Fluorescent tRNA

The broad field of nucleic acid structure and dynamics has undergone remarkable development during the past decade. Especially in regard to dynamics, modem fluorescence methods have yielded some of the most important advances. This chapter concerns primarily the application of time-resolved fluorescence techniques to study the dynamics of nucleic acid/dye complexes, and the inferences regarding rotational mobilities, deformation potentials, and alternate structures of nucleic acids that follow from such experiments. Emphasis is mainly on the use of time-resolved fluorescence polarization anisotropy (FPA), although results obtained using other techniques are also noted. This chapter is devoted mainly to free DNAs and tRNAs, but DNAs in nucleosomes, chromatin, viruses, and sperm are also briefly discussed. 

The reader is referred to other reviews for detailed discussions of the electronic states and luminescence of nucleic acids and their constituents/0 fluorescence correlation spectroscopy/2) spectroscopy of dye/DNA complexes/0 and ethidium fluorescence assays/4,0 A brief review of early work on DNA dynamics as well as a review of tRNA kinetics and dynamics have also appeared. The diverse and voluminous literature on the use of fluorescence techniques to assay the binding of proteins and antitumor drugs to nucleic acids and on the use of fluorescent DNA/dye complexes in cytometry and cytochemistry lies entirely outside the scope of this chapter. 

An excellent review of tRNA structures and dynamics was presented in 1983 08°) o y subsequent fluorescence decay and FPA studies are reviewed here. The use of excitation transfer to measure intramolecular distances1 and the use of fluorescence as a probe of protein/tRNA interactions082 l85) lie outside the scope of this chapter. 

Since tRNA is more varied structurally than DNA, ethidium could reside in pockets as well as intercalate into double-strand regions. The fluorescence decay provides information about the type, or types, of binding sites occupied by ethidium. It is currently believed that the excited state of ethidium is quenched by proton transfer to the solvent0 86-1 and that its lifetime is reduced with increasing solvent exposure. If ethidium occupies two or more kinds of sites with different degrees of exposure to solvent, then its fluorescence decay is expected to be multiexponential. 

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... 

The fluorescence decay is multiexponential.(199 200) This is unequivocal evidence that the wyebutine base can be bound in at least two different conformations with different solvent shielding. Wells and Lakowicz(200) resolved two exponential components. They measured the normalized amplitudes and lifetimes for the wyebutine fluorescence at two different concentrations of added Mg2+ S° = 0.50, t, = 1.7 ns, = 0.50, and t2 = 5.9 ns with no added Mg2+ present, and S°i = 0.16, t, =0.6 ns, S2 = 0.84, and r2 = 6.0ns with 10 nM Mg2+. Since the 6ns component is the longest lifetime present, it must represent the conformation that shields the wyebutine to the greatest extent and is generally believed to involve a 3 stack of bases 34-38.w 199-2011 The fraction of the tRNA in this conformation increases when Mg2 + is added to the solution. This structure is also observed in crystal structures which include Mg2+.(202 204) In the other conformation(s), the wyebutine is more exposed to the solvent. A 5 stack, which does not include bases 37 and 38, is one possibility. The wyebutine base would be more exposed to the solvent and have a shorter fluorescence lifetime as a result. However, both NMR data(205 206) and chemical modification studies(207) are inconsistent with a 5 stack. For the moment, this matter is unresolved. 

The intersubunit rotation is required for translocation as ribosomes trapped in the nonrotated state by an engineered intersubunit disulfide bridge fail in tRNA-mRNA movement. Real-time observation of intersubunit movement by fluorescence resonance energy transfer (FRET) showed that intersubunit movement occurs concomitantly with hybrid state formation, and that the rotated state can be trapped by the antibiotic viomycin. Similarly to the fluctuation of tRNAs between classical and hybrid states, single-molecule studies have detected spontaneous intersubunit movement where the 3 OS subunit fluctuates between a rotated... 

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. ... 

Figure 6 A general method for expressing prokaryotic tRNAs in mammalian cells, (a) A type-3 Pol III promoter, the H1 promoter, was combined with other gene elements in different ways to express the Escherichia coii amber suppressor tRNA . The GFP gene containing the TAG mutation at a permissive site serves as a fluorescence reporter for amber suppression. Translation of full-length GFP generates green fluorescence and indicates the expression of functional Escherichia coii amber suppressor tRNA in cells, (b) Total fluorescence intensity of cells transfected with constructs shown in (a). The combination in tRNA4 yields the most efficient tRNA expression.

An effective approach to study tRNA dynamics that is amenable to measurements of changes in both kinetics and amplitude is by fluorescence labeling at a site-specific position as a probe for local environment changes, either within the tRNA molecule or in the contact with a binding partner. The clear advantage of the fluorescence approach is the ability to monitor environmental changes continuously and in real time. Such fluorescendy labeled tRNAs were first used to study tRNA reactions on the ribosome... 

This chapter describes methods of fluorophore labeling to D residues with improvements over the previous ones, and methods to introduce fluorophores to the CCA sequence. Both sequence motifs are general to tRNAs and are located in the hot spots for binding interactions, suggesting that these site-specific fluorescent labeling methods will have a broad range of utility in generating important information on tRNA dynamics. 

Fluorescent labeling of D residues in native and transcripts of tRNAs. 

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