mutagenesis and preparation of rna in vitro transcripts a plasmid encoding adenovirus type 2 ( ad2 ) va rnai was created with a 5-t7 rna polymerase ( t7 rnap ) promoter and 3-hepatitis delta virus ribozyme sequence followed by a drai restriction site for run - off transcription , as described previously ( 27 ) .
va rnai mutants were generated in this plasmid by quikchange site - directed mutagenesis ( stratagene ) and confirmed by automated dna sequencing .
the va rnai terminal stem ( ts ) was first deleted in three successive segments of 5 bp each to create the mutants ts5 , ts10 , and ts15 . for these rnas ,
the 3 terminal bp of the wild - type sequence were maintained to provide a strong promoter for t7 rna polymerase ( t7 rnap ) .
a final deletion of the entire terminal stem , including the asymmetric bulge of nucleotides 2130 , created the construct ts21 . in ts21 the t7 rnap promoter was generated by reversal of the new terminal base pair , g  c . a final mutation in the apical stem of full - length va rnai , 78cu , generated base pair rna .
drai - linearized plasmid dna templates ( 100 g / ml ) were used in run - off transcription reactions ( 0.5 or 1.0 ml volume ) under optimal conditions for va rnai ( 28 ) using t7 rnap expressed from plasmid pt7 - 911 and purified by ni affinity chromatography ( 29 ) .
rna transcripts were purified by preparative denaturing page with gels containing 50% urea and 8% acrylamide .
va rnai bands were identified by uv shadowing , excised from the gel , and eluted using a biotrap device ( schleicher & schuell ) before ethanol precipitation and resuspension in te buffer .
samples contained 2025 g of rna in a solution containing 10 mm mops buffer , ph 7.0 , and 50 mm kcl . for experiments with va rnai ( ts21 ) at different ph values ,
the following buffers were used in otherwise identical conditions : mes ( ph 5.5 , 6.0 , and 6.5 ) , mops ( 6.5 , 7.0 , and 7.5 ) , and hepes ( 7.5 and 8.0 ) . experiments performed with different buffers at the same ph gave identical results .
uv melting curves were collected on a varian cary 400 uv - visible spectrophotometer with a 6-cell multichanger , running in dual beam mode .
up to five melting curves were collected in each experiment with the sixth cell containing only buffer and fitted with an in - sample temperature probe .
3a ) , referred to as  melting profiles ,  were calculated using a savitsky - golay algorithm as implemented in the program od deriv .
pkr protein expression and purification  pkr was expressed in escherichia coli in a nonphosphorylated form using plasmids encoding both the kinase and -protein phosphatase ( 30 , 31 ) .
purification was accomplished using established procedures as follows : chitin affinity chromatography for pkr expressed from plasmid ptyb2-pkr(-pp ) ( 30 ) or sequential purification by heparin affinity , poly(i)poly(c ) rna - sepharose affinity , and gel filtration chromatographies on an ktapurifier 100 system for untagged pkr expressed from ppet - pkr / ppase ( 31 ) .
poly(i)poly(c ) rna - sepharose was prepared using single - stranded poly(c ) ( 4 mg / ml ; ge healthcare ) and poly(i ) ( 4 mg / ml ; sigma ) rnas coupled to cnbr - activated sepharose 4b ff ( ge healthcare ) as described by wagner et al .
pkr produced from ppet - pkr / protein phosphatase was used for all other experiments .
pkr autophosphorylation inhibition assays  purified pkr was dialyzed into 2 reaction buffer ( 100 mm tris , ph 7.8 , 100 mm kcl , 10% glycerol , 5 mm dithiothreitol ) .
wild - type va rnai and each terminal stem deletion mutant rna were diluted into the same buffer to generate a 5 stock for each point in the inhibition assay ( see fig .
pkr ( 2 l ; 0.1 g ) was preincubated with the va rnai ( 3 l ) at room temperature for 5 min .
an equal volume of 2 pkr / activation reaction mixture was added , and each sample was incubated at room temperature for a further 10 min .
the 2 pkr / activation reaction mixture contained 0.6 g / ml poly(i)poly(c ) , 4 m mgcl2 , 40 m atp , and 0.2 mci / ml [ p]atp ( 6000 ci / mmol , 10 mci / ml ; perkinelmer life sciences ) .
samples were heated at 90 c for 25 min and fractionated by 10% acrylamide sds - page at 150200 v for 1 h. gels were fixed , dried , and exposed to an imaging plate ( ge healthcare ) and viewed using typhoon 8600 phosphorimager .
control experiments without poly(i)poly(c)-activating rna showed that none of the va rnai samples activated pkr .
isothermal titration calorimetry ( itc)the thermodynamics of va rnai - pkr interactions were measured using a vp - itc microcalorimeter following established procedures for va rnai ( 33 , 34 ) .
rna and protein were dialyzed exhaustively into 10 mm sodium phosphate , ph 6.5 , 100 mm nacl , and 5 mm -mercaptoethanol .
the sample cell contained ts21 rna at a concentration of 3 m , and the injection syringe contained pkr at 60 m .
titration experiments were performed at 30 c and involved a single 2-l injection followed by 28  10-l injections of 24 s duration with 360 s spacing .
titration curves were fit by a nonlinear least squares method in microcal origin software using a model for one or two binding sites . a model with two binding sites
was found to give the optimal fit and was used to extract thermodynamic parameters kd , h , s , and n   table 1thermodynamic parameters of pkr - rna interactionrnakdnhsgnmkcal / molcal / molkkcal / mol ts21   83  20.1   1.16   6.7   10.4   9.8   va rna1 79  9   nr 11.3   4.8   9.8 adata are from ref .
38bnr indicates not reported   thermodynamic parameters of pkr - rna interaction data are from ref .
gel filtration experiments were performed using a superdex 10/300 gl column ( ge healthcare ) attached to an ktapurifier 100 system and equilibrated with 10 mm sodium phosphate buffer , ph 6.5 , and 100 mm nacl .
the pkr concentration was 3 m for all experiments that contained protein , whereas the rna concentration was 0.75 m ( 4:1 protein to rna ratio ) , 1.5 m ( 2:1 ) , 3 m ( rna alone and 1:1 ) , or 6 m ( 1:2 ) .
to examine its role in creating or maintaining a functional va rnai structure that efficiently inhibits pkr , the ad2 va rnai terminal stem ( ts ) was progressively shortened .
three successive deletions of 5 bp each and a final deletion of the entire terminal stem , including the asymmetric bulge of nucleotides 2130 , were made to create ts5 , ts10 , ts15 , and ts21 rnas , respectively ( fig .
1 ) . pkr kinase inhibition assays  pkr autophosphorylation correlates well with subsequent activity against its primary cellular target eif2 ( 35 ) and was used as a convenient assay of efficacy of our mutant va rnai inhibitors .
we anticipated that the activity of va rnai would decrease with each successive deletion to the terminal stem .
the ability of each mutant rna to inhibit pkr autophosphorylation upon addition of dsrna activator rna was therefore measured and compared with wild - type va rnai ( fig .
wild - type va rnai inhibited pkr completely at a concentration of < 5 g / ml , in agreement with previous studies ( 23 , 36 ) .
as expected , the mutant rnas showed a decrease in activity for each successive deletion of 5 bp from the terminal stem , with 5 , 10 , and 50 g / ml rna required for full pkr inhibition by the ts5 , ts10 , and ts15 rnas , respectively ( fig .
this most severely deleted rna displayed wild - type activity , completely inhibiting pkr autophosphorylation at a concentration of < 5 g / ml . to confirm this ,
the experiment was repeated at equimolar concentrations of wild - type and ts21 rnas ( because for an equal mass of rna in each reaction , the shorter va rnai ( ts21 ) would contain 50% more molecules ) .
quantitation of these assays confirmed that va rnai ( ts21 ) inhibits pkr autophosphorylation at least as efficiently as wild - type rna despite lacking the entire terminal stem ( fig .
impact of terminal stem deletions on va rnai structure rna uv melting analysis can be used to assess the impact of mutations upon the folding and stability of an rna molecule .
the unfolding of large rnas can , however , be a complex process that occurs in a number of overlapping transitions ( 37 ) .
we therefore describe unfolding transitions in the melting profile , the first derivative of the uv melting curve , as  apparent transitions ,  each with an associated apparent melting temperature ( tm ) . we have shown that wild - type va rnai unfolds in two independent apparent transitions ( fig .
3a ) and assigned the lower ( 60 c ) and higher ( 85 c ) temperature apparent transitions to the unfolding of the terminal stem / central domain and apical stem , respectively ( 25 ) .
this assignment is further confirmed by the present data as deletions in the terminal stem affect only the lower temperature apparent transition ( fig .
the second unfolding transition , corresponding to the apical stem unfolding , is identical to that of wild - type va rnai in each case .  
proposed secondary structure for ad2 va rnai ( 16 ) , including a highly conserved pair of complementary tetranucleotide sequences ( outline typeface ) . shaded
boxed regions indicate helical segments progressively deleted from the terminal stem to create rnas : ts5 ( nts 48 and 148152 ) , ts10 ( nts 413 and 143152 ) , ts15 ( nts 418 and 138152 ) , and ts21 ( nts 132 and 131155 ) .
the white boxed region ( nts 13 and 153155 ) was retained in ts5 , ts10 , and ts15 rnas to maintain a strong promoter for in vitro transcription by t7 rnap ; in ts21 , base pair c  g was reversed for this purpose , as indicated .
a final point mutation , 79cu , was made to create an apical stem mutant rna ( bp rna ) with a watson - crick a 
proposed secondary structure for ad2 va rnai ( 16 ) , including a highly conserved pair of complementary tetranucleotide sequences ( outline typeface ) . shaded
boxed regions indicate helical segments progressively deleted from the terminal stem to create rnas : ts5 ( nts 48 and 148152 ) , ts10 ( nts 413 and 143152 ) , ts15 ( nts 418 and 138152 ) , and ts21 ( nts 132 and 131155 ) .
the white boxed region ( nts 13 and 153155 ) was retained in ts5 , ts10 , and ts15 rnas to maintain a strong promoter for in vitro transcription by t7 rnap ; in ts21 , base pair c  g was reversed for this purpose , as indicated .
a final point mutation , 79cu , was made to create an apical stem mutant rna ( bp rna ) with a watson - crick a 
a , assays of pkr autophosphorylation inhibition by wild - type va rnai and terminal stem deletion mutants ts5 , ts10 , ts15 , and ts21 . pkr ( 0.1 g ) was preincubated with 0 , 0.5 , 1 , 5 , 10 , and 50 g / ml of va rna prior to activation by poly(i)poly(c ) rna ( 0.3 g / ml ) .
a progressive reduction in inhibitory activity is observed as the terminal stem is shortened , but this trend is sharply reversed for the largest deletion ts21 .
b , assays of pkr autophosphorylation inhibition by wild - type ( wt ) va rnai and ts21 rna performed as in a but using equimolar rna concentrations ( 0 , 0.01 , 0.02 , 0.04 , 0.1 , 1.0 , and 10 m ) .
c , quantification of the data shown in b. each experiment in a and b was repeated in at least three independent experiments .
va rnai ts21 is an active inhibitor of pkr autophosphorylation . a , assays of pkr autophosphorylation inhibition by wild - type va rnai and terminal stem deletion mutants ts5 , ts10 , ts15 , and ts21 . pkr ( 0.1 g )
was preincubated with 0 , 0.5 , 1 , 5 , 10 , and 50 g / ml of va rna prior to activation by poly(i)poly(c ) rna ( 0.3 g / ml ) . a progressive reduction in inhibitory activity
is observed as the terminal stem is shortened , but this trend is sharply reversed for the largest deletion ts21 .
b , assays of pkr autophosphorylation inhibition by wild - type ( wt ) va rnai and ts21 rna performed as in a but using equimolar rna concentrations ( 0 , 0.01 , 0.02 , 0.04 , 0.1 , 1.0 , and 10 m ) .
c , quantification of the data shown in b. each experiment in a and b was repeated in at least three independent experiments .
the unfolding of the wild - type va rnai terminal stem and central domain are coupled and highly cooperative and result in the sharp apparent transition in the melting profile at 61 c ( fig .
our previous studies demonstrated that even very subtle alterations in sequence , such as compensatory base pair exchanges , can be sufficient to uncouple these unfolding events into two or more apparent transitions ( 25 ) .
such uncoupling of the first apparent transitions is observed for all the terminal stem deletion rna mutants ( fig .
the ts5 rna terminal stem and central domain unfold as two apparent transitions , one at the original apparent tm ( 60 c ) and one at lower temperature ( tm  50 c ) .
both ts10 and ts15 rna have a similar profile shape but , surprisingly , in addition to uncoupling the unfolding process , the apparent tm of each new apparent transition is also increased with the highest stability of the remaining terminal stem / central domain observed for ts15 rna . the melting profile for ts21 mutant is the most remarkable and distinct from wild - type va rnai .
deletion of the terminal stem creates an rna with a very  loose  central domain structure that unfolds in two very broad apparent transitions centered around 45 and 65 c ( fig .
3b ) . a range of uv melting experiments conducted in the presence of various concentrations and types of monovalent cation or with mg did not indicate any specific stabilization of any domain of the rna ( data not shown ) .
however , significant stabilization of the first broad apparent transition ( tm  45 c ) is observed at low ph ; over the range 7.5 to 5.5 a stabilization of 11 c is observed ( fig .
3c ) . in sharp contrast , the second broad transition ( tm  65 c ) corresponding to the remaining central domain structure is unaffected by changes in ph .
3d ) show that the central domain stability is also dependent upon ph in this context , although the tm is smaller , presumably as the structure is already stabilized close to the maximum extent possible by the intact terminal stem .
specific protonation - dependent stabilization within the central domain most likely arises from a component of tertiary structure within the rna ( see  discussion  ) .
the apical stem is also moderately stabilized at lower ph with a tm  4 c over the same range for both ts21 and wild - type va rnai ( fig .
we hypothesized that the ph dependence of apical stem unfolding might arise from the presence of an ( ac ) mismatch pair predicted from the current secondary structure model ( fig .
this was confirmed using a 79cu mutation to create a watson - crick a - u base pair at this position (  bp rna  ) , removing the potential protonation site .
this resulted in an apical stem structure that was most stable at neutral ph ( fig .
analysis of wild - type and ts21 va rnai interaction with pkr  we next examined the interaction of ts21 rna with pkr to explore potential differences in molecular recognition between this new  mini - va rnai  and the wild - type sequence .
analytical gel filtration chromatography of pkr , va rnai , and pkr - va rnai complexes at various molar ratios was used to investigate the nature and stoichiometry of binding ( fig .
wild - type va rnai eluted considerably earlier from the column than ts21 rna as expected based on the molecular size ( fig .
pkr and both rnas in isolation showed a single major peak in the chromatogram characterized by strong 230 nm / no 260 nm and strong 260 nm / weak 230 nm absorbances , respectively .
the protein and rna content of each peak for various complex ratios could therefore be determined using the relative absorbances . at an input molar ratio of 1:2 pkr to ts21 rna ,
a single peak corresponding to a complex of 1:1 stoichiometry is observed well resolved from a second later eluting peak corresponding to free rna .
wild - type va rnai exhibits the same behavior , but the peaks are not well resolved because of the larger size of the rna . at an input ratio of 1:1 , for both rnas a single major peak is observed that elutes at the volume corresponding to the 1:1 complex .
in contrast , at higher protein to rna ratios a difference in binding behavior is observed . for ts21 , although some higher molecular weight species are visible as a leading shoulder , a significant portion of the protein - rna complex still elutes at the volume corresponding to the complex of 1:1 stoichiometry ( fig .
4 , marked by dotted vertical line between 1:1 and 2:1 panels in the right column ) . in contrast , with a 2:1 input ratio , the pkr - wild - type va rnai complex elutes significantly earlier , corresponding to higher molecular weight complex , with no 1:1 stoichiometry complex remaining . for both rnas , a further increase in protein to rna input ratio decreases the elution volume for both complexes ( or mixtures of complexes ) , but at no concentration
a , uv melting curves ( dashed lines ) and melting profiles ( first derivatives of the melting curves ; solid lines ) for wild - type va rnai collected at 260 ( blue ) and 280 nm ( black ) .
the assignment shown of rna domain unfolding to each region of the melting profile was determined using compensatory base pair changes in each helical region of the rna ( 25 ) .
b , melting profiles of wild - type va rnai ( black ) , ts5(blue ) , ts10 ( green ) , ts15 ( orange ) , and ts21 ( red ) at 280 nm . all terminal stem mutations affect only the first apparent transition ( tm  60 c ) as expected .
ts21 rna unfolds in three apparent transitions with the first two corresponding to the remaining central domain structure . the first central domain apparent unfolding transition is dramatically stabilized by low ph ( tm  11 c between ph 5.5 and 7.5 ) , whereas the second apparent transition is unaffected ( tm  0 ) .
the final apparent transition ( tm  85 c ) corresponds to the apical stem and shows a small increase in stability at lower ph ( tm  4 c ) .
d , comparison of uv melting profiles at 280 nm for full - length wild - type va rnai and bp rna . as for ts21 rna , both the central domain ( tm  60 c ) and apical stem ( tm  85 c ) unfolding exhibit dependence upon ph in the context of the full - length wild - type rna , with greater stability at lower ph . in contrast
, the ph dependence is reversed for bp rna , which lacks the ( ac ) mismatch pair ( fig .
a , uv melting curves ( dashed lines ) and melting profiles ( first derivatives of the melting curves ; solid lines ) for wild - type va rnai collected at 260 ( blue ) and 280 nm ( black ) .
the assignment shown of rna domain unfolding to each region of the melting profile was determined using compensatory base pair changes in each helical region of the rna ( 25 ) .
b , melting profiles of wild - type va rnai ( black ) , ts5(blue ) , ts10 ( green ) , ts15 ( orange ) , and ts21 ( red ) at 280 nm . all terminal stem mutations affect only the first apparent transition ( tm  60 c ) as expected .
ts21 rna unfolds in three apparent transitions with the first two corresponding to the remaining central domain structure .
the first central domain apparent unfolding transition is dramatically stabilized by low ph ( tm  11 c between ph 5.5 and 7.5 ) , whereas the second apparent transition is unaffected ( tm  0 ) .
the final apparent transition ( tm  85 c ) corresponds to the apical stem and shows a small increase in stability at lower ph ( tm  4 c ) .
d , comparison of uv melting profiles at 280 nm for full - length wild - type va rnai and bp rna . as for ts21 rna , both the central domain ( tm  60 c ) and apical stem ( tm  85 c ) unfolding exhibit dependence upon ph in the context of the full - length wild - type rna , with greater stability at lower ph . in contrast
, the ph dependence is reversed for bp rna , which lacks the ( ac ) mismatch pair ( fig .
5 ) under conditions used previously to characterize the binding of various rnas to pkr ( 34 , 38 ) . in line with previous observations ,
the titration curve for ts21 rna was best fit using a model for two binding sites , and the values derived for parameters associated with the  high affinity  binding site are given in table 1 .
most strikingly , although the enthalpic and entropic contributions to binding differ dramatically for ts21 and wild - type va rnai , the resulting binding affinity and overall free energy change are identical .
va rnai has long been recognized as a potent inhibitor of the cellular anti - viral defenses mediated by pkr .
a large body of evidence has identified the apical stem of va rnai as the primary binding site and its complex central domain as the major determinant of inhibitory activity ( 1324 ) .
although far less extensively examined , prior mutagenesis suggested that an intact terminal stem structure , particularly adjacent to the central domain , might also contribute to inhibition of pkr ( 21 ) .
our previous studies of va rnai unfolding supported this idea because the central domain stability was found to be coupled to and dependent upon that of the terminal stem ( 25 ) .
thus , in addition to carrying promoter sequences , the terminal stem might play an indirect role in va rnai activity by acting as a structural clamp to stabilize the functional rna tertiary fold of the central domain or to protect against rna unwinding or nuclease attack .  
elution of wild - type ( wt ) va rnai ( left column ) and ts21 rna ( right column ) from a gel filtration column , alone and with pkr at various molar ratios ( indicated in the top right of each panel as input protein : rna ratio ) .
uv absorbance was measured at both 260 and 230 nm to allow the relative protein and rna content of each peak to be determined .
elution of wild - type ( wt ) va rnai ( left column ) and ts21 rna ( right column ) from a gel filtration column , alone and with pkr at various molar ratios ( indicated in the top right of each panel as input protein : rna ratio ) .
uv absorbance was measured at both 260 and 230 nm to allow the relative protein and rna content of each peak to be determined .
to directly assess its contribution to va rnai structure and pkr inhibition , we used site - directed mutagenesis to systematically shorten the terminal stem helix . in doing so
, we also hoped to identify an rna with a significantly shortened terminal stem that might be beneficial for in vitro biochemical and biophysical analyses of pkr - rna interactions .
for example , deletion of a significant length of the helix would remove the potential for the terminal stem to provide secondary , or 
creation of a minimal va rnai inhibitor of pkr  the terminal stem of va rnai was deleted in three sequential steps of 5 bp and a final deletion of the entire helix ( fig .
we anticipated that a progressive reduction in pkr inhibitory activity would be observed but that it might be possible to identify a point at which the terminal stem could be deleted without significant loss of function .
for the first three rnas , ts5 , ts10 , and ts15 , this expectation was met , with each requiring more inhibitor rna to provide complete inhibition of pkr .
remarkably , however , the trend was completely reversed for ts21 rna , which exhibited wild - type activity ( fig .
2 ) despite completely lacking a terminal stem domain . as the apical stem in isolation is a weak activator of pkr and the two conserved tetranucleotide sequences in the central domain immediately adjacent to the 21 deletion are critical for efficient inhibition , the ts21 rna created here is the shortest possible va rna inhibitor of pkr ( mini - va rnai ) .
structural impact of terminal stem deletions  uv melting experiments were used to assess the global impact on the rna structure of each terminal stem deletion . in line with previous observations ( 25 ) , deletions in the terminal stem affected only the lower temperature apparent transition that corresponds to the coupled unfolding of the terminal stem and central domain .
even the smallest deletion , ts5 , uncouples the unfolding of these domains such that two apparent transitions are observed , one at lower apparent tm and the other at the original tm .
similarly shaped profiles are observed for ts10 and ts15 rnas but with variation in both the hypochromicity and tm of both apparent transitions ( fig .
the changes observed in apparent tm might reflect a reorganization of the base pairing within the terminal stem , e.g. to incorporate nucleotides in the 2430 loop as the lower stem is shortened ( and presumably destabilized ) .
terminal stem structures corresponding to the wild - type and each deletion mutant sequence , but with a short stable stem - loop above base pair 39120 in place of the central domain and apical stem , were assessed using mfold ( 39 ) .
two identical low energy structures were identified for each rna , with the secondary structure of fig . 1 corresponding to a marginally less stable structure ( data not shown ) .
it is therefore plausible that the changes observed in the melting profiles correspond to a switch in the pairing scheme to the alternative structure identified as the stem is shortened .
regardless of the precise pairing , however , such alterations clearly do not have a major impact on the remaining central domain structure ; for deletions up to ts15 , the melting profiles indicate a folded structure and the steady decrease in pkr inhibition activity correlates with the size of the deletion .  
itc analysis of rna - protein interaction for titration of pkr ( 60 m ) into ts21rna ( 3 m ) .
the titration heats measured on binding ( top panel ) were integrated to produce the binding isotherm ( lower panel ) , and this fit to a model for two binding sites using the origin software package .
the thermodynamic parameters determined from the fit for the first , high affinity , binding site are shown in table 1 .  
itc analysis of rna - protein interaction for titration of pkr ( 60 m ) into ts21rna ( 3 m ) .
the titration heats measured on binding ( top panel ) were integrated to produce the binding isotherm ( lower panel ) , and this fit to a model for two binding sites using the origin software package .
the thermodynamic parameters determined from the fit for the first , high affinity , binding site are shown in table 1 .
the melting profile for the remaining central domain structure of ts21 rna is , however , dramatically different with two very broad peaks of low hypochromicity ( fig .
we initially took this result to indicate that the central domain of va rnai was largely unfolded in the absence of an intact terminal stem , supporting the idea of its role as a structural clamp to secure the functional central domain structure ( 19 ) .
although this may be true , from the high activity of the ts21 rna the terminal stem is clearly not essential for pkr inhibition .
it is also interesting to note that in the itc analysis of the ts21 rna - pkr interaction , there is a large change in the enthalpic and entropic contributions to binding despite an overall identical g and binding affinity as for wild - type - va rnai .
although we are cautious in attempting to interpret the physical meaning of these changes , both the uv melting profiles and other spectroscopic analyses we have conducted with ts21 rna5 point to a flexible ,  loosely  folded central domain structure .
earlier structure probing experiments on the full - length rna have shown that both pkr and mg can induce similar conformational changes upon binding ( 15 ) .
these observations suggest that va rnai has a dynamic flexible structure , most pronounced in the absence of a terminal stem in ts21 rna , that is folded or stabilized into the correct functional tertiary structure upon interaction with pkr .
the va rnai central domain contains a ph - dependent tertiary structure  removal of the terminal stem allowed us to assess in more detail other factors that might influence the va rnai central domain tertiary folding . as for wild - type va rnai ( 25 )
, no significant specific stabilization of any apparent unfolding transition was observed in melting experiments containing various concentrations of mg or different monovalent ions ( data not shown ) .
one component of the central domain was , however , specifically stabilized by low ph with a striking difference in apparent tm of 11 c over the ph range 5.57.5 .
this result provides clear evidence for the involvement of a protonated base in creating the va rnai central domain structure , such as a protonated cytosine in forming base triple interactions as observed in dna ( 40 , 41 ) and rna triplexes ( 42 , 43 ) , and other rna tertiary structures ( 4446 ) . the most obvious candidates for the site(s ) of protonation within the central domain are cytosines 104105 , 107 , or 116 in an interaction with base pairs in the lower stem of the rna ( nts 120130 ) .
interestingly , c is immediately adjacent to the conserved pair of tetranucleotides also proposed to be involved in the rna tertiary structure ( 20 ) .
however , point mutations at each of these nucleotides ( to either g or a ) did not dramatically reduce the inhibitory function of the resulting rna ( 24 ) . therefore , confirmation of the site of protonation and whether this is critical for central domain tertiary structure folding and/or pkr inhibition will require further detailed investigation .
implications for analysis of pkr - rna interaction  the terminal stem is an imperfectly paired helix that could potentially provide a secondary binding site for the dsrna binding domain of pkr .
our gel filtration data shows differences in complex formation with excess pkr between wild - type and ts21 rnas . for both rnas similar large ( nonspecific ) complexes
are ultimately formed , but unlike the wild - type rna , ts21 rna initially maintains a predominantly 1:1 complex stoichiometry suggesting that it does lack some lower affinity binding site for pkr .
itc data for va rnai and other inhibitor and activator rnas are best fit using a model for two sites , where the lower affinity site is ascribed to nonspecific binding and otherwise ignored ( 33 , 34 , 38 ) .
our itc analysis of ts21 rna , which lacks the terminal stem , shows two distinct binding events .
the kd and g values for the high affinity site are in excellent agreement with previous values for va rnai ( table 1 ) although , as noted above , with large compensating changes in h and s .
more curiously , even though the second  weak affinity  binding site is well resolved in the titration and the data apparently well fit by a two - site model , the thermodynamic parameters obtained for this site are highly unrealistic .
the shape of the titration suggests a binding site of moderate affinity , but a kd of 0.5 nm is obtained .
we hypothesize that this  binding site  in fact arises from conformational heterogeneity in the va rnai apical stem ( 20 ) .
rather than a single rna molecule with two binding sites , the data reflect a mixed population of rna structures with the fully extended apical stem predominant and producing the high affinity site .
we predict that rnas with modified apical stems that remove this heterogeneity will produce a pkr binding titration curve fit optimally with a single high affinity site , and experiments to test this hypothesis are currently underway .  
va rnai is processed by dicer to produce short single - stranded rnas from the ts that are incorporated into risc , and an apical stem - central domain ( as - cd ) rna , equivalent to ts21 rna , that remains fully active against pkr .
thus , each transcript from the va rnai gene is able to both saturate cellular rna interference mechanisms and block the pkr - mediated down - regulation of general translation of cellular and viral proteins via eif2 phosphorylation .
abbreviations used for proteins are as follows : pkr , dsrna binding motifs ( 1 and 2 ) ; kd , kinase domain
va rnai is processed by dicer to produce short single - stranded rnas from the ts that are incorporated into risc , and an apical stem - central domain ( as - cd ) rna , equivalent to ts21 rna , that remains fully active against pkr .
thus , each transcript from the va rnai gene is able to both saturate cellular rna interference mechanisms and block the pkr - mediated down - regulation of general translation of cellular and viral proteins via eif2 phosphorylation .
abbreviations used for proteins are as follows : pkr , dsrna binding motifs ( 1 and 2 ) ; kd , kinase domain .
va rnai function in vivo  the va rnai terminal stem has been suggested to play a role in stabilizing or protecting the critical central domain against degradation in vivo ( 21 ) . although our results do agree that the terminal stem provides a large degree of structural stabilization , it is clearly not necessary for pkr inhibition .
the active mini - va rnai we have produced in vitro is strikingly similar to the product of dicer processing of the full - length rna in vivo ( 26 ) .
thus , adenovirus may both inhibit pkr and saturate dicer using a single gene product where each rna transcript contributes to both mechanisms ( fig .
va rnai may therefore be a truly bifunctional rna and exemplify a remarkably parsimonious use of short rna transcripts to counteract host cell defenses against viral infection .
 adenoviruses use the short noncoding rna transcript virus - associated ( va ) rnai to counteract two critical elements of the host cell defense system , innate cellular immunity and rna interference , mediated by the double - stranded rna - activated protein kinase ( pkr ) and dicer / rna - induced silencing complex , respectively . 
 we progressively shortened the va rnai terminal stem to examine its necessity for inhibition of pkr . 
 each deletion , up to 15 bp into the terminal stem , resulted in a cumulative decrease in pkr inhibitory activity . remarkably , however , despite significant apparent destabilization of the rna structure , the final rna mutant that lacked the entire terminal stem ( ts21 rna ) efficiently bound pkr and exhibited wild - type inhibitory activity . 
 ts21 rna stability was strongly influenced by solution ph , indicating the involvement of a protonated base within the va rnai central domain tertiary structure . 
 gel filtration chromatography and isothermal titration calorimetry analysis indicated that wild - type va rnai and ts21 rna form similar 1:1 complexes with pkr but that the latter lacks secondary binding site(s ) that might be provided by the terminal stem . 
 although ts21 rna bound pkr with wild - type kd , and overall change in free energy ( g ) , the thermodynamics of binding ( h and s ) were significantly altered . 
 these results demonstrate that the va rnai terminal stem is entirely dispensable for inhibition of pkr . 
 potentially , va rnai is therefore a truly bi - functional rna ; dicer processing of the va rnai terminal stem saturates the rna interference system while generating a  mini - va rnai  molecule that remains fully active against pkr . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION