Yeast tRNA from

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]. 

For most of the history of mankind, unraveling the nucleotide sequence of even a quite small nucleic acid was a formidable undertaking. Following 7 years of labor, Robert Holley solved the first such structure, that for an alanine tRNA from yeast, in 1961. This molecule contains a linear chain of 76 nucleotides and includes some unusual bases, which actually help in base sequence determination. For this achievement, Holley shared the Nobel Prize in Physiology or Medicine in 1968. 

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). 

Yeast alanine tRNA (tRNA 3), the first nucleic acid to be completely sequenced (Fig. 27-11), contains 76 nucleotide residues, 10 of which have modified bases. Comparisons of tRNAs from various species have revealed many common denominators of structure (Fig. 27-12). Eight or more of the nucleotide residues have modified bases and sugars, many of which are methylated derivatives of the principal bases. Most tRNAs have a guanylate (pG) residue at the 5 end, and all have the trinucleotide sequence CCA(3 ) at the 3 end. When... 

In all tRNAs the bases can be paired to form "clover-leaf" structures with three hairpin loops and sometimes a fourth as is indicated in Fig. 5-30.329 331 This structure can be folded into the L-shape shown in Fig. 5-31. The structure of a phenylalanine-carrying tRNA of yeast, the first tRNA whose structure was determined to atomic resolution by X-ray diffraction, is shown.170/332 334 An aspartic acid-specific tRNA from yeast,335 and an E. coli chain-initiating tRNA, which places N-formyl-methionine into the N-terminal position of proteins,336,337 have similar structures. These molecules are irregular bodies as complex in conformation as globular proteins. Numerous NMR studies show that the basic... 

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.

A third elongation factor, eEF3, which is an ATPase, is required by yeast and fungi.408-410 The 1044-residue yeast protein may be required for ATP-dependent release of deacylated tRNA from the exit site. 

FIGURE 28.11 Phenylalanine tRNA from yeast, (a) A schematic drawing showing the sequence of bases. Transfer RNAs usually contain a number of modified bases ( gray circles). One of these is a modified guanosine (G ) in the anticodon. Hydrogen bonds, where present, are shown as dashed lines, (b) The structure of yeast tRNAphe as determined by X-ray crystallography. 

In one of the earliest examples of the application of AUC to study RNA folding, Henley et al. (1966) demonstrated that the s2o,w values of tRNA molecules (unfractionated tRNAs from yeast) decreased with... 

Figure 10.29 Diagram of tRNA from yeast, specific for alanine. I, inosine V pseudouridine mG, methylguanosine m2G, dimethylguanosine T, ribothymidine hU, dihydrouridine ml, methylinosine.

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]. 

Both fluorescence and NMR techniques have been used in studies involving lanthanide-RNA interactions. Fluorescence enhancement of several hundred fold was observed on interaction of Tb3+ and Eu3+ with tRNA from E. coli. Enhancement was not observed in the case tRNA from yeast. From the fluorescence measurements the conclusion [105] that there are four tight equivalent sites for Eu3+ per tRNA molecule containing approximately 80 nucleotides with Kd of 6 x 10-6 M. X-ray studies [61] also indicate four lanthanide binding sites on tRNA. 

Fig. 20.8. Three-dimensional structure of phenylalanine specific tRNA from yeast. Watson-Crick type base pairs indicated by slabs, nonstandard base-base interactions that stabilize the tertiary structure are denoted a to h. Invariant and semi-invariant nucleotides are shaded, the four double helical regions are indicated by a a-(amino add) arm, Tarm, D arm, a.c. (anticodon arm [696]...

Total tRNA and 2% tRNA from yeast and bull s liver were obtained according to a previously described procedme [5]. An enriched fraction of specific tRNA was obtained by preparative electrophoresis in polyacrylamide gel [6]. The enriched tRNA preparations contained from 75 to 150 nmoles of tRNA. 

FIGURE 3.20 The structure of yeast tRNA (a) the cloverleaf form of the base sequence tertiary base-pairing interactions are represented by thin red lines connecting the participating bases. Bases that are conserved in all tRNAs are circled by solid and dashed lines respectively. The different parts of the structure are colour-coded, (b) The X-ray structure showing how the different base-paired stems are arranged to form an L-shaped molecule. The sugar-phosphate backbone is represented as a ribbon with the same colour scheme as in (a). (From Voet Voet, 2004. Reproduced with permission from John Wiley and Sons, Inc.)... 

As for dot blot hybridization, RNA probes can be used to detect RNA targets but these probes could not be stripped from the membrane due to the higher stability of RNA RNA hybrids (Srivastava and Schonfeld, 1991). Moreover, RNA probes make very stringent washes mandatory (e.g., (pre)hybridization at 60°C and in 5 X SSPE, 50% formamide, 0.2% SDS, 200 xg/ml denatured carrier DNA and 200 (ig/ml yeast tRNA and washes, after a hot rinse , in 0.1 X SSPE, 0.1% SDS at 60°C) and may still lead to spurious binding of probes to rRNA. Normalization of blots by rehybridizing with a probe to a cellular function suffers from the variation in the level of expression of some genes. Instead, jS-actin or 28S rRNA probes (Barbu and Dautry, 1989) are suggested. 

Figure 4.1 depicts the cloverleaf structure of a tRNA the bars represent base pairs in the stems. There are four arms and three loops - the acceptor, D, T pseudouridine C, and anticodon arms, and D, T pseudouridine C, and anticodon loops. Sometimes tRNA molecules have an extra or variable loop (shown in yellow in Fig. 4.1). The synthesis of transfer RNA proceeds in two steps. The body of the tRNA is transcribed from a tRNA gene. The acceptor stem is the same for all tRNA molecules and added after the synthesis of the main body. It is replaced often during lifetime of a tRNA molecule. The 3-D structure of a yeast tRNA molecule, which can code for the amino acid serine, shows how the molecule is folded with the...

Binding buffer Selection buffer supplemented with 2pg/ mL of yeast tRNA (Invitrogen) and 125 pg/mL of BSA (Bovine serum albumin from Sigma-Aldrich). 

In addition to the variety of modifications found on the heterocyclic base discussed above, many nucleosides are also methylated at the 2 -hydroxyl of the ribose moiety and make up approximately 8 % of existing tRNA modifications. One interesting example of the 2 - 0-ribose modification was studied in S. cerevisiae, where the yeast tRNA molecules corresponding to His, Pro, and Gly(G-C-C) contain a 2 -0-methylated nucleoside at position 4 in the acceptor stem. A methylated cytosine is found in tRNA ° and tRNA, and the modified Am nucleoside is found in tRNA . Modifications in a duplex region of tRNA are very rare, yet modification at this position ( 4) is conserved in eukaryotes. A yeast knock-out strain of the gene trmlS was produced, and it was determined by HPLC and primer extension analysis that tRNAs purified from this organism did not exhibit the 2 - 0-methyl modification at position 4. 

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