosine (Q) in tRNA(Asn) and tRNA(His), mannosyl-queuosine (manQ) in tRNA(Asp), and galactosyl-queuosine (galQ) in tRNA(Tyr). An analytical procedure based on the combined means of purified tRNA isolation from liver cells and ribonucleoside analysis by reverse-phase high performance liquid chromatography coupled with real-time UV-spectrometry (RPLC-UV) was developed for the quantitative analysis of the three Q-derivatives present in total tRNA from liver tissues and liver cell cultures. Using this analytical procedure, the rates of Q-tRNA modification were studied in total tRNAs…

The primary structure of cytoplasmic initiator tRNAMetfromSchizosaccharomyces pombe

Presence and coding properties of 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um) in the wobble position of the anticodon of tRNA(Leu) (U*AA) from brewer's yeast.

AbstractThe unknown modified nucleoside U* has been isolated by enzymatic and HPLC protocols from tRNALeu(U*AA) recently discovered in brewer's yeast. The pure U* nucleoside has been characterized by electron impact mass spectroscopy, and comparison of its chromatographic and UV-absorption properties with those of appropriate synthetic compounds. The structure of U* was established as 2′-O-methyl-5-carbamoylmethyluridine (ncm5Um). The yeast tRNALeu (U*AA) is the only tRNA so far sequenced which has been shown to contain ncm5Um. The location of such a modified uridine at the first position of the anticodon restricts the decoding property to A of the leucine UUA codon.

2'-O-methyl-5-formylcytidine (f5Cm), a new modified nucleotide at the 'wobble' of two cytoplasmic tRNAs Leu (NAA) from bovine liver.

The nucleotide analysis of a cytoplasmic tRNA(Leu) isolated from bovine liver revealed the presence of an unknown modified nucleotide N. The corresponding N nucleoside was isolated by different enzymatic and chromatographic protocols from a partially purified preparation of this tRNA(Leu). Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2'-O-methyl-5-formylcytidine (f5CM), and its reduced derivative as 2'-O-methyl-5-hydroxy-methylcytidine (om5Cm). By sequ…

Eukaryotic tRNAs(Pro): primary structure of the anticodon loop; presence of 5-carbamoylmethyluridine or inosine as the first nucleoside of the anticodon.

The modified nucleoside U*, located in the first position of the anticodon of yeast, chicken liver and bovine liver tRNA(Pro) (anticodon U*GG), has been determined by means of TLC, HPLC, ultraviolet spectrum and gas chromatography-mass spectrometry. The structure was established as 5-carbamoylmethyluridine (ncm5U). In addition, we report on the primary structures of the above-mentioned tRNAs as well as those which have the IGG anticodon. In yeast, the two tRNA(Pro) (anticodons U*GG and IGG) differ by eight nucleotides, whereas in chicken and in bovine liver, both anticodons are carried by the same 'body tRNA' with one posttranscriptional exception at position 32, where pseudouridine is asso…

Identification and structural characterization of O-beta-ribosyl-(1"----2')-adenosine-5"-phosphate in yeast methionine initiator tRNA.

We report in this paper on the complete structure determination of the modified nucleotide A*, now called Ar(p), that was previously identified in yeast methionine initiator tRNA as an isomeric form of O-ribosyl-adenosine bearing an additional phosphoryl-monoester group on its ribose2 moiety. By using the chemical procedure of periodate oxidation and subsequent beta-elimination with cyclohexylamine on mono- and dinucleotides containing Ar(p), we characterized the location of the phosphate group on the C-5" of the ribose2 moiety, and the linkage between the two riboses as a (1"----2')-glycosidic bond. Since the structural difference between phosphatase treated Ar(p) and authentic O-alpha-rib…

Identification of modifications in microbial, native tRNA that suppress immunostimulatory activity

2′-O-methylation of guanosine 18 is a naturally occurring tRNA modification that can suppress immune TLR7 responses.