TRNA E. coli

Cusack, S., Yaremchuk, A., and Tukalo, M. (1996). The crystal structures of T. thermophilus lysyl-tRNA synthetase complexed with E. coli tRNA(Lys) and a T. thermophilus tRNA (Lys) transcript anticodon recognition and conformational changes upon binding of a lysyl-adenylate analogue. EM BO J. 15, 6321—6334. 

Satisfactory fits of the fluorescence decays for ethidium bound to yeast tRNAPhe and E. coli tRNA al require (at least) two exponentials in the sum response S(t) [cf. Eq. (4.56)] under all conditions studied.0 870 88) The normalized amplitudes and lifetimes for tRNA 1 (extrapolated to zero concentration) are S° = 0.917, r, = 25.6ns and S02 = 0.082, t2 = 5.6 ns.(187) The results for tRNAPhe are similar.(188) This requirement for two (or more) exponentials is unequivocal evidence for at least two ethidium binding sites. The dominant component has a lifetime similar to, but slightly longer than, that of ethidium intercalated in DNA and is taken to represent ethidium... 

Removal of endogeneous Mg2+ by rigorous treatment (heating to 80 °C in the presence of 10 mM EDTA) introduces a fundamental difference between yeast tRNAPhe and E. coli tRNA]781, although their xR values remain... 

The first orthogonal E. coli tRNA-synthetase pair generated from archaeal bacteria was derived from the tyrosyl pair taken from Methanococcus janmschii In vitro experiments showed that the major recognition elements of M. jannaschii tRNA" include the discriminator base A73 and the first base pair, C1-G72, in the acceptor stem (Figure 2(a)). The anticodon triplet participates only weakly in identity determination. By contrast, E. coli uses A73, G1-C72, a long variable arm, and the anticodon as identity elements. The M. 

This selection scheme was used to evolve the orthogonal E. coli tRNA u -TyrRS pair in yeast. A synthetase library (10 in size) was similarly constructed by randomizing five active-site residues in E. coli TyrRS corresponding to the five residues randomized in the Af/TyrRS. Mutant synthetases were identified after several rounds of positive and negative selection that incorporate a number of unnatural amino acids into proteins, albeit with rather low protein yields (about 0.05 mgl A similar approach has been used to evolve orthogonal E. coli leucyl tRNAcuA LeuRS pairs that selectively incorporate photochromic and fluorescent amino acids into proteins in yeast. ... 

A new method to efficiently express prokaryotic tRNAs in yeast involves an external Pol III promoter containing the consensus A- and B-box sequences (Figure 5(c)). When placed upstream of the E. coli tRNA (without the 3 -CCA trinucleotide), the promoter drives transcription of a primary RNA transcript consisting... 

Fig. 4. Comparison of E. coli tRNA and semi-synthetic suppressor tRNA y. The residues that have been changed are highlighted...

Figure 5-51 (A) The low-field region of the one-dimensional H NMR spectrum of E. coli tRNAjVal at 27°C in H20. Resonances are identified by letters A - X. (B) NOESY spectrum of the same tRNA under similar conditions showing the imino-imino NOEs. In the lower right sector the connectivity traces of the acceptor helix and dihydrouridine helix are shown as solid and dotted lines, respectively. In the NOESY sample the two protons in peak EF are partially resolved whereas the two protons in peak T have coalesced. (C) NOESY spectrum of E. coli tRNA,Val at 32°C showing the imino and aromatic proton regions. AU-type imino protons have been connected horizontally by a dotted line to the cross-peak of their proximal C2-H or C8-H in the 7 to 9 ppm region, which has been labeled with the corresponding lower-case letter. From Hare et al.669 Courtesy of Brian Reid.

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 synthesis and incorporation into RNA of three hypermodified tRNA nucleosides has been reported. The presence of the N -isopentyl modified adenosine (68, i A) in the anticodon stem-loop of E.coli tRNA alters metal ion binding compared to the unmodified stem-loop sequence.The presence of the N -lysine modified derivative (69), ms t A, found in tRNA of the specific RNA primer for HIV-1 RT has been shown to increase the stability of the hairpin.1-Methyladenosine has also been incorporated into RNA. ... 

RNA is precipitated from solution by adding one-tenth the volume of 2.5 M sodium acetate (pH 6.0) and 2.5 times the aqueous volume of ethanol. Mix well and leave on dry ice for 5 min, followed by centrifugation at 10,000 g for 15 min at 4°C. If a small amount of RNA (less than 0.1 i.g/ml) is going to be recovered, inclusion of either 20 p.g/ml E. coli tRNA or glycogen as a carrier is recommended. Wash the precipitate with 70% ethanol and centrifuge at 10,000 g for 5 min. Let remaining traces of ethanol evaporate (or use a speed-vac briefly), before the precipitate is dissolved in double-distilled H20 or TE buffer. 

E. coli tRNA (100x 5 p.g/p.1 RNase free Boehringer Mannheim) Bovine serum albumin (50x 10 xg/pl)... 

Purified RNA is redissolved at about 50 fmol/p.1 with 20 ng/ xl E. coli tRNA carrier. 

E. coli tRNA (RNase free 5 xg/ il) (Boehringer Mannheim)... 

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