anticodon and 5-methylaminomethyluridine

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNA

Topics: Anticodon; Bacteriocins; Base Pairing; Binding Sites; Colicins; Escherichia coli; Gene Expression Regulation, Bacterial; Molecular Docking Simulation; Nucleic Acid Conformation; Peptide Elongation Factor Tu; Plasmids; Protein Binding; Protein Biosynthesis; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Ribosomes; RNA, Bacterial; RNA, Transfer, Arg; Substrate Specificity; Thiouridine; Uridine

Specificity of the codon-anticodon interaction is often modulated by post-transcriptional modification at the first position of the anticodon (position 34) of the tRNA molecules. The modification of U(34) into 5-aminomethyluridine derivatives facilitates the pairing of the base with a G at the third position of the codon (position III). It was proposed that this wobble pairing is dependent on the deprotonation of the uridine derivative [Takai and Yokoyama, Nucleic Acids Res., 31, 6383-6391 (2003)]. This seemed to explain many results from biochemical and genetic experiments. On the other hand, however, the geometry of the base pair between 5-methylaminomethyluridine and G(III) in a crystal of the ribosomal 30S subunit was quite different from the predicted one [Murphy IV et al., Nat. Struct. Mol. Biol., 11, 1186-1191 (2004)]. It is obvious that the deprotonation, if any, should be inefficient at the pH at which the crystal was made. Therefore, the crystal structure does not exclude the possibility that the pairs are primarily dependent on the deprotonation of the modified uridines at the physiological pH.

Topics: Anticodon; Base Pairing; Codon; Guanine; Models, Molecular; Nucleic Acid Conformation; Uridine

Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA(Lys3)(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL(Lys3)(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoyl-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contributed structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(Lys3)(UUU) to bind poly-A programmed ribosomes. Neither ASL(Lys3)(UUU)-t(6)A(37) nor ASL(Lys3)(UUU)-mnm(5)U(34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(Lys3)(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA(Lys). The completely unmodified anticodon stem and loop of human tRNA(Lys1,2)(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t(6)A(37). The data suggest that tRNA(Lys)(UUU) species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.

Topics: Adenosine; Anticodon; Binding Sites; Humans; Nucleic Acid Conformation; Protein Binding; Pseudouridine; Ribosomal Proteins; RNA, Ribosomal, 16S; RNA, Transfer, Lys; Structure-Activity Relationship; Thermodynamics; Uridine

5-Methylaminomethyluridine (mnm5U) exists in the first position of the anticodon (position 34) of Escherichia coli tRNA4Arg for codons AGA/AGG. In the present study, the temperature dependence of the ribose-puckering equilibrium of pmnm5U was analyzed by proton NMR spectroscopy. Thus, the enthalpy difference (delta H) between the C2'-endo and C3'-endo forms was obtained at 0.65 kcal.mol-1. By comparison of the delta H values of pU and pmnm5U, the 5-substitution was found to increase the relative stability of the C3'-endo form over the C2'-endo form significantly (by 0.56 kcal.mol-1). Furthermore, this conformational "rigidity" was concluded to depend on the 5'-phosphate group, because nucleoside U exhibits only a negligible change in the ribose-puckering equilibrium upon the 5-methylaminomethyl substitution. Further NMR analyses and molecular dynamics calculations revealed that interactions between the 5-methylaminomethyl and 5'-phosphate groups of pmnm5U restrict the conformation about the glycosidic bond to a low anti form, enhancing steric repulsion between the 2-carbonyl and 2'-hydroxyl groups in the C2'-endo form. This intrinsic conformational rigidity of the mnm5U residue in position 34 may contribute to the correct codon recognition.

Topics: Anticodon; Computer Graphics; Escherichia coli; Magnetic Resonance Spectroscopy; Models, Molecular; Nucleic Acid Conformation; Ribose; RNA, Transfer, Arg; Thermodynamics; Uridine