(A) Polyadenylation reaction with radiolabeled (asterisk) tRNA_i^Met (0.5 nM tRNA_i^Met, 150 nM TRAMP, 2 mM equimolar ATP-Mg²⁺). Aliquots were removed at 1 min intervals and resolved on denaturing PAGE. Added adenosines are marked on the right.
(B) Left: contour plot of the fraction of the adenylated intermediates (A_i) vs. reaction time for the timecourse in panel A. The color bar shows the color progression from A_i = 0 to 0.2 (contours: A_i = 0.035, 0.070, 0.105, 0.140, 0.175). Right: contour plot for a simulated reaction with equal rate constants for each adenylation step (k = 1.5 min⁻¹).
(C) Quantitative analysis of individual adenylation steps. Kinetic scheme for the polyadenylation reaction. For corresponding equations and fitting of the data set see Materials and Methods. Plots show representative timecourses for selected species (A₀, A₁, A₂, A₁₁) from the reaction displayed in panel A. Lines indicate the fit.
(D) Observed rate constants for individual adenylation steps. Points represent averages for multiple independent experiments as shown in panel A. The error bars mark one standard deviation. The modulation of individual observed rate constants was independent of the order of addition of TRAMP, RNA, and ATP (Fig.S1C–E).
(E) Rate constants at TRAMP and ATP saturation (k_max) for individual adenylation steps Rate constants were determined from multiple reactions with increasing TRAMP and ATP concentrations. Error bars mark the deviation of values obtained at ATP and TRAMP saturation (Fig.S1F–L).
(F) Apparent ATP affinity (K_1/2^ATP) for individual adenylation steps. Values were determined from multiple reactions with increasing ATP concentrations (Fig.S1F–L). Error bars indicate the standard deviation.

(A) Polyadenylation of an RNA substrate consisting of a 16 bp duplex with 1 nt 3' terminal overhang (100 nM TRAMP, 2 mM ATP-Mg²⁺ and 0.5 nM RNA). The asterisk marks the radiolabel. The 16 nt top strand contained a 3' terminal 2',3'-dideoxy residue to prevent adenylation. Plots show rate constants at TRAMP and ATP saturation (k_max) and the apparent ATP affinity (K_1/2^ATP) for individual adenylation steps. Values were determined from multiple reactions with increasing TRAMP and ATP concentrations (Figs.S1F–L). Error bars indicate the standard deviation.
(B) Polyadenylation of a 23 bp RNA duplex with 1 nt 3’ overhang (100 nM TRAMP, 2 mM ATP-Mg²⁺ and 0.5 nM RNA, top strand with 3' terminal 2',3'-dideoxy residue). Plots correspond to those in panel A.
(C) Polyadenylation of a 24 nt ssRNA substrate (100 nM TRAMP, 2 mM ATP-Mg²⁺ and 0.5 nM RNA). Plots correspond to those in panel A.

(A) Polyadenylation of a 24 nt ssRNA substrate with four terminal adenosines (filled symbols) by TRAMP. For comparison, the identical substrate without the terminal adenosines is shown (open symbols, values identical to those in Fig.3C). Asterisks mark the radiolabel. Values were determined from multiple reactions with increasing TRAMP and ATP concentrations, error bars indicate the standard deviation. The dashed lines mark k₁ and k₅, A₁ and A₅, the arrows emphasize the shift of the peaks for adenylation rate constants and apparent ATP affinities by four nucleotides.
(B) Polyadenylation of a 24 nt ssRNA substrate with four consecutive adenosines 5 nt removed from the 3’ terminus (filled symbols). For comparison, values for the identical substrate without the terminal adenosines are shown (open symbols, same as panel A). Plots correspond to those in panel A.

(A) Reaction scheme illustrating the principle of processivity (P_1…n) for individual adenylation steps (T: TRAMP, A_0…n: adenylated RNA species, TA_0…n: TRAMP bound to the respective adenylated species, k_f^1…n: adenylation rate constant for individual step, k_diss^1…n: dissociation rate constant for individual step). For more experimental details see Materials and Methods, Supplementary Material, and Fig.S7.
(B) Processivity of TRAMP for individual adenylation steps with the 24 nt ssRNA substrate. The average number of steps (N), shown on the right, corresponds to the processivity according to: P = (N-1)/N (Ali and Lohman, 1997). The dotted line marks P = 0.5, N = 2. Processivity values are the average from multiple independent measurements, the error bars mark one standard deviation.
(C) Processivity of Trf4p/Air2p for individual adenylation steps of the 24 nt ssRNA substrate. Values are the average from multiple independent measurements, the error bars mark one standard deviation.
(D) Actual adenylation rate constants of TRAMP (filled circles) and Trf4p/Air2p (open circles) for individual adenylation steps with the 24 nt ssRNA substrate. Rate constants were calculated according to Eq.S1 with k_fⁿ + k_dissⁿ = k_maxⁿ (Ali and Lohman, 1997). Values shown were calculated from the data in Figs.3C, 4A and panels B, C, error bars mark one standard deviation.
(E) Dissociation rate constants of TRAMP (filled circles) and Trf4p/Air2p (open circles) for individual adenylation steps with the 24 nt ssRNA substrate. Rate constants were calculated with Eq.S7, using the values for Pⁿ and k_fⁿ determined in panels B–D. Error bars mark one corresponding standard deviation.
(F) Free activation enthalpies (ΔG^‡) for adenylation (upper panels) and dissociation (middle panels), and the free energy for ATP affinities (ΔG°, lower panels) for individual adenylation steps for TRAMP (left panels) and Trf4p/Air2p (right panels), measured for the 24 nt ssRNA substrate. Free activation enthalpies were calculated according to ΔG^‡ = −RT·ln(hk/k_bT) (R: gas constant, T: temperature, h: Planck constant, k rate constants determined in panels D,E, k_b: Boltzmann constant). Free energies for functional ATP affinities were calculated according to ΔG°= −RT·ln(1/K_1/2^ATP), using the ATP affinities (K_1/2^ATP) determined in Fig.3C (TRAMP) and Fig.4A (Trf4p/Air2p).
(G) Mtr4p effects on free activation enthalpies for adenylation (upper panel) and dissociation (middle panel), and on the free energies of functional ATP affinities (lower panels) for individual adenylation steps. The effect is expressed as difference in the respective free activation enthalpies and free energies shown in panel F, e.g., ΔΔG^‡ = ΔG^‡(TRAMP) − ΔG^‡(Trf4p/Air2p). The arrows on the right show how energy differences correspond to slower/faster rate constants and weaker/tighter ATP binding for each adenylation step.

(A) Experimental scheme to measure poly(A) tail lengths of pre-tRNA_i^Met in vivo by Sanger sequencing. The heterogeneous 3’ termini of pre-tRNA_i^Met (IMT1~4) are displayed, together with the sequence of the control substrate.
(B) Left panel: in vitro transcribed tRNA_i^Met with three 3'-terminal uridines, polyadenylated by TRAMP. The number of appended adenosines is marked. Right panel: Representative Sanger sequencing chromatogram for this RNA after the poly(A)-tail lengths measurement procedure shown in panel A. The dashed line at A₈ indicates the start of the decrease in the A signal and the increase in G signal.
(C) Representative sequencing chromatogram for the cellular pre-tRNA_i^Met sample. The dashed line at A₄ indicates the start of the decrease in A signal and the increase in G signal. Experiments were repeated multiple times and virtually identical chromatograms were obtained.

(A) Polyadenylation of the 24 nt ssRNA substrate (asterisk: radiolabel) with Trf4p/Air2p (100 nM Trf4p/Air2p, 2 mM ATP-Mg²⁺ and 0.5 nM RNA). Plots correspond to those in Fig.3. Values were determined from multiple reactions with increasing TRAMP and ATP concentrations (Figs. S1F–L), error bars indicate the standard deviation. As reference, the dashed line marks A₄.
(B) Polyadenylation of the 23 bp RNA duplex with 1 nt 3’ overhang by Trf4p/Air2p. The y-axis for the plot of apparent ATP affinities was broken to enable direct comparison of the identical reaction with wtTRAMP (Fig.3B).
(C) Polyadenylation of the 24 nt ssRNA substrate by TRAMP^Mtr4-20p (100 nM TRAMP^Mtr4-20p, 2 mM ATP-Mg²⁺ and 0.5 nM RNA).
(D) Polyadenylation of the 23 bp RNA duplex (1 nt 3’ overhang) by TRAMP^Mtr4-20p. The inset shows the data with 10-fold magnification in the y-axis, to enable direct comparison of the identical reaction with wtTRAMP (Fig. 3C).
(E) Polyadenylation of the 16 bp duplex (1 nt 3'-terminal overhang) by TRAMP^Mtr4-20p.

(A) Domain structure of Mtr4p (Weir et al., 2010). Domain names are shown. The blue bar represents E947.
(B) Crystal structure of Mtr4p in complex with ADP and 5 nt oligo(A). Molecule B from Weir et al. (2010) is shown. The domains are colored as in panel A. E947 is shown in blue and RNA in orange. The dashed circle marks the area magnified in panel C.
(C) Close up view of E947 and the 5 nt oligo(A). For clarity, only resides 945–1026 in the helical bundle domain are shown (gray). E947 is positioned to contact N6 of the 4^th adenine from the 5’ end (Weir et al., 2010).
(D) Polyadenylation of the 24 nt ssRNA substrate by TRAMP^Mtr4p(E947A) (100 nM TRAMP^Mtr4p(E947A), 2 mM ATP-Mg²⁺, 0.5 nM RNA).
(E) Rate constants at TRAMP^Mtr4p(E947A) and ATP saturation (k_max, upper panel), and apparent ATP affinity (K_1/2^ATP, lower panel) for individual adenylation steps. For comparison, values for wtTRAMP are shown (open shapes). Values were determined from multiple independent reactions, error bars indicate the standard deviation.