TRNA"" spectra

This chapter describes the application of P-NMR spectroscopy to the study of the conformation of transfer ribonucleic acids (tRNAs). The P-NMR spectrum of pure acceptor tRNA species has been shown to contain considerable fine structure (Figs. 1 and 2). High-resolution ip-NMR spectra by Gueron and Shulman (1975) and later by Salemink et al. (1979) and Gorenstein and Luxon (1979) revealed 16 individual phosphate resonances spread over 7 ppm that were not observed in earlier P tRNA spectra (Gueron, 1971 Weiner et al., 1974 Gorenstein and Kar, 1975). 

The temperature dependence of the chemical shifts for tRNA (Figs. 3 and 6) differs somewhat from tRNA". Peak C shows almost no temperature dependence of its chemical shift between 20 and 60°C (melting temperature 65°Q. Peak D shifts slightly upfield at higher temperature and peaks E and F both shift upfield with increasing temperature (as opposed to F shifting downfield in tRNA ). The E and F resonances also appear further upfield than in the tRNA" spectra peak E is at —0.2 ppm at 25 C and peak F is at —0.45 ppm. There are three peaks far upheld, T, U, and U, that are all temperature-dependent. 

The P spectra of tRNA plus Et ion at 31 C are shown in Fig. 9. The chemical shifts of the scattered peaks (see discussion of tRNA spectra in Gorenstein, Chapter 9) are virtually unaffected with one possible exception. However, a number of peaks undergo significant line broadening as a function of added Et (Fig. 10). The only peak in the downfield portion of the spectrum (+3.3 to —0.453 ppm) that broadens is peak E at 0.245 ppm. The linewidth of this peak goes from —IS Hz at an Et/tRNA ratio of 0 to >25 Hz when the Et/tRNA ratio is increased to 1.32. At an Et/tRNA ratio of 2.0, the peak cannot be detected as a separate peak. It is possible that the chemical drift of peak E moves upheld to coincide with peak F at —0.077 ppm. However, the intensity of pe F with added Et increases only slightly. Tbis can be explained by the superposition of a broadened peak E under the sharper peak F. The entire upheld portion of the spectrum (— 1.85 to—4.53 ppm) broadens and loses resolution as the Et/tRNA ratio increases. Peaks... 

Beyer, D., Kroll, H. P., Endermann, R., Schiller, G., Siegel, S., Bauser, M., Pohlmann, J., Brands, M., Ziegelbauer, K., Haebich, D., Eymann, C., and Brotz-Oesterhelt, H. (2004). New class of bacterial phenylalanyl-tRNA synthetase inhibitors with high potency and broad-spectrum activity. Antimicrob. Agents Chemother. 48, 525—532. 

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.

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.

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. 

The purine ring system is undoubtedly among the most ubiquitous of all the heterocyclic compounds. This arises not only from the universal occurrence of adenine and guanine in DNA and RNA and of additional modified derivatives in the various tRNAs but also from the subsidiary uses of the ring system in very many biochemical systems Indeed across the whole spectrum of biochemical reactions in living systems there is hardly a reaction sequence which does not involve in some way a purine derivative such as the adenosine or guanosine mono-, di- and tri-phosphates, associated cyclic phosphates and nucleotide coenzymes. 

B) A similar spectrum for a 35-residue "minihelix" that contains the acceptor stem of the tRNA and seven fluorouracils. 

The planar structure, except the geometry of a C8 double bond, was initially proposed shown in Fig. (9) by the detailed analysis of ID and 2D NMR (COSY, HMQC and HNBC) data and by comparison of its and NMR spectra with those of structurally related compounds such as aerothionin (18), purealin (19) and others [30-41]. Although the DQF-COSY spectrum of 17 showed few H- H correlations, the HMBC spectrum was very useful for elucidating the planar structure of 17. The geometiy of C8 double bond was elucidated by a NOESY H7 /NH correlation to be trnas. The relative stereochemistry of 17 was clarified by... 

The tetracyclines block the attachment of aminoacyl tRNA to the acceptor site on the bacterial ribosome. They are broad-spectrum drugs with good activity against chlamydial and mycoplasmal species, as well as against other indicated bacteria. Doxycycline is of particular use in the treatment of prostatitis, minocycline is useful for treating meningococcal carrier states, and demeclocycline is useful for treating the syndrome of inappropriate secretion of ADH (SIADH). Their biodisposition and side effects are discussed. 

---