anticodon and 5-methylcytidine

The contributions of the natural modified nucleosides to RNA identity in protein/RNA interactions are not understood. We had demonstrated that 15 amino acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, anticodon domain of yeast tRNA(Phe) (ASL(Phe)). Affinity and specificity of the selected peptides for the modified ASL(Phe) have been characterized by fluorescence spectroscopy of the peptides' tryptophans. One of the peptides selected, peptide t(F)2, exhibited the highest specificity and most significant affinity for ASL(Phe) modified with 2'-O-methylated cytidine-32 and guanosine-34 (Cm(32) and Gm(34)) and 5-methylated cytidine-40 (m(5)C(40)) (K(d) = 1.3 +/- 0.4 microM) and a doubly modified ASL(Phe)-Gm(34),m(5)C(40) and native yeast tRNA(Phe) (K(d) congruent with 2.3 and 3.8 microM, respectively) in comparison to that for the unmodified ASL(Phe) (K(d) = 70.1 +/- 12.3 microM). Affinity was reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide t(F)2's higher affinity for the ASL(Phe)-Cm(32),Gm(34),m(5)C(40) hairpin and fluorescence resonance energy transfer from its tryptophan to the hypermodified wybutosine-37 in the native tRNA(Phe) placed the peptide across the anticodon loop and onto the 3'-side of the stem. Inhibition of purified yeast phenylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast tRNA(Phe) corroborated the peptide's binding to the anticodon domain. The phage-selected peptide t(F)2 has three of the four amino acids crucial to G(34) recognition by the beta-structure of the anticodon-binding domain of Thermus thermophilus FRS and exhibited circular dichroism spectral properties characteristic of beta-structure. Thus, modifications as simple as methylations contribute identity elements that a selected peptide specifically recognizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacylation.

Topics: Anticodon; Binding Sites; Cytidine; Guanosine; Models, Chemical; Nucleic Acid Conformation; Nucleosides; Peptide Library; Peptides; Protein Binding; RNA, Fungal; RNA, Transfer, Phe

The role of modified nucleosides in tRNA structure and ion binding has been investigated with chemically synthesized RNAs corresponding to the yeast tRNA(Phe) anticodon stem and loop (tRNA(ACPhe). Incorporation of d(m5C) at position 14 of the stem of tRNA(ACPhe)-d(m5C14), CCAGACUGAAGAU-d(m5C14)-UGG, analogous to m5C40 in native tRNA(Phe), introduced a strong Mg2+ binding at a site distant from the m5C. A Mg(2+)-induced structural transition, detected by circular dichroism spectroscopy, was similar to that observed for the DNA analog of tRNA(ACPhe) (Guenther et al., 1992; Dao et al., 1992). In contrast, Mg2+ had little effect on unmodified tRNA(ACPhe)-rC14 or tRNA(ACPhe)-d(C14). Modified tRNA(ACPhe)-d(m5C14) bound two Mg2+ ions, and the binding was cooperative. The dissociation constant of the two Mg2+ ions from tRNA(ACPhe)-d(m5C14), 2.5 x 10(-9) M2, is the result of an RNA structure significantly stabilized by Mg2+ binding, delta G = -11.7 kcal/mol. The tRNA(ACPhe)-d(m5C14) structure, investigated by 1H NMR, had a double stranded stem of five base pairs and two additional base pairs across what was a seven membered loop in the unmodified tRNA(Phe)AC. Methylation of cytidine in the yeast tRNA(ACPhe) enables the molecule to form more than one conformation through a process regulated by Mg2+ concentration. Thus, the simplest of posttranscriptional modifications of tRNA, a methylation, is involved in a somewhat distant, internal-site Mg2+ binding and stabilization of tRNA structure, especially that of the anticodon stem and loop.

Topics: Anticodon; Base Sequence; Circular Dichroism; Cytidine; Kinetics; Magnesium; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Nucleic Acid Conformation; Oligoribonucleotides; RNA, Transfer, Phe; Saccharomyces cerevisiae

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Anticodon; Base Sequence; Cations, Divalent; Circular Dichroism; Cytidine; DNA; Hot Temperature; Magnesium; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Denaturation; RNA, Transfer, Phe; Saccharomyces cerevisiae; Spectrophotometry, Ultraviolet; Substrate Specificity