BACKGROUND
rna ligases have become useful tools to label, circularize, or to perform intermolecular ligation of rna  <cit> .
bacteriophage t <dig> encodes  <dig> proteins with ligase activity: t <dig> rna ligase  <dig> , and the more recently discovered t <dig> rna ligase  <dig>   <cit> .
the t <dig> rna ligases are able to join adjacent 3'-oh and 5'-po <dig> polynucleotides as a result of a three-step reaction .
in the first step, the enzyme becomes adenylated on an active site lysine generating an amp-ligase intermediate and inorganic pyrophosphate.
in the second step, the amp is transferred from the enzyme to 5'-po <dig> rna to produce a 5'-adenylated polynucleotide.
the final step is the formation of a phosphodiester bond between the 3'-oh ligation acceptor and 5'-adenylated polynucleotide ligation donor.
the reaction is promoted by the attack on the 5'-phosphorus by the 3'-oh and results in amp release  <cit> .
during a ligation reaction, t <dig> rna ligases have the potential to produce undesired products such as concatemers or circles.
these products arise due to the presence of atp and polynucleotides with 5'-po <dig>  and 3'-oh.
after steps  <dig> and  <dig> of the ligation reaction, the resulting 5'-adenylated polynucleotide has the ability to ligate to another 3'-oh polynucleotide or to its own 3'-oh end.
these ligation side products may be troublesome for analysis and quantification.
extensive structural and functional studies of t <dig> rnl <dig> have established roles for domains and key residues necessary for ligation activity of the enzyme.
for instance, the n-terminal domain  is essential for the ligase adenylation  and for the phosphodiester bond formation  while the c-terminal  domain is dispensable for these steps, but is involved in the transfer of amp from the enzyme-amp complex to 5'-po <dig> donor  <cit> .
interestingly t <dig> rnl <dig>   is ten times more active than the full length t <dig> rnl <dig> in 5'-adenylated rna ligation  <cit> .
these features make t <dig> rnl2tr an attractive tool for attaching adapters or labels to rna 3'-ends due to the inability of the enzyme to adenylate 5'-po <dig> donor molecules which should reduce the formation of unwanted concatemers in ligation reactions.
three rnl <dig> active site residues  have properties of interest to us when conservatively substituted in the context of the full length t <dig> rnl <dig>  r55k, k227q, k225r retained the ability to perform ligation step  <dig> -- phosphodiester bond formation between the 3'-oh rna and an adenylated rna  <cit> , yet have decreased ability to form enzyme adenylate complexes .
we wondered whether reducing enzyme-adenylate complex formation in a ligase compromised for donor adenylation would produce fewer unwanted side products even when ligation is performed in the absence of atp.
to answer this question, a series of t <dig> rnl2tr mutants was developed incorporating combinations of r55k, k225r and k227q mutations.
the mutants were expressed and purified to characterize their utility for ligation of adenylated oligodeoxynucleotide adapters to small rnas.
we demonstrate that some enzyme-adenlyation-deficient mutants of the truncated t <dig> rnl <dig> retain the ability to form concatemer side-ligation products.
we hypothesize that t <dig> rna ligases and truncated ligases form ligation side products because of the reverse reaction of ligation step  <dig>  that is, amp is transferred from 5'-adenylated adapters to 5'-po <dig> rna, thus producing undesired adenylated substrates that form unexpected products by ligation with 3'-oh rna.
we provide direct evidence for the reversal of ligation reaction step  <dig> by t <dig> rna ligases and correlate that activity with the formation of ligation side-products that include concatemers.
furthermore, we present data that implicate k <dig> in truncated t <dig> rnl <dig> as key for the reverse ligation reaction step  <dig>  together these data demonstrate the utility of the k227q variant of t <dig> rnl2tr as a tool for molecular biology, and provide insight into the molecular basis of ligation side product formation.
RESULTS
formation of ligation side products by t <dig> rna ligases
attachment of adapters to rna 3'-ends is a common first step for rna quantification and discovery by rt-pcr and high-throughput sequencing.
ligations are commonly performed using an rna ligase in the absence of atp to prevent the formation of rna circles and concatemers  <cit> .
in this case, adapters are 3'- blocked and 5'- adenylated so that they are, in essence, ligation donor reaction intermediates.
rna acceptors are commonly either fragmented from larger rnas or small rnas that have 3'-oh and may have 5'-po <dig> groups.
therefore, promotion of phosphodiester bond formation  is the only required enzymatic activity for a strand-joining reaction containing an adenylated adapter.
we monitored the formation of ligation products using commercially available rna ligases and defined  rna acceptor rnas and  dna donors.
in addition to products of the expected size, we detected ligation products that migrated with longer apparent length when treated with rnl <dig>  rnl <dig>  or rnl2tr .
this finding was surprising since our ligation reaction conditions should have prevented concatamerization of rna substrates.
the absence of atp in the reactions should prevent enzyme adenylation in all cases, and the truncation of amino acids 250- <dig> of t <dig> rnl2tr should additionally prevent the transfer of the adenylyl group to any available oligonucleotide 5'-po <dig> groups.
these observations led us to test the activity of a number of conservative point mutants of t <dig> rnl <dig> when placed in the context of the truncated enzyme.
we chose to examine k227q, k225r, and r55k since previous studies established that these mutations, in the context of the full-length ligase preserved strand-joining activity , but were deficient in enzyme adenylation.
we reasoned that preventing enzyme adenylation might further reduce the formation of side-ligation products in the context of the truncated ligase.
the use of t <dig> rnl2tr k227q for small rna cloning has been reported  <cit> .
purification and strand-joining activity of t <dig> rnl2tr conservative mutants
the indicated mutations were introduced into t <dig> rnl2tr singly or in combination.
mutant ligases were produced as n-terminal maltose binding protein  fusion proteins in e. coli and purified by amylose and q sepharose chromatography .
variant truncated ligases were examined for their ability to promote the intermolecular ligation of 5'-adenylated 17-mer dna adapters to 5'-fam-labeled 31-mer rna oligonucleotides.
this experiment measures ligation step  <dig> - phosphodiester bond formation.
all mutants had strand-joining activity, except the r55k k225r ligase .
t <dig> rnl2tr, t <dig> rnl2tr fused to the mbp tag , and r55k displayed the same extent of strand-joining after one hour of incubation, at which ~10% of fam-rna was ligated.
the extent of strand-joining  promoted by the k227q and k225r mutants of the fusion construct was significantly less that of the wild-type t <dig> rnl2tr.
the double mutants r55k k227q and k225r k227q gave the same or a greater yield of the strand joining reaction product  than did their respective single mutants, and the activity of the r55k k227q mutant was similar to t <dig> rnl2tr .
in contrast, the r55k k225r mutant failed to perform the reaction .
surprisingly, the triple mutant retained the ability to ligate its substrates and had a similar efficiency to the k225r k227q ligase .
effect of ph on strand-joining activity
we next examined whether the conservative active site mutations that we had introduced changed the ph optimum for intermolecular strand joining activity .
we performed ligation reactions in buffers from ph  <dig>  to ph  <dig>  containing  <dig> mm mg2+,  <dig> mm dtt and either  <dig> mm tris-hcl or  <dig> mm tris-acetate.
the substrates for the assays were 5'- fam-labeled 31-mer rna and 5'-adenylated 17-mer dna.
under multiple turnover conditions, all proteins were active , but their ph optima differed .
we observed that the optimal ph for t <dig> rnl2tr is  <dig> , where  <dig> pmol of enzyme ligated 17% of the fam-labeled rna, .
all mutations, with the exception of r55k exhibited reduced yield of ligation products under all ph conditions tested as compared to t <dig> rnl2tr at its optimum.
variants containing k227q, or k225r either singly  or in combination with other substitutions  had optima shifted toward higher ph.
as shown in figure 4d, the triple mutant had an optimum efficiency between ph  <dig>  and  <dig>  and the lowest strand-joining activity .
single turnover assays  were performed to mimic common usage conditions for t <dig> rnl2tr .
the assays were identical to the multiple turnover reactions, except for the amount of ligase was increased to  <dig>  pmol.
t <dig> rnl2tr was active over a large ph range from  <dig>  to  <dig> .
this was in contrast to the narrower range observed under multiple turnover conditions .
t <dig> rnl2tr+mbp had the same profile .
as shown in figure 4f, the single mutants had differing ph ranges in which they were maximally active: r55k was most efficient between ph  <dig>  to  <dig> , while k225r was most efficient between ph  <dig> - <dig> .
the k227q mutant was most active between ph  <dig>  and  <dig> .
as we observed under single turnover conditions, the double and triple mutants had higher strand-joining activity in higher ph conditions as compared to t <dig> rnl2tr  with the least accumulation of ligation product observed with the triple mutant .
from a practical standpoint, these data are instructive in that all of the ligases perform the strand-joining reaction efficiently when in high concentration in buffer with ph  <dig> - <dig> .
effect of peg  <dig> on single turnover strand-joining reactions
polyethylene glycol  is known to stimulate ligation reactions for the t <dig> rna ligases  <cit> .
we examined the activity of the mutants at different peg  <dig> concentrations for intermolecular ligation of 5'-adenylated dna to 3'-oh rna .
in agreement with our previous observations, strand joining of 5'-fam-labeled 31-mer rna to 5'-adenylated 17-mer dna adapter was stimulated as peg  <dig> concentration increased up to 25% for t <dig> rnl2tr  <cit> .
t <dig> rnl2tr+mbp showed an identical response up to 25% peg .
we did not observe further stimulation when peg concentration was increased beyond 25%, and handling concentrations of peg greater than 25% was difficult because of the high viscosity of the reactions.
overall, the single and multiple mutants displayed stimulated strand joining activity with increased concentration of peg  <dig> .
under maximal peg stimulation, all variants tested were able to convert nearly 100% of the substrate to ligated form.
strand-joining activity over time
we performed time course experiments to monitor the progression of intermolecular strand-joining reactions under multiple turnover conditions using a 5'-fam-labeled 31-mer rna and a 17-mer 5'-adenylated dna adapter .
the amount of ligated product was calculated  at different time points between  <dig> and  <dig> hours.
all ligase variants tested, except k225r, ligated ~75% of the input rna after  <dig> h. the k225r variant accumulated ~36% of ligated product.
given the input concentration of ligase, these experiments indicate an average of  <dig> ligation events per enzyme molecule over the course of the reaction for all variants, except k225r which catalyzed  <dig> events.
ligation reactions containing k225r accumulated significantly less ligated products than t <dig> rnl2tr .
after one hour of ligation, ligated products accumulated to  <dig>  +/-1% of maximum when incubated with k225r as compared to  <dig> +/-  <dig> % of maximum when incubated with t <dig> rnl2tr .
after  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> hours, ligated products accumulated to  <dig> +/- 1%,  <dig> +/- 2%,  <dig> +/- 4%,  <dig> +/- 5%,  <dig> +/- 3%, and  <dig> +/- 8% when incubated with k225r as compared to.
<dig> +/-3%,  <dig> +/- 3%,  <dig> +/- 3%,  <dig> +/- 3%,  <dig> +/- 3%,  <dig> +/- 2%, and  <dig> +/- 3% when incubated with t <dig> rnl2tr .
mean levels of rna substrate ligated by t <dig> rnl2tr mutants that are significantly different are lower than the corresponding wt t <dig> rnl2tr values .
ns = no significant difference detected, * = means differ p <  <dig> , ** = means differ p <  <dig> , *** = means differ p <  <dig>  as determined by two-way anova followed by bonferroni post-tests for comparison to t <dig> rnl2tr.
we noted that k227q accumulated ligation products significantly more slowly than rnl2tr, or rnl2tr+mbp, but accumulated to the same degree after  <dig> hours of incubation.
after one hour, ligated products accumulated to  <dig>  +/- <dig> % of maximum when incubated with k227q vs.  <dig> +/-  <dig> % of maximum when incubated with t <dig> rnl2tr .
after  <dig>   <dig>   <dig>  and  <dig> hours, ligated products accumulated to  <dig> +/- 1%,  <dig> +/- 2%,  <dig> +/- 4%, and  <dig> +/- 5% when incubated with k227q vs.  <dig> +/-3%,  <dig> +/- 3%,  <dig> +/- 3%,  <dig> +/- 3%, and  <dig> +/- 3% when incubated with t <dig> rnl2tr .
after  <dig> hours, ligated products accumulated to  <dig> +/- 2% of maximum when incubated with k227q, while products accumulated to  <dig> +/- 3% of maximum for t <dig> rnl2tr .
accumulated ligation products were not significantly different after  <dig> of incubation  <dig> +/- 2% vs.  <dig> +/- 3%, for k227q vs. t <dig> rnl2tr  .
interestingly, combining r55k and k227q increased the accumulation of ligated rna at earlier time points, and we could detect no difference at any time point comparing r55k k227q to rnl2tr.
combining k225r with k227q increased ligation product accumulation at earlier time points and the total product accumulated after  <dig> h as compared to k225r alone .
the accumulated product for this mutant was significantly lower after  <dig> and  <dig> hours of ligation, but not before of after these time points.
after  <dig>  and  <dig> hours of ligation with the k225r k227q mutant, ligation products had accumulated to  <dig> +/- 2%, and  <dig> +/- 3% if maximum, compared to  <dig> +/- 3%, and  <dig> +/- 3% of maximum with t <dig> rnl2tr .
the triple mutant displayed a similar profile to the k227q mutant for the accumulation of ligation products .
interestingly, t <dig> rnl2tr, t <dig> rnl2tr+mbp, the r55k and the k225r mutants did not accumulate additional ligation products after twelve hours of reaction.
in contrast, the k227q, the r55k k227q and the k225r k227q, and triple mutants continue to accumulate ligated products over the entire course of the experiment.
by the end of the experiment, only the k225r mutant had accumulated significantly less ligated substrate than wild-type t <dig> rnl2tr
considered together, the results so far established that the introduction of conservative mutations, singly and in combination at positions  <dig>   <dig>  and  <dig>  in the context of the truncated t <dig> rna ligase  <dig> yielded ligases that could reasonably be used as tools for molecular biology.
we next sought to determine whether these ligases had increased performance with respect to the formation of unwanted ligation products.
concatemer formation by t <dig> rnl2tr variants
ligation side products such as concatemers and circles are problematic for ligase applications such as high-throughput sequencing library construction.
we tested the ability of each ligase to produce desired and undesired products in intermolecular ligation reactions.
ligation reactions were performed using 5'-po <dig> 22-mer rna acceptor and 5'-adenylated 17-mer dna donor.
the dna donor was blocked at the 3'-end by the addition of an -nh <dig> group.
thus the 5'-po <dig> end of rna could serve as a ligation donor substrate for joining to the 3'-oh of another rna 22-mer.
reactions were performed in buffer containing  <dig> mm tris hcl ph  <dig> ,  <dig> mm mg2+,  <dig> mm dtt and  <dig> % peg  <dig>  to maximize ligation efficiency.
expected products were  <dig> nt in length, and ligation side products were predicted to be 39+22n or 22+22n  nt in length.
the two wt ligases , and the wild-type truncated rnl <dig>  formed the final ligation product , but also formed higher molecular weight species of ~  <dig> and  <dig> nt.
for these ligases, the 5'-po <dig> rna band was completely absent at the end of the reaction indicating that all the substrate was consumed.
the single k227q mutation produced only the desired ligation product, whereas ligation side products were observed for r55k and k225r.
the r55k+k225r mutant showed low levels of accumulated ligation product of the correct size, consistent with the low activity we observed in earlier experiments.
other multiple mutants, all of which contained the k227q mutation, formed dramatically reduced levels of undesired ligation products.
together, these observations correlate k <dig> with the formation of undesired ligation products.
adapter deadenylation
our ligation conditions did not contain atp, yet we continually observed the accumulation of ligation side products that could only be explained by the concatemerization of rna inputs that would require the adenylation of 5'-po <dig> ends.
in the absence of exogenous atp, one possible source of adenylyl groups in our experimental system is ligation reaction  <dig> - transfer of amp from the ligase active site to the 5'-po <dig> of the donor oligonucleotide - running in reverse.
that is, the transfer of adenylyl groups from the adenylated donor substrate to the catalytic lysine in the active site of the ligase.
we tested the ability of t <dig> rnl2tr and variants to remove the amp from 5'-adenylated dna oligonucleotides by incubating these substrates overnight in the absence of acceptor under single turnover ligation reaction conditions containing  <dig> % peg  <dig> .
after incubation, reactions lacking enzyme had only one band corresponding to the 5'-adenylated dna oligonucleotide indicating that it was stable during the assay.
in contrast, when incubated with t <dig> rnl <dig>  we observed a single band with lower molecular weight that co-migrated with 5'-po <dig> dna adapter.
we interpreted this result to indicate that the amp was completely removed from the 5'-adenylated dna adapter.
adenylated substrates incubated with t <dig> rnl <dig> wt, rnl2tr and t <dig> rnl2tr+mbp migrated as two bands; one corresponding to the input, and the other that migrated at the same position as the 5'-po <dig> adapter.
we interpret this to indicate that these enzymes were able to remove the adenylyl groups from some of the substrate.
r55k and k225r mutants similarly converted the adenylated substrates into  <dig> species.
on the other hand, the adenylated dna adapters incubated with k227q migrated as the higher molecular weight intact species.
adenylated dna adapters incubated overnight with the multiply mutated t <dig> rnl2tr variants were largely unchanged.
these results correlate the deadenylation activity of t <dig> rnl <dig> with k <dig>
amp transfer by variant ligases
the observation that incubation of t <dig> rna ligases with adenylated oligonucleotides could result in changes in their migration consistent with deadenylation led us to directly monitor the fate of the amp group in question.
to do so we followed amp transfer during the ligation reaction using 32p-α-amp-labeled adenylated dna substrates and 5'-po <dig> rna.
concatemers
intermolecular ligation reactions were carried out using  <dig> pmol of 5'-α-32p-adenylated dna adapter and  <dig> pmol of 5'-po <dig> rna overnight with one pmole of ligase .
when resolved by urea page, the negative control reaction containing no enzyme displayed a single band that migrated at  <dig> nucleotides indicating the stability of the α- 32p-amp attached to the dna adapter  over the course of the experiment.
the majority of the radioactivity detected was concentrated at the bottom of the gel for all of the reactions containing ligase except for the r55k k225r reaction where the majority of signal was observed to co-migrate with a 22-mer length.
this was consistent with the inactivity of this variant observed in other experiments.
we interpret the results from the other ligase variants to indicate that, by the end of the reaction, the radioactive amp had been released as a nucleotide into the reaction mixture.
these results are consistent either with ligation of the adenylated dna to the intended 3'-oh end of the rna, or with amp addition to the unintended 5'-po <dig> rna substrate that results in concatemers, followed by phosphodiester bond formation.
in both scenarios, α-32p-amp would be released into the reaction mixture.
deadenylation
we performed deadenylation activity assays of the mutants by performing ligation reactions in the absence of the rna acceptor .
in control reactions containing no-enzyme, only the input 22-mer oligonucleotide  was evident, indicative of the stability of the 5'-adenylated dna adapter.
radioactive signal that migrated with the adenylated adapter was absent from the reaction incubated with t <dig> rnl <dig>  instead, the entirety of the signal co-migrated with free amp.
we interpret this to mean that t <dig> rnl <dig> has the ability to both remove the α-32p amp from the adenylated adapter and to also release it into the reaction mixture.
when reactions containing t <dig> rnl <dig>  rnl2tr and rnl2tr+mbp were resolved, they had reduced signal that co-migrated with the intact adenylated adapter.
full-length t <dig> rnl <dig> had an increased radioactive signal that co-migrated with amp.
we interpret this result to indicate that full-length t <dig> rnl <dig>  like t <dig> rnl <dig> has the ability to remove the amp from the adenylated adapter and to release it into solution.
in contrast to t <dig> rnl <dig>  in reactions that contained t <dig> rnl <dig>  rnl2tr, and rnl2tr+mbp, we observed radioactive signal that migrated with higher molecular weight than the intact adenylated adapter, as well as a smaller amount of signal that co-migrated with amp.
the high molecular weight signal that we observed was consistent with the covalent attachment of α-32p amp to the ligase itself .
adenylated substrate incubated with t <dig> rnl2tr k227q remained largely unchanged, and we did not observe radioactive signal that co-migrated with amp or with high molecular weight.
reactions containing r55k and k225r yielded signals co-migrating with the adenylated dna adapter and with higher molecular weight species.
as with the unmutated ligases, we interpret this result to indicate that these variants are able to remove the adenylyl group from the adenylated adapters and that it remains attached to the ligase.
as found for k227q, reactions containing the ligases with multiple mutations, showed the majority of 32p signal co-migrating with the adenylated adapter.
treatment of the ligation reactions with proteinase k shifted the high molecular weight radioactive signal so that it migrated with lower molecular weight, suggesting that the higher mw band is indeed ligase-32p-amp since ligases are sensitive to proteinase k digestion.
.
amp transfer from ligation donor to rna 5'-po4
to establish that amp could be transferred from an adenylated dna adapter to an rna 5'-end, we incubated radioactively adenylated dna adapters  with 5'-po <dig> rna that was blocked at its 3'-end .
blocking the 3'-end of the rna prevented strand-joining.
in the absence of enzyme, we observed radioactive signal corresponding to the adenylated adapter.
in reactions that contained rnl <dig>  the entirety of the radioactive signal migrated with low molecular weight corresponding with amp.
in reactions incubated with rnl <dig>  rnl2tr, rnl2tr+mbp, and r55k, we observed radioactive signal that co-migrated with 28-mer 3'-blocked rna.
we additionally observed higher molecular weight amp*-ligase adducts for rnl2tr, rnl2tr+mbp, r55k and k225r.
we did not observe radioactive signal corresponding to the 28-mer rna for the inactive ligase r55k+k225r, or for the active ligases containing k227q.
taken together, we interpret these results to directly demonstrate the reversal of ligation reaction step  <dig> - transfer of adenylyl groups from adenylated oligonucleotide donors to the ligase, and from the ligase to rna 5'-po <dig> ends, or to generate free amp.
furthermore our observations are consistent with the requirement of a lysine residue at position  <dig> for this reverse reaction to occur.
discussion
in this work, we introduced conservative active-site mutations in t <dig> rnl2tr to determine their characteristics, especially as they relate to formation of unwanted ligation products.
we could detect no apparent difference in the strand-joining activity of t <dig> rnl2tr and the t <dig> rnl2tr+mbp, and interpret this to mean that the mbp tag does not influence strand-joining activity.
t <dig> rnl <dig> has six characteristic motifs in the n-terminus which are involved in catalysis: motif i, ia, iii, iiia, iv and v  <cit> .
during ligation step  <dig>  the amp is covalently bound to the k <dig> in motif i. the adenine of the adenylyl group is close to f <dig> , while the ribose interacts with e <dig>  and r <dig>  via hydrogen bonds with the 3' and 2' carbons.
the phosphate group is also hydrogen-bonded with k <dig> .
between step  <dig> and  <dig>  the active site is remodeled: the amp phosphate is now coordinated with k <dig>  and k <dig> .
a conformational change takes place so that r <dig> no longer coordinates the ribose o3' but gains contacts with the two oxygens of the 5'-po <dig> rna.
r <dig> is involved in the orientation and the recognition of the 5'-po <dig> end  <cit> .
based on mutational studies of full length t <dig> rnl <dig>  <cit> , and structural studies of the adenylyl transferase domain  <cit> , r <dig>  k <dig>  and k <dig> were implicated as key catalytic residues for the enzyme adenylation step.
we have extended these findings by demonstrating that of the three, only k <dig> is necessary for the reverse reaction of ligation step  <dig> .
thus k <dig> is a catalytic residue for adenylyltransferase both in the forward and backward direction.
co-crystal studies revealed two structural forms containing t <dig> rnl <dig> with 5'-adenylated ligation donor and a 3'- end ligation acceptor  <cit> .
significant remodeling of the active site between step  <dig> product formation and the step  <dig> substrate binding was observed.
r <dig> no longer interacts with the ligation donor 5'-po <dig> oxygen, and k <dig> and k <dig> cease to interact with the amp phosphate.
the phosphoanhydride region of the ligation donor changes its orientation to interact with the 3'-oh rna with more optimal geometry for strand joining  <cit> .
since k <dig> also plays a role in coordinating the amp oxygen and in step 2- <dig> structural rearrangements, we infer that substitution of k227q explains both the lack of reverse step  <dig> and the slow phosphodiester bond formation .
the slower rate observed for step  <dig> may occur because substitution of the side chain of lysine at position  <dig> with glutamine interferes with re-orienting the appdna relative to the rna 3'-oh to a configuration conducive to phosphodiester bond formation.
all of the t <dig> rnl2tr mutants were active in strand-joining reactions except the r55k k225r ligase.
this variant was also inactive for reversal of ligation step  <dig>  we did not detect any evidence of protein instability for this mutant.
however, neither the r55k, or k225r single mutants nor the r55k k225r k227q triple mutant showed a loss of activity similar to the r55k k225r double mutant.
this r55k k225r double mutation may either severely disrupt the function of the active site by modifying the interaction between the substrates, or cause protein misfolding.
previous studies demonstrated equivalent strand-joining activity when r55k was compared to full-length wt t <dig> rnl <dig>  <cit> .
we report similar observations in the context of t <dig> rnl2tr.
in our studies k227q and k225r ligases displayed reduced strand-joining activity, also consistent with that study.
curiously, the r55k k227q had similar strand joining activity to t <dig> rnl2tr, suggesting that the r55k mutation is able to compensate for the reduced activity of the k227q mutant.
studying substrate turnover for the ligase variants illustrated that on a molar basis, except for the k225r mutant, all ligase variants could perform the strand-joining reaction to the same extent as t <dig> rnl2tr.
this suggests to us that the mutations do not decrease the final efficiency of the ligases; rather the speed of the strand-joining reaction is altered for these mutants.
the k225r mutation seems to be detrimental to the enzyme since its turnover was reduced in comparison to the other mutants.
however, the addition of peg  <dig> demonstrated that it possible to restore k225r activity, since we observed strand joining of more than 80% of fam-labeled substrate with high concentrations of the crowding agent.
peg  <dig> appeared to increase the rate of the strand joining reaction for all variants tested.
the reduced total turnover observed for the k225r variant may therefore have resulted from the combined effects of slow turnover and enzyme stability in our time course experiments.
we studied the ability of the variant ligases to form concatemers and circles from 5'-po <dig> rna and the ability to deadenylate a 5'-adenylated dna adapter.
our results demonstrate that only the k227q and the r55k k227q could prevent the formation of concatemers while retaining the ability to perform the strand-joining reaction.
other ligases formed ligation side products by the end of 24-hour experiments.
the k225r k227q and r55k k225r k227q ligases produced fewer side products than the t <dig> rnl2tr.
the k227q and the r55k k227q mutants are the only active variants that completely prevented adapter deadenylation, whereas the k225r k227q and the triple mutants have deadenylation activity, albeit reduced.
thus, the ligases that have impaired or no ability to remove the amp from the 5'-adenylated dna adapter also produce fewer ligation side products.
by following α-32p-amp in the ligation reaction we sought to determine whether the reverse reaction of ligation step  <dig> could occur.
our results show that amp was released into the reaction mixture for all of the active ligases.
however in the absence of a ligation acceptor, radioactive signal from adenylated dna was markedly reduced .
this was evident for all ligases, except for active variants that had k227q.
for some ligases, a higher molecular weight signal was observed.
since the reaction mixture contained only radioactive adapter, buffer and ligase, we attribute the high molecular weight signal to 32p amp-protein intermediate.
digestion of the reactions with proteinase k confirmed this assertion.
we conclude that the reverse reaction occurred in the mixture for all of the active t <dig> rnl2tr variants except for the k227q and the r55k k227q mutants.
by introducing 5'-po <dig>  3'-blocked rna into the ligation reactions, we directly followed the radioactive adenylate transfer from adenylated dna adapter to 5'-po <dig> rna.
thus concatemers and circles may be formed by t <dig> rnl2tr because of its ability to transfer the amp from the 5'-adenylated dna adapter to itself, and then to a new 5'-po <dig> rna.
moreover, the deletion of the c-terminal domain from the full length t <dig> rnl <dig> is not sufficient to prevent the transfer of the amp: only the k227q mutation completely prevented this reaction in the context of the truncated ligase.
CONCLUSIONS
t <dig> rnl <dig> is now commonly used in rna research, notably for the identification of small rnas by generating libraries for sequencing with high-throughput sequencing technology.
the mutations that we tested in the context of the truncated enzyme have established the utility of variants including k227q  <cit> , in reducing unwanted side products, and shed light on the roles of important active site residues and their formation.
t <dig> rnl2tr k227q produces different results in small rna profiling experiments by microarrays as compared to t <dig> rnl <dig> or t <dig> rnl2tr  <cit> .
it seems likely that the reduction of ligation side products should improve rna sequencing library quality, perhaps resulting in the generation of data that more accurately reflects the composition of starting rna pools.

 rna ligases have become useful tools to label, circularize, or to perform intermolecular ligation of rna. bacteriophage t encodes proteins with ligase activity: t rna ligase, and the more recently discovered t rna ligase. the t rna ligases are able to join adjacent 3'-oh and 5'-po polynucleotides as a result of a three-step reaction. in the first step, the enzyme becomes adenylated on an active site lysine generating an amp-ligase intermediate and inorganic pyrophosphate. in the second step, the amp is transferred from the enzyme to 5'-po rna to produce a 5'-adenylated polynucleotide. the final step is the formation of a phosphodiester bond between the 3'-oh ligation acceptor and 5'-adenylated polynucleotide ligation donor. the reaction is promoted by the attack on the 5'-phosphorus by the 3'-oh and results in amp release. during a ligation reaction, t rna ligases have the potential to produce undesired products such as concatemers or circles. these products arise due to the presence of atp and polynucleotides with 5'-po and 3'-oh. after steps and of the ligation reaction, the resulting 5'-adenylated polynucleotide has the ability to ligate to another 3'-oh polynucleotide or to its own 3'-oh end. these ligation side products may be troublesome for analysis and quantification. extensive structural and functional studies of t rnl have established roles for domains and key residues necessary for ligation activity of the enzyme. for instance, the n-terminal domain is essential for the ligase adenylation and for the phosphodiester bond formation while the c-terminal domain is dispensable for these steps, but is involved in the transfer of amp from the enzyme-amp complex to 5'-po donor. interestingly t rnl is ten times more active than the full length t rnl in 5'-adenylated rna ligation. these features make t rnl2tr an attractive tool for attaching adapters or labels to rna 3'-ends due to the inability of the enzyme to adenylate 5'-po donor molecules which should reduce the formation of unwanted concatemers in ligation reactions. three rnl active site residues have properties of interest to us when conservatively substituted in the context of the full length t rnl r55k, k227q, k225r retained the ability to perform ligation step -- phosphodiester bond formation between the 3'-oh rna and an adenylated rna, yet have decreased ability to form enzyme adenylate complexes. we wondered whether reducing enzyme-adenylate complex formation in a ligase compromised for donor adenylation would produce fewer unwanted side products even when ligation is performed in the absence of atp. to answer this question, a series of t rnl2tr mutants was developed incorporating combinations of r55k, k225r and k227q mutations. the mutants were expressed and purified to characterize their utility for ligation of adenylated oligodeoxynucleotide adapters to small rnas. we demonstrate that some enzyme-adenlyation-deficient mutants of the truncated t rnl retain the ability to form concatemer side-ligation products. we hypothesize that t rna ligases and truncated ligases form ligation side products because of the reverse reaction of ligation step that is, amp is transferred from 5'-adenylated adapters to 5'-po rna, thus producing undesired adenylated substrates that form unexpected products by ligation with 3'-oh rna. we provide direct evidence for the reversal of ligation reaction step by t rna ligases and correlate that activity with the formation of ligation side-products that include concatemers. furthermore, we present data that implicate k in truncated t rnl as key for the reverse ligation reaction step together these data demonstrate the utility of the k227q variant of t rnl2tr as a tool for molecular biology, and provide insight into the molecular basis of ligation side product formation.  formation of ligation side products by t rna ligases attachment of adapters to rna 3'-ends is a common first step for rna quantification and discovery by rt-pcr and high-throughput sequencing. ligations are commonly performed using an rna ligase in the absence of atp to prevent the formation of rna circles and concatemers. in this case, adapters are 3'- blocked and 5'- adenylated so that they are, in essence, ligation donor reaction intermediates. rna acceptors are commonly either fragmented from larger rnas or small rnas that have 3'-oh and may have 5'-po groups. therefore, promotion of phosphodiester bond formation is the only required enzymatic activity for a strand-joining reaction containing an adenylated adapter. we monitored the formation of ligation products using commercially available rna ligases and defined rna acceptor rnas and dna donors. in addition to products of the expected size, we detected ligation products that migrated with longer apparent length when treated with rnl rnl or rnl2tr. this finding was surprising since our ligation reaction conditions should have prevented concatamerization of rna substrates. the absence of atp in the reactions should prevent enzyme adenylation in all cases, and the truncation of amino acids 250- of t rnl2tr should additionally prevent the transfer of the adenylyl group to any available oligonucleotide 5'-po groups. these observations led us to test the activity of a number of conservative point mutants of t rnl when placed in the context of the truncated enzyme. we chose to examine k227q, k225r, and r55k since previous studies established that these mutations, in the context of the full-length ligase preserved strand-joining activity, but were deficient in enzyme adenylation. we reasoned that preventing enzyme adenylation might further reduce the formation of side-ligation products in the context of the truncated ligase. the use of t rnl2tr k227q for small rna cloning has been reported. purification and strand-joining activity of t rnl2tr conservative mutants the indicated mutations were introduced into t rnl2tr singly or in combination. mutant ligases were produced as n-terminal maltose binding protein fusion proteins in e. coli and purified by amylose and q sepharose chromatography. variant truncated ligases were examined for their ability to promote the intermolecular ligation of 5'-adenylated 17-mer dna adapters to 5'-fam-labeled 31-mer rna oligonucleotides. this experiment measures ligation step - phosphodiester bond formation. all mutants had strand-joining activity, except the r55k k225r ligase. t rnl2tr, t rnl2tr fused to the mbp tag, and r55k displayed the same extent of strand-joining after one hour of incubation, at which ~10% of fam-rna was ligated. the extent of strand-joining promoted by the k227q and k225r mutants of the fusion construct was significantly less that of the wild-type t rnl2tr. the double mutants r55k k227q and k225r k227q gave the same or a greater yield of the strand joining reaction product than did their respective single mutants, and the activity of the r55k k227q mutant was similar to t rnl2tr. in contrast, the r55k k225r mutant failed to perform the reaction. surprisingly, the triple mutant retained the ability to ligate its substrates and had a similar efficiency to the k225r k227q ligase. effect of ph on strand-joining activity we next examined whether the conservative active site mutations that we had introduced changed the ph optimum for intermolecular strand joining activity. we performed ligation reactions in buffers from ph to ph containing mm mg2+, mm dtt and either mm tris-hcl or mm tris-acetate. the substrates for the assays were 5'- fam-labeled 31-mer rna and 5'-adenylated 17-mer dna. under multiple turnover conditions, all proteins were active, but their ph optima differed. we observed that the optimal ph for t rnl2tr is, where pmol of enzyme ligated 17% of the fam-labeled rna,. all mutations, with the exception of r55k exhibited reduced yield of ligation products under all ph conditions tested as compared to t rnl2tr at its optimum. variants containing k227q, or k225r either singly or in combination with other substitutions had optima shifted toward higher ph. as shown in figure 4d, the triple mutant had an optimum efficiency between ph and and the lowest strand-joining activity. single turnover assays were performed to mimic common usage conditions for t rnl2tr. the assays were identical to the multiple turnover reactions, except for the amount of ligase was increased to pmol. t rnl2tr was active over a large ph range from to. this was in contrast to the narrower range observed under multiple turnover conditions. t rnl2tr+mbp had the same profile. as shown in figure 4f, the single mutants had differing ph ranges in which they were maximally active: r55k was most efficient between ph to, while k225r was most efficient between ph -. the k227q mutant was most active between ph and. as we observed under single turnover conditions, the double and triple mutants had higher strand-joining activity in higher ph conditions as compared to t rnl2tr with the least accumulation of ligation product observed with the triple mutant. from a practical standpoint, these data are instructive in that all of the ligases perform the strand-joining reaction efficiently when in high concentration in buffer with ph -. effect of peg on single turnover strand-joining reactions polyethylene glycol is known to stimulate ligation reactions for the t rna ligases. we examined the activity of the mutants at different peg concentrations for intermolecular ligation of 5'-adenylated dna to 3'-oh rna. in agreement with our previous observations, strand joining of 5'-fam-labeled 31-mer rna to 5'-adenylated 17-mer dna adapter was stimulated as peg concentration increased up to 25% for t rnl2tr. t rnl2tr+mbp showed an identical response up to 25% peg. we did not observe further stimulation when peg concentration was increased beyond 25%, and handling concentrations of peg greater than 25% was difficult because of the high viscosity of the reactions. overall, the single and multiple mutants displayed stimulated strand joining activity with increased concentration of peg. under maximal peg stimulation, all variants tested were able to convert nearly 100% of the substrate to ligated form. strand-joining activity over time we performed time course experiments to monitor the progression of intermolecular strand-joining reactions under multiple turnover conditions using a 5'-fam-labeled 31-mer rna and a 17-mer 5'-adenylated dna adapter. the amount of ligated product was calculated at different time points between and hours. all ligase variants tested, except k225r, ligated ~75% of the input rna after h. the k225r variant accumulated ~36% of ligated product. given the input concentration of ligase, these experiments indicate an average of ligation events per enzyme molecule over the course of the reaction for all variants, except k225r which catalyzed events. ligation reactions containing k225r accumulated significantly less ligated products than t rnl2tr. after one hour of ligation, ligated products accumulated to +/-1% of maximum when incubated with k225r as compared to +/- % of maximum when incubated with t rnl2tr. after and hours, ligated products accumulated to +/- 1%, +/- 2%, +/- 4%, +/- 5%, +/- 3%, and +/- 8% when incubated with k225r as compared to. +/-3%, +/- 3%, +/- 3%, +/- 3%, +/- 3%, +/- 2%, and +/- 3% when incubated with t rnl2tr. mean levels of rna substrate ligated by t rnl2tr mutants that are significantly different are lower than the corresponding wt t rnl2tr values. ns = no significant difference detected, * = means differ p <, ** = means differ p <, *** = means differ p < as determined by two-way anova followed by bonferroni post-tests for comparison to t rnl2tr. we noted that k227q accumulated ligation products significantly more slowly than rnl2tr, or rnl2tr+mbp, but accumulated to the same degree after hours of incubation. after one hour, ligated products accumulated to +/- % of maximum when incubated with k227q vs. +/- % of maximum when incubated with t rnl2tr. after and hours, ligated products accumulated to +/- 1%, +/- 2%, +/- 4%, and +/- 5% when incubated with k227q vs. +/-3%, +/- 3%, +/- 3%, +/- 3%, and +/- 3% when incubated with t rnl2tr. after hours, ligated products accumulated to +/- 2% of maximum when incubated with k227q, while products accumulated to +/- 3% of maximum for t rnl2tr. accumulated ligation products were not significantly different after of incubation +/- 2% vs. +/- 3%, for k227q vs. t rnl2tr. interestingly, combining r55k and k227q increased the accumulation of ligated rna at earlier time points, and we could detect no difference at any time point comparing r55k k227q to rnl2tr. combining k225r with k227q increased ligation product accumulation at earlier time points and the total product accumulated after h as compared to k225r alone. the accumulated product for this mutant was significantly lower after and hours of ligation, but not before of after these time points. after and hours of ligation with the k225r k227q mutant, ligation products had accumulated to +/- 2%, and +/- 3% if maximum, compared to +/- 3%, and +/- 3% of maximum with t rnl2tr. the triple mutant displayed a similar profile to the k227q mutant for the accumulation of ligation products. interestingly, t rnl2tr, t rnl2tr+mbp, the r55k and the k225r mutants did not accumulate additional ligation products after twelve hours of reaction. in contrast, the k227q, the r55k k227q and the k225r k227q, and triple mutants continue to accumulate ligated products over the entire course of the experiment. by the end of the experiment, only the k225r mutant had accumulated significantly less ligated substrate than wild-type t rnl2tr considered together, the results so far established that the introduction of conservative mutations, singly and in combination at positions and in the context of the truncated t rna ligase yielded ligases that could reasonably be used as tools for molecular biology. we next sought to determine whether these ligases had increased performance with respect to the formation of unwanted ligation products. concatemer formation by t rnl2tr variants ligation side products such as concatemers and circles are problematic for ligase applications such as high-throughput sequencing library construction. we tested the ability of each ligase to produce desired and undesired products in intermolecular ligation reactions. ligation reactions were performed using 5'-po 22-mer rna acceptor and 5'-adenylated 17-mer dna donor. the dna donor was blocked at the 3'-end by the addition of an -nh group. thus the 5'-po end of rna could serve as a ligation donor substrate for joining to the 3'-oh of another rna 22-mer. reactions were performed in buffer containing mm tris hcl ph, mm mg2+, mm dtt and % peg to maximize ligation efficiency. expected products were nt in length, and ligation side products were predicted to be 39+22n or 22+22n nt in length. the two wt ligases, and the wild-type truncated rnl formed the final ligation product, but also formed higher molecular weight species of ~ and nt. for these ligases, the 5'-po rna band was completely absent at the end of the reaction indicating that all the substrate was consumed. the single k227q mutation produced only the desired ligation product, whereas ligation side products were observed for r55k and k225r. the r55k+k225r mutant showed low levels of accumulated ligation product of the correct size, consistent with the low activity we observed in earlier experiments. other multiple mutants, all of which contained the k227q mutation, formed dramatically reduced levels of undesired ligation products. together, these observations correlate k with the formation of undesired ligation products. adapter deadenylation our ligation conditions did not contain atp, yet we continually observed the accumulation of ligation side products that could only be explained by the concatemerization of rna inputs that would require the adenylation of 5'-po ends. in the absence of exogenous atp, one possible source of adenylyl groups in our experimental system is ligation reaction - transfer of amp from the ligase active site to the 5'-po of the donor oligonucleotide - running in reverse. that is, the transfer of adenylyl groups from the adenylated donor substrate to the catalytic lysine in the active site of the ligase. we tested the ability of t rnl2tr and variants to remove the amp from 5'-adenylated dna oligonucleotides by incubating these substrates overnight in the absence of acceptor under single turnover ligation reaction conditions containing % peg. after incubation, reactions lacking enzyme had only one band corresponding to the 5'-adenylated dna oligonucleotide indicating that it was stable during the assay. in contrast, when incubated with t rnl we observed a single band with lower molecular weight that co-migrated with 5'-po dna adapter. we interpreted this result to indicate that the amp was completely removed from the 5'-adenylated dna adapter. adenylated substrates incubated with t rnl wt, rnl2tr and t rnl2tr+mbp migrated as two bands; one corresponding to the input, and the other that migrated at the same position as the 5'-po adapter. we interpret this to indicate that these enzymes were able to remove the adenylyl groups from some of the substrate. r55k and k225r mutants similarly converted the adenylated substrates into species. on the other hand, the adenylated dna adapters incubated with k227q migrated as the higher molecular weight intact species. adenylated dna adapters incubated overnight with the multiply mutated t rnl2tr variants were largely unchanged. these results correlate the deadenylation activity of t rnl with k amp transfer by variant ligases the observation that incubation of t rna ligases with adenylated oligonucleotides could result in changes in their migration consistent with deadenylation led us to directly monitor the fate of the amp group in question. to do so we followed amp transfer during the ligation reaction using 32p-α-amp-labeled adenylated dna substrates and 5'-po rna. concatemers intermolecular ligation reactions were carried out using pmol of 5'-α-32p-adenylated dna adapter and pmol of 5'-po rna overnight with one pmole of ligase. when resolved by urea page, the negative control reaction containing no enzyme displayed a single band that migrated at nucleotides indicating the stability of the α- 32p-amp attached to the dna adapter over the course of the experiment. the majority of the radioactivity detected was concentrated at the bottom of the gel for all of the reactions containing ligase except for the r55k k225r reaction where the majority of signal was observed to co-migrate with a 22-mer length. this was consistent with the inactivity of this variant observed in other experiments. we interpret the results from the other ligase variants to indicate that, by the end of the reaction, the radioactive amp had been released as a nucleotide into the reaction mixture. these results are consistent either with ligation of the adenylated dna to the intended 3'-oh end of the rna, or with amp addition to the unintended 5'-po rna substrate that results in concatemers, followed by phosphodiester bond formation. in both scenarios, α-32p-amp would be released into the reaction mixture. deadenylation we performed deadenylation activity assays of the mutants by performing ligation reactions in the absence of the rna acceptor. in control reactions containing no-enzyme, only the input 22-mer oligonucleotide was evident, indicative of the stability of the 5'-adenylated dna adapter. radioactive signal that migrated with the adenylated adapter was absent from the reaction incubated with t rnl instead, the entirety of the signal co-migrated with free amp. we interpret this to mean that t rnl has the ability to both remove the α-32p amp from the adenylated adapter and to also release it into the reaction mixture. when reactions containing t rnl rnl2tr and rnl2tr+mbp were resolved, they had reduced signal that co-migrated with the intact adenylated adapter. full-length t rnl had an increased radioactive signal that co-migrated with amp. we interpret this result to indicate that full-length t rnl like t rnl has the ability to remove the amp from the adenylated adapter and to release it into solution. in contrast to t rnl in reactions that contained t rnl rnl2tr, and rnl2tr+mbp, we observed radioactive signal that migrated with higher molecular weight than the intact adenylated adapter, as well as a smaller amount of signal that co-migrated with amp. the high molecular weight signal that we observed was consistent with the covalent attachment of α-32p amp to the ligase itself. adenylated substrate incubated with t rnl2tr k227q remained largely unchanged, and we did not observe radioactive signal that co-migrated with amp or with high molecular weight. reactions containing r55k and k225r yielded signals co-migrating with the adenylated dna adapter and with higher molecular weight species. as with the unmutated ligases, we interpret this result to indicate that these variants are able to remove the adenylyl group from the adenylated adapters and that it remains attached to the ligase. as found for k227q, reactions containing the ligases with multiple mutations, showed the majority of 32p signal co-migrating with the adenylated adapter. treatment of the ligation reactions with proteinase k shifted the high molecular weight radioactive signal so that it migrated with lower molecular weight, suggesting that the higher mw band is indeed ligase-32p-amp since ligases are sensitive to proteinase k digestion.. amp transfer from ligation donor to rna 5'-po4 to establish that amp could be transferred from an adenylated dna adapter to an rna 5'-end, we incubated radioactively adenylated dna adapters with 5'-po rna that was blocked at its 3'-end. blocking the 3'-end of the rna prevented strand-joining. in the absence of enzyme, we observed radioactive signal corresponding to the adenylated adapter. in reactions that contained rnl the entirety of the radioactive signal migrated with low molecular weight corresponding with amp. in reactions incubated with rnl rnl2tr, rnl2tr+mbp, and r55k, we observed radioactive signal that co-migrated with 28-mer 3'-blocked rna. we additionally observed higher molecular weight amp*-ligase adducts for rnl2tr, rnl2tr+mbp, r55k and k225r. we did not observe radioactive signal corresponding to the 28-mer rna for the inactive ligase r55k+k225r, or for the active ligases containing k227q. taken together, we interpret these results to directly demonstrate the reversal of ligation reaction step - transfer of adenylyl groups from adenylated oligonucleotide donors to the ligase, and from the ligase to rna 5'-po ends, or to generate free amp. furthermore our observations are consistent with the requirement of a lysine residue at position for this reverse reaction to occur. discussion in this work, we introduced conservative active-site mutations in t rnl2tr to determine their characteristics, especially as they relate to formation of unwanted ligation products. we could detect no apparent difference in the strand-joining activity of t rnl2tr and the t rnl2tr+mbp, and interpret this to mean that the mbp tag does not influence strand-joining activity. t rnl has six characteristic motifs in the n-terminus which are involved in catalysis: motif i, ia, iii, iiia, iv and v. during ligation step the amp is covalently bound to the k in motif i. the adenine of the adenylyl group is close to f, while the ribose interacts with e and r via hydrogen bonds with the 3' and 2' carbons. the phosphate group is also hydrogen-bonded with k. between step and the active site is remodeled: the amp phosphate is now coordinated with k and k. a conformational change takes place so that r no longer coordinates the ribose o3' but gains contacts with the two oxygens of the 5'-po rna. r is involved in the orientation and the recognition of the 5'-po end. based on mutational studies of full length t rnl, and structural studies of the adenylyl transferase domain, r k and k were implicated as key catalytic residues for the enzyme adenylation step. we have extended these findings by demonstrating that of the three, only k is necessary for the reverse reaction of ligation step. thus k is a catalytic residue for adenylyltransferase both in the forward and backward direction. co-crystal studies revealed two structural forms containing t rnl with 5'-adenylated ligation donor and a 3'- end ligation acceptor. significant remodeling of the active site between step product formation and the step substrate binding was observed. r no longer interacts with the ligation donor 5'-po oxygen, and k and k cease to interact with the amp phosphate. the phosphoanhydride region of the ligation donor changes its orientation to interact with the 3'-oh rna with more optimal geometry for strand joining. since k also plays a role in coordinating the amp oxygen and in step 2- structural rearrangements, we infer that substitution of k227q explains both the lack of reverse step and the slow phosphodiester bond formation. the slower rate observed for step may occur because substitution of the side chain of lysine at position with glutamine interferes with re-orienting the appdna relative to the rna 3'-oh to a configuration conducive to phosphodiester bond formation. all of the t rnl2tr mutants were active in strand-joining reactions except the r55k k225r ligase. this variant was also inactive for reversal of ligation step we did not detect any evidence of protein instability for this mutant. however, neither the r55k, or k225r single mutants nor the r55k k225r k227q triple mutant showed a loss of activity similar to the r55k k225r double mutant. this r55k k225r double mutation may either severely disrupt the function of the active site by modifying the interaction between the substrates, or cause protein misfolding. previous studies demonstrated equivalent strand-joining activity when r55k was compared to full-length wt t rnl. we report similar observations in the context of t rnl2tr. in our studies k227q and k225r ligases displayed reduced strand-joining activity, also consistent with that study. curiously, the r55k k227q had similar strand joining activity to t rnl2tr, suggesting that the r55k mutation is able to compensate for the reduced activity of the k227q mutant. studying substrate turnover for the ligase variants illustrated that on a molar basis, except for the k225r mutant, all ligase variants could perform the strand-joining reaction to the same extent as t rnl2tr. this suggests to us that the mutations do not decrease the final efficiency of the ligases; rather the speed of the strand-joining reaction is altered for these mutants. the k225r mutation seems to be detrimental to the enzyme since its turnover was reduced in comparison to the other mutants. however, the addition of peg demonstrated that it possible to restore k225r activity, since we observed strand joining of more than 80% of fam-labeled substrate with high concentrations of the crowding agent. peg appeared to increase the rate of the strand joining reaction for all variants tested. the reduced total turnover observed for the k225r variant may therefore have resulted from the combined effects of slow turnover and enzyme stability in our time course experiments. we studied the ability of the variant ligases to form concatemers and circles from 5'-po rna and the ability to deadenylate a 5'-adenylated dna adapter. our results demonstrate that only the k227q and the r55k k227q could prevent the formation of concatemers while retaining the ability to perform the strand-joining reaction. other ligases formed ligation side products by the end of 24-hour experiments. the k225r k227q and r55k k225r k227q ligases produced fewer side products than the t rnl2tr. the k227q and the r55k k227q mutants are the only active variants that completely prevented adapter deadenylation, whereas the k225r k227q and the triple mutants have deadenylation activity, albeit reduced. thus, the ligases that have impaired or no ability to remove the amp from the 5'-adenylated dna adapter also produce fewer ligation side products. by following α-32p-amp in the ligation reaction we sought to determine whether the reverse reaction of ligation step could occur. our results show that amp was released into the reaction mixture for all of the active ligases. however in the absence of a ligation acceptor, radioactive signal from adenylated dna was markedly reduced. this was evident for all ligases, except for active variants that had k227q. for some ligases, a higher molecular weight signal was observed. since the reaction mixture contained only radioactive adapter, buffer and ligase, we attribute the high molecular weight signal to 32p amp-protein intermediate. digestion of the reactions with proteinase k confirmed this assertion. we conclude that the reverse reaction occurred in the mixture for all of the active t rnl2tr variants except for the k227q and the r55k k227q mutants. by introducing 5'-po 3'-blocked rna into the ligation reactions, we directly followed the radioactive adenylate transfer from adenylated dna adapter to 5'-po rna. thus concatemers and circles may be formed by t rnl2tr because of its ability to transfer the amp from the 5'-adenylated dna adapter to itself, and then to a new 5'-po rna. moreover, the deletion of the c-terminal domain from the full length t rnl is not sufficient to prevent the transfer of the amp: only the k227q mutation completely prevented this reaction in the context of the truncated ligase.  t rnl is now commonly used in rna research, notably for the identification of small rnas by generating libraries for sequencing with high-throughput sequencing technology. the mutations that we tested in the context of the truncated enzyme have established the utility of variants including k227q, in reducing unwanted side products, and shed light on the roles of important active site residues and their formation. t rnl2tr k227q produces different results in small rna profiling experiments by microarrays as compared to t rnl or t rnl2tr. it seems likely that the reduction of ligation side products should improve rna sequencing library quality, perhaps resulting in the generation of data that more accurately reflects the composition of starting rna pools. 
with the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of t rnl2tr. this reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. thus, t rn2tr mutants containing the k227q mutation are useful for reducing undesired ligation products. we characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction. t rna ligases and are useful tools for rna analysis. the truncated form of t rna ligase comprising amino acids 1-, is an attractive tool for attachment of adapters or labels to rna 3'-ends. compared to t rna ligase t rnl2tr has a decreased ability to ligate 5'-po ends in single-stranded rna ligations, and compared to the full-length t rnl the t rnl2tr has an increased activity for joining 5'-adenylated adapters to rna 3'-ends. the combination of these properties allows adapter attachment to rna 3'-ends with reduced circularization and concatemerization of substrate rna.
 t rna ligases and are useful tools for rna analysis. their use upstream of rna analyses such as high-throughput rna sequencing and microarrays has recently increased their importance. the truncated form of t rna ligase comprising amino acids 1-, is an attractive tool for attachment of adapters or labels to rna 3'-ends. compared to t rna ligase t rnl2tr has a decreased ability to ligate 5'-po ends in single-stranded rna ligations, and compared to the full-length t rnl the t rnl2tr has an increased activity for joining 5'-adenylated adapters to rna 3'-ends. the combination of these properties allows adapter attachment to rna 3'-ends with reduced circularization and concatemerization of substrate rna.  with the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of t rnl2tr. we characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction.  our data demonstrate that lysine is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. this reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. thus, t rn2tr mutants containing the k227q mutation are useful for reducing undesired ligation products. we furthermore report optimal conditions for the use of these improved t rnl2tr variants. 