TRAMP displays modulated polyadenylation activity
To quantitatively characterize polyadenylation by TRAMP from S. cerevisiae
, we reconstituted the complex from recombinant components (Fig.S1A,B
). Polyadenylation activity of the reconstituted TRAMP was first measured using tRNAiMet
(). This RNA resembles one of the physiological targets of TRAMP, and had been previously used to detect polyadenylation activity of TRAMP obtained from yeast (Kadaba et al., 2004
; Wang et al., 2008
). Reactions were performed under pre-steady state conditions (i.e., enzyme excess over the substrate), because the kinetic description of this reaction regime contains fewer parameters than steady-state regimes and thus provides the most accurate quantitative data. Polyadenylation timecourses were analyzed by denaturing PAGE, to resolve polyadenylated species at single nucleotide resolution ().
Modulated polyadenylation activity by TRAMP
Plots of the fractions of polyadenylated species vs. reaction time revealed accumulation of species with 3 to 5 adenosines in a time window from approximately 1 to 3 minutes (). Over longer reaction times, the poly(A) tail grew to 15 nt and longer. The temporary accumulation of species with 3 – 5 adenosines suggested a modulation of the polyadenylation activity in response to the number of added nucleotides. To test this assertion, we determined rate constants for individual adenylation steps. A simple kinetic scheme consisting of a series of irreversible, pseudo-first order reactions faithfully described the experimental data (). The observed rate constants for individual adenylation steps (kobs) represent multiple physical processes, including adenylation and dissociation of TRAMP from the RNA.
Plots of rate constants vs. the corresponding number of added adenosines revealed a clear modulation of the polyadenylation activity. Rate constants increased for the first three steps, and then decreased to a fairly constant level (). The resulting peak in rate constants explains the temporary accumulation of RNA species with 3 – 5 adenosines in a straightforward manner: TRAMP forms these species relatively fast, but extends them only slowly.
To gain further insight into the molecular basis of the modulated polyadenylation activity, we examined the dependence of individual rate constants on the ATP concentration. We determined the functional affinity for ATP (K1/2,ATP
) for each adenylation step and each adenylation rate constant at ATP and TRAMP saturation (kmax
, , Fig.S1F–L
). The observed peak in polyadenylation rate constants at A3
broadened slightly at ATP saturation (). Most notable was a pronounced peak of low ATP affinity at A5/
(). This peak indicates an approximately 20-fold decrease in ATP affinity for the polyadenylation reaction at A5/
, compared to earlier and later adenylation steps. This drop in ATP affinity occurs immediately after the peak for the highest adenylation rate constant at A3
, revealing that the decrease in adenylation rate constants is accompanied by a marked reduction in ATP affinity. Thus, modulation of ATP affinity and adenylation rate constants synergistically favor the temporal accumulation of species with 3 – 5 adenosines.
Hypomethylated pre-tRNAiMet, a prototypical TRAMP target, accumulates poly(A) tails with approximately 4 adenosines in vivo
We next examined whether the physiological TRAMP target, hypomethylated tRNAiMet
) (Kadaba et al., 2004
; Kadaba et al., 2006
), accumulated similarly short poly(A) tails in vivo
. To measure the poly(A) tail lengths of cellular pre-tRNAiMet
with single base resolution, we adopted a 3’ RACE strategy (). We isolated total RNA from the yeast trm6-504
strain, where non-functional tRNA m1
A methylase Trm6p leads to accumulation of hypomethylated pre-tRNAiMet
, which is targeted by TRAMP (Kadaba et al., 2004
). Following the extension of the RNA 3' ends with guanosine-inosine tails, polyadenylated pre-tRNAiMet
were specifically amplified by RT-PCR. We accounted for the heterogeneity in the 3' ends of pre-tRNAiMet
(Kadaba et al., 2004
) by processing of the PCR products with the restriction enzyme Mse
I, which was possible because all precursors end with at least two 3' Us (). The isolated pre-tRNAiMet
poly(A) tails were ligated to a piece of synthetic DNA, amplified and subjected to Sanger sequencing to delineate the number of added adenosines ().
Accumulation of poly(A) tails with approximately 4 adenosines on hypomethylated pre-tRNAiMet in vivo
The method was calibrated with a tRNAiMet
processed in vitro
(gel panel in ). The corresponding sequencing chromatogram shows excellent agreement between input and final sequencing result (). The robustness of the method was further tested with longer, in vitro
generated poly(A) tail lengths, and similar agreements were seen (H.J. et al.
, unpublished results). We then measured the lengths of the poly(A) tails of pre-tRNAiMet
appended in vivo
(). The corresponding Sanger chromatogram indicates accumulation of RNA species with roughly 4 adenosines (), in excellent agreement with our polyadenylation measurements in vitro
(). Accumulation of similarly short poly(A) tails on other TRAMP targets in vivo
had been observed by others (Grzechnik and Kufel, 2008
; Lebreton et al., 2008
; Wlotzka et al., 2011
). The striking correlation between the poly(A) tail lengths distribution of TRAMP targets in vivo
and the temporary accumulation of short poly(A) tails in vitro
is consistent with the notion that TRAMP displays modulated polyadenylation activity in the cell as well.
Modulated polyadenylation activity with generic model substrates
To investigate how polyadenylation activity by TRAMP was modulated, we next examined in vitro whether the modulation was specific for physiological TRAMP targets, or if TRAMP also polyadenylated simple model RNAs in a similar fashion. First, we tested a substrate consisting of a 16 bp duplex with a single nucleotide overhang at the 3’ end (). The protruding nucleotide was necessary to obtain appreciable levels of polyadenylation. On a 16 bp blunt end duplex, TRAMP displayed exceedingly low, unquantifiable activity (H.J. et al., unpublished results).
Modulated polyadenylation activity with generic model substrates
Adenylation rate constants (kmax) for the RNA duplex with the single nucleotide overhang displayed a clear peak, and ATP affinities for individual adenylation steps (K1/2,ATP) showed a pronounced peak of low ATP affinity (), as seen for the tRNAiMet substrate (). While both peaks were slightly shifted, compared to the tRNAiMet substrate, the sharp decrease in ATP affinity coincided again with the decrease in adenylation rate constants (). Extending the duplex to 23 bp had little effects on overall adenylation rate constants, presence of the characteristic peak in adenylation rate constants, and the corresponding decrease in ATP affinity, although the peaks were slightly shifted, compared to the 16 bp duplex (). The data obtained with these simplified model substrates clearly indicated that modulated polyadenylation activity is not restricted to physiological targets, but an inherent feature of TRAMP.
The slight shifts of the peaks for polyadenylation rate constants and ATP affinities for the different substrates suggested potential effects of RNA structure on the modulation. It had been shown that RNA structure affects Mtr4p binding (Weir et al., 2010
), but it was also possible that the modulation with the tested substrates was caused by Mtr4p-mediated duplex unwinding. To test whether the modulation of polyadenylation depended on duplex unwinding, we measured polyadenylation of a single-stranded RNA. If modulation required unwinding, then the absence of duplexes would eliminate or drastically change the modulation. While TRAMP displayed only weak, unquantifiable activity on a 17 nt ssRNA (Fig.S3D,E
), a 24 nt ssRNA was robustly adenylated (). Both adenylation rate constants (kmax
) and ATP affinities for the individual adenylation steps (K1/2,ATP
) displayed the peaks indicating modulated polyadenylation (). Similar modulation was seen for other ssRNAs longer than 17 nt (H.J. et al.
, unpublished results). The modulated polyadenylation activity on single stranded RNA indicates that the modulation is not based on duplex unwinding by TRAMP. Notwithstanding, unwinding could still contribute to a small extent to the observed slight influence of RNA secondary structure on TRAMP activity.
The modulation of polyadenylation activity depends on Mtr4p
If duplex unwinding was not causing the modulation of polyadenylation activity, did the helicase Mtr4p affect the modulation at all? To answer this question, we measured polyadenylation by a TRAMP complex without Mtr4p (Trf4p/Air2p). With the 16 bp duplex substrate described above, Trf4p/Air2p showed only low, unquantifiable levels of polyadenylation activity (Fig.S4A,B
). The 24 nt ssRNA and the 23 bp substrates were robustly polyadenylated (). With both substrates, Trf4p/Air2p produced longer poly(A) tails than TRAMP over comparable timeframes (). Adenylation rate constants increased for the first two steps, but did not produce the characteristic peak seen with complete TRAMP (). Similarly absent was the peak for ATP affinities (). These observations demonstrate that Trf4p/Air2p does not display the modulated polyadenylation activity seen with complete TRAMP, thus indicating a critical role of Mtr4p in the modulation. In striking correlation with our observations, Mtr4p depletion in vivo
causes hyperadenylation of TRAMP targets (Houseley and Tollervey, 2006
Removal or mutation of Mtr4p diminishes modulation of polyadenylation activity
To illuminate how Mtr4p contributed to the modulation, we examined a TRAMP complex with a mutated Mtr4p (TRAMPMtr4-20p
). The Mtr4-20p mutation, located in the helicase motif VI, strongly decreases unwinding and RNA-stimulated ATPase activities of Mtr4p, but TRAMPMtr4-20p
retains polyadenylation activity (Wang et al., 2008
). With all substrates tested, TRAMPMtr4-20p
generated longer poly(A) tails than wtTRAMP at comparable reaction times (). With the 24 nt ssRNA and the 23 bp substrates, polyadenylation rate constants and ATP affinities showed only very broad peaks, and there was only little, if any, coordination between changes in rate constants and ATP affinity that was seen with wtTRAMP (). With the 16 bp duplex substrate, no peaks in adenylation rate constants or ATP affinities were seen (). These results indicate that the Mtr4-20p mutation causes a precipitous loss in the capacity of TRAMP to modulate polyadenylation activity. This observation provides further evidence that Mtr4p plays a critical role in modulating TRAMP polyadenylation activity. Moreover, the data reveal that the presence of Mtr4p in TRAMP alone is not sufficient to modulate polyadenylation. The modulation apparently requires Mtr4p with intact coordination between RNA and ATP binding sites, which is impaired in the Mtr4-20p mutant (Jackson et al., 2010
; Wang et al., 2008
; Weir et al., 2010
The modulation of polyadenylation activity depends on the number of 3’-terminal adenosines
Having implicated Mtr4p in the modulation of polyadenylation by TRAMP, we next asked how TRAMP determined at which steps to decrease adenylation rate constants and ATP affinities. A central point in this regard was whether TRAMP adjusted its activity only for adenosines that it appended or also for adenosines already present in the RNA. To distinguish between these possibilities, we measured rate constants and ATP affinities for individual adenylation steps with a 24 nt ssRNA substrate containing four 3' terminal adenosine residues (). Rate constants did not display the characteristic peak at A4, but were remarkably similar to those measured for steps > 4 for the substrate without 3’-terminal adenosines (). ATP affinities showed a peak shifted by 4 positions, compared to the substrate without the 3’ terminal adenosines (, lower panel). This characteristic shift was not seen with a 24 nt ssRNA substrate containing four consecutive adenosines within the sequence (). The data demonstrate that TRAMP adjusts its activity based on the presence of a critical number of 3' terminal adenosines, but irrespective of whether or not they are appended by TRAMP.
TRAMP adjusts polyadenylation activity based on the number of 3' terminal adenosines
Residues outside the helicase domain of Mtr4p participate in the detection of 3’-terminal nucleotides
Since Mtr4p modulated polyadenylation, we next probed whether and how the 3’-terminal nucleotides were detected by Mtr4p during polyadenylation. A recent crystal structure of Mtr4p indicated a potential base recognition site, outside the helicase core (Molecule B in Weir et al., 2010
, ). This structure suggested that E947, which is highly conserved in Mtr4p orthologs, contacts adenosine-specific groups on the fourth base from the 5’ end of the RNA bound in the structure (). Reasoning that E947 might be involved in the identification of the critical number of 3’ terminal adenosines, we replaced E947 with an alanine. TRAMP with Mtr4p(E947A) (TRAMPMtr4p(E947A)
) produced longer poly(A) tails than wtTRAMP over identical reactions times (). The peak in adenylation rate constants seen with TRAMPMtr4p(E947A)
was significantly broader than with wtTRAMP. No clear peak at all was seen for ATP affinities, which also were much lower for later steps (7–10) than for wtTRAMP (). Thus, TRAMPMtr4p(E947A)
markedly diminished the modulation of polyadenylation, similar to the Mtr4-20p mutation in TRAMPMtr4-20p
). We conclude that E947 is important for modulating polyadenylation. This notion is consistent with a scenario where Mtr4p directly binds the 3’ terminal nucleotides, and upon detection of 3' terminal adenosines, alters the polyadenylation activity of Trf4p.
E947 in Mtr4p is critical for the modulation of polyadenylation
Generation of short poly(A) tails involves multiple cycles of TRAMP binding and dissociation
To further understand how the polyadenylation activity was modulated, it was important to examine whether multiple binding and dissociation events were required until 4 adenosines were added. To answer this question, we determined the processivity of TRAMP for individual adenylation steps (Fig.S7
). The processivity is the probability of TRAMP adding the next adenosine vs. dissociating from the substrate (). This probability is directly related to the average number of steps per binding event (Ali and Lohman, 1997
TRAMP processivity and Mtr4p effects on multiple reaction parameters
Plots of processivity vs. number of added adenosines revealed a steady increase in processivity until P = 0.64 ± 0.10 at A4, followed by a slight decrease to P = 0.40 ± 0.03 at A10 (). These data indicate that TRAMP dissociates roughly four times faster than it adds the adenosine for the first step, thus using about 5 binding events to add the first adenosine. For subsequent steps, dissociation and adenylation are roughly equally fast, i.e., TRAMP adds roughly 2 nucleotides per binding event (). The data show that TRAMP undergoes multiple binding and dissociation cycles to append 4 to 5 adenosines.
Mtr4p modulates Trf4p activity through multiple, energetically small effects
We next examined the effects of Mtr4p on TRAMP processivity. TRAMP without Mtr4p (Tr4p/Air2p) showed lower processivity than wtTRAMP for the first four steps. Subsequent steps displayed a slightly higher processivity than wtTRAMP (). To understand the influence of Mtr4p on a more quantitative level, we calculated forward and dissociation rate constants for each adenylation step for TRAMP with and without Mtr4p (). Mtr4p enhances polyadenylation rate constants for steps 1 – 3, and then slows these rate constants for subsequent steps (). In addition, Mtr4p enhances TRAMP dissociation for the first step. For subsequent steps, Mtr4p decreases dissociation rate constants, thus prolonging the time TRAMP remains bound to the RNA ().
To visualize the multifaceted, coordinated effects of Mtr4p on the Trf4p activity, we calculated energetic contributions of Mtr4p to adenosine addition, TRAMP dissociation and ATP affinity for individual adenylation steps (). Compared to the reaction without Mtr4p, the helicase enhances adenylation rate constants and promotes tighter ATP binding for the first two steps (). For steps 4 and higher Mtr4p slows adenylation rate constants and weakens ATP binding (). Mtr4p slows TRAMP dissociation from the RNA, except for the first step (). In energetic terms, the impact of Mtr4p is greatest on ATP affinities and adenylation rate constants. In general, however, Mtr4p imparts rather small changes on the individual rate constants. Yet, numerous small effects multiply over many steps and thus significantly alter the polyadenylation pattern, compared to the reaction without or with impaired Mtr4p. The coordination between changes in rate constants and changes in ATP affinity provides additional synergy to favor a temporal accumulation of short poly(A) tails ().