PMC

Effect of base replacements at position 37 on the stability of A-site binding of pept-tRNA. Dissociation from the A site was measured at 37°C in buffer A (10 mM MgCl₂). Wild-type pept-tRNA (Y37) (•), pept-tRNA(Y37G) (▪), Y37A (□), Y37U (▴), Y37C (▵).

(A) Structure of the Y base (commonly used symbol for wybutine, systematic symbol yW; The RNA Modification Database, http://medlib.med.utah.edu/RNAmods) compared to G. (B) Arrangement of the Y base (Y37) in the complex of yeast tRNA^Phe with U6-mRNA in the A site of Thermus thermophilus ribosomes (); P-site tRNA is also shown. Y37 and U₆-mRNA are highlighted.

Mg²⁺ dependence of association and dissociation rate constants of A-site binding. (A) Dissociation rate constants, k_off, measured with wt pept-tRNA(Y37) (•) and with pept-tRNA(Y37G) (▪), Y37A (□), Y37U (▴), Y37C (▵) at 37°C. (B) Association rate constants, k_on calculated for 1 μM total concentration of ligands. Symbols as in A.

Mg²⁺ dependence of the anticodon–anticodon duplex formation between tRNA^Phe and tRNA^Glu2. (A) Time courses of complex formation between tRNA^Phe (yeast, anticodon 3′-AAG-5′) and tRNA^Glu2 (E. coli, anticodon 5′-mnm⁵s²UUC-3′) measured by stopped-flow monitoring the fluorescence of Y37 in tRNA^Phe (transient 4) and the dissociation of the complex with addition of tRNA^Phe (E. coli, anticodon 3′-AAG-5′) (transient 2) or with twofold buffer dilution (transient 3). Transients 1 and 5, controls measured by mixing yeast tRNA^Phe with noncomplementary E. coli tRNA^Gly (anticodon 5′-CCG-3′) or nonfluorescent E. coli tRNA^Phe with tRNA^Glu2, respectively. (B) Mg²⁺ dependence of the K_d of the tRNA^Phe(Y37)•tRNA^Glu2 complex formation (open circles) or of A-site binding (cf. Fig. 5C; closed circles).

Temperature dependence of the rate constants of A-site binding. (A) Rate constants with different pept-tRNA constructs measured at 10 mM Mg²⁺ and 37°C. Black bars, k_off; gray bars, k_on. (B) Temperature dependence of the dissociation rate constants, k_off, of wt pept-tRNA(Y37) at 6 mM Mg²⁺ (•) or pept-tRNA(−Y) at 20 mM Mg²⁺(○) and pept-tRNA(Y37G) (▪), Y37A (□), Y37U (▴), Y37C (▵) at 10 mM MgCl₂. (C) Temperature dependence of the association rate constants, k_on, calculated for 1 μM total concentration of the ligands. Symbols as in B.

Mg²⁺ dependence of the K_d of A-site binding. (A) Time courses of dissociation of pept-tRNA(Y37A) at 37°C. From top to bottom: 25, 20, 14, 10, 8 mM Mg²⁺. (B) Time courses of dissociation of pept-tRNA(Y37C) at 37°C. From top to bottom: 30, 26, 23, 18, 14, 12 mM Mg²⁺. (C) log(K_d) versus log[Mg²⁺] plot for wt pept-tRNA(Y37) (•), pept-tRNAs(Y37G) (▪), Y37A (□), Y37U (▴), Y37C (▵), −Y (○), and Y37C in the presence of paromomycin (⋄). The slopes of the plots give the numbers of Mg²⁺ ions involved in the interaction between pept-tRNA and the A site.

Temperature dependence of the K_d of A-site binding. (A) Time courses of dissociation of wt pept-tRNA(Y37) at 6 mM MgCl₂. From top to bottom: 0°C, 29°C, 31°C, 33°C, 35°C, 37°C, and 39°C. (B) Time courses of dissociation of pept-tRNA(Y37U) at 10 mM MgCl₂. From top to bottom: 15°C, 20°C, 23°C, 26°C, 30°C, 34°C, 37°C. (C) Temperature dependence of the K_d values of wt pept-tRNA^Phe(Y37) at 6 mM MgCl₂ (•) and extrapolated to 10 mM MgCl₂ from the linear Mg²⁺ dependence of K_d (Fig. 5) (★); pept-tRNA(−Y) at 20 mM MgCl₂ (○) and extrapolated to 10 mM MgCl₂ (⋄); pept-tRNA(Y37G)(▪), Y37A (□), Y37U (▴), Y37C (▵) at 10 mM MgCl₂.