Conformational dynamics of the anticodon loop in yeast tRNAPhe as sensed by the fluorescence of wybutine.
Conformational and dynamic properties of the anticodon loop of yeast tRNAPhe were investigated by analyzing the time resolved fluorescence of wybutine serving as a local structural probe adjacent to the anticodon GmAA on its 3' side. The influence of Mg2+, important for stabilizing the tertiary structure of tRNA, and of the complementary anticodon s2UUC of E. coli tRNA2Glu were investigated. Fluorescence lifetimes and anisotropies were measured with ps time resolution using time correlated single photon counting and a mode locked synchronously pumped and frequency doubled dye laser as excitation source. From the analysis of lifetimes (tau) and rotational relaxation times (tau R) we conclude that wybutine occurs in various structural states: one stacked conformation where the base has no free mobility and the only rotational motion reflects the mobility of the whole tRNA molecule (tau = 6 ns, tau R = 19 ns), an unstacked conformation where the base can freely rotate (tau = 100 ps, tau R = 370 ps) and an intermediary state (tau = 2 ns, tau R = 1.6 ns). Under biological conditions, i.e. in the presence of Mg2+ and neutral salts, wybutine is found in a stacked and immobile state which is consistent with the crystallographic picture. In the presence of the complementary codon however, as exemplified by the E. coli-tRNA2Glu anticodon, our analysis indicates that the codon-anticodon complex exists in an equilibrium of structural states with different rotational mobility of wybutine. The conformation with wybutine freely mobile is the predominant one and suggests that this conformation of the codon-anticodon structure differs from the canonical 3'-5' stack.
D000926:Anticodon; D004926:Escherichia coli; D006147:Guanine / Q000031:analogs & derivatives; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer; D012346:RNA, Transfer, Amino Acyl; D012441:Saccharomyces cerevisiae; D013050:Spectrometry, Fluorescence
D000926:Anticodon; D012346:RNA, Transfer, Amino Acyl; C012099:wye; D006147:Guanine; D012343:RNA, Transfer