Yeast tRNA

FIGURE 12.34 A general diagram for the structure of tRNA. The positions of invariant bases as well as bases that seldom vary are shown in color. The numbering system is based on yeast tRNA R = purine Y= pyrimidine. Dotted lines denote sites in the D loop and variable loop regions where varying numbers of nucleotides are found in different tRNAs. 

Lavery, R., A, Pullman, and B. Pullman. 1980. The Electrostatic Molecular Potential of Yeast tRNA. pheIII. The Molecular Potential and the Steric Accessibility Associated with the Phosphate Groups. Theor. Chim. Acta 57, 233. 

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

The Lester and Dougherty labs, which have collaborated to extend the suppression mutagenesis technique to Xenopus oocytes with remarkable success [30, 31], began with a suppressor tRNA ( MN3 ) designed for in vivo use and demonstrated that it functioned more effectively in the oocyte system than a yeast tRNA -derived suppressor tRNA. They have since developed an alternative suppressor based on tRNA " from Tetrahymena thermophila that has proven to be considerably more versatile, efficient and accurate in the oocyte system [32], as well as showing good suppression efficiency in E. coli transcription-translation reactions [33]. 

The base sequence and the tertiary structure of the yeast tRNA specific for phenylalanine (tRNA " ) is typical of all tRNAs. The molecule (see also p.86) contains a high proportion of unusual and modified components (shaded in dark green in Fig. 1). These include pseudouridine (T), dihydrouridine (D), thymidine (T), which otherwise only occurs in DNA, and many methylated nucleotides such as 7-methylguanidine (m G) and—in the anticodon—2 -0-methylguanidine (m G). Numerous base pairs, sometimes deviating from the usual pattern, stabilize the molecule s conformation (2). 

Figure 5-54 (A) An 19F NMR spectrum of the 76-residue E. coli tRNAVal containing 5-fluorouracil in 14 positions. Recorded at 47°C. The numbers above the resonances indicate the position in the sequence. (The sequence is not identical to that for the yeast tRNA shown in Fig. 5-30.) Modified from Chu et al.i9i Courtesy of Jack Horowitz.

Soon after the discovery of RNase Ti, it was suggested (70, 71) that it would become an important tool for the elucidation of nucleotide sequence in RNA. Indeed, since 1962 several workers have tried to use the enzyme for the nucleotide sequence analysis of RNA, especially in highly purified specific tRNA s. Finally, the brilliant research of Holley and his associates in 1965 resulted in the first elucidation of the complete nucleotide sequence of an RNA, alanine specific yeast tRNA, using RNase Ti as a main tool (29). Since then many successful elucidations of nucleotide sequence of various RNA s, using RNase Ti as a main tool, followed, and now the enzyme is well-known as an essential tool for the structural analysis of RNA. 

Friederich, M. W., and Hagerman, P. J. (1997). The angle between the anticodon and aminoacyl acceptor stems of yeast tRNA(Phe) is strongly modulated by magnesium ions. Biochemistry 36, 6090—6099. 

Other solutions yeast tRNA (1 pg/pL), S.O.C. medium (Gibco-BRL), LB medium, 1.2% agar-LB plate containing 100 pg/mL ampicillin. 

The deprotected oligonucleotide synthetic product is precipitated twice in ethanol, and a 0.5 fig/fd solution in water is prepared (concentration is measured from a UV absorption spectrum). One microliter of the oligo-deoxynucleotide solution is mixed with 2 fd of 10X PL, 5 fd of [y-32P]ATP (or [y-35S]ATP), 1 fd of T4 polynucleotide kinase, and 11 fd water. After incubation at 37 ° (for 45 min with [y-32P]ATP or for 2 hr with [y-35S]ATP), the reaction is stopped by the addition of 150 [A of 5 M ammonium acetate, pH 5.5, and 130 fd water and 10 fd of the yeast tRNA solution are added to the mixture before precipitation with 1 ml ethanol. After chilling at —70° for at least 15 min, the precipitate is collected by centrifugation (12,000 g, 15 min), redissolved, and submitted to two additional cycles of precipitation-redissolution. Finally, the precipitate is redissolved in 20 fd of gel loading mix and the mixture analyzed on a 8% acrylamide-7 Af urea slab gel in IX electrophoresis buffer, until the bromphenol blue has reached the middle of the gel. 

It is useful to note that lanthanides act as catalysts in some biochemical reactions like (i) depolymerization of RNA [14], (ii) cleavage of yeast tRNA [15], (iii) cleavage of phosphate group [14] from ATP in presence of Ce3+, (iv) increase in amide proton exchange in aspartyl-phenylalanine [16]. 

Figure 6 Sites of specific binding of Mg + ions in yeast tRNA . (a) secondary structure. (Ref i49. Reproduced by permission of American Society for Biochemistry Molecular Biology) (b) tertiary structure. (Ref 150. Reproduced by permission of Oxford University Press)...

Typical examples of the mixed outer-sphere interactions are present in the crystal structure of yeast tRNA . In contrast to a number of outer-sphere H-bonds between the metal-coordinated ligands and tRNA residues, these site-specific complexes contain only a few direct, inner-sphere bonds either to phosphate alone (Mg +), to phosphate and bases (Mn +), or to guanine alone (Pt +) (Figme 14). 

Figure 28.6 Structure of a tRNA molecule. The tRNA is a roughly cloverleaf-shaped molecule containing an anticoaon triplet on one "leaf" and an amino acid unit attached covalently at its 3 end. The example shown is a yeast tRNA that codes for phenylalanine. The nucleotides not specifically identified are chemically modified analogs of the four common nucleotides.

Wyosine (Nucleoside Yt) (Yeast tRNA " ) Doridosine Tedania digitata and Anisodoris nobilis) 78TL2579 ... 

Unique architectural features of tRNAs also can serve as identity elements (2). For example, the long variable loop of IRNA " interacts specifically with SerRS. In addition, the tertiary G15 G48 Levitt base pair in E. coli tRNA , and the triplet interaction in tRNA P, is formed between G45, and the G10 U25 parr confers identity. Occasionally, modified nucleotides can act as determinants, as in the case of coli tRNA , tRNA , tRNA T , and yeast tRNA . All of these tRNAs contain modifications in the anticodon loop. 

The potential utility of N-labelcd oligonucleotides to probe unique nucleic acid structure, drug binding, and nucleic acid- protein interactions has led to considerable interest in the development of routes to the requisite N-labeled monomers. In the purine series, chemically synthesized N 1-labeled hypoxanthine was incorporated into a yeast tRNA by fermentation and... 

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