BACKGROUND
eukaryotic transcriptional activators that stimulate transcription initiation of a particular set of target genes usually consist of a sequence-specific dna-binding domain  and a transcription activation domain .
the dbd targets these activators to a specific location in the promoter region of a gene, and the ad mediates transcription initiation by recruiting gene-specific factors, chromatin-remodeling factors, mediator complexes, and general transcription factors  <cit> .
dbds are classified into several distinct patterns or motifs according to their sequences and structural similarities.
in contrast, ads do not share significant sequence homologies, and therefore no specific motif has been defined.
despite this, several classes of ads with distinctive sequence features were identified, including acidic activators  <cit> , glutamine-rich activators  <cit> , and proline-rich activators  <cit> .
while an increasing number of transcriptional activators have been identified, the direct targets or interacting partners of most transcriptional activators and the detailed mechanisms through which these interacting partners induce transcription initiation remain largely unknown.
to date, only a few protein factors have been explicitly identified as direct targets of transcriptional activators, including the tata box-binding protein , tfiib, tfiih, and a few others  <cit> .
one of the earliest and best-studied models for transcriptional regulation in eukaryotes is that of yeast gal <dig>  which is involved in regulating galactose metabolism in response to changes in the concentration of the carbohydrate  <cit> .
the gal <dig> system  consists of four structural  and three regulatory  genes.
protein products of the structural genes are required for transport and metabolism of galactose, and protein products of the regulatory genes control expression of the structural genes.
gal <dig> is an acidic transcriptional activator comprised of two functionally independent domains: an n-terminal sequence-specific dbd and a c-terminal ad.
induction of the gal4-responsive genes by galactose is mediated through the specific binding of the gal4-dbd to an upstream activating sequence in their promoter regions and subsequent recruitment of the general transcription apparatus by the gal4-ad.
under non-inducing conditions in the absence of galactose, gal <dig> activity is repressed by the interaction of gal <dig> with the gal4-ad  <cit> .
under inducing conditions when galactose is present, the repressor, gal <dig>  is taken away by gal <dig> from the gal4-ad, which is then able to recruit the transcription machinery and initiate transcription  <cit> .
in prokaryotes, there are typically  <dig> aminoacyl-trna synthetases, one for each amino acid  <cit> .
in eukaryotes, protein synthesis occurs in the cytoplasm, and also in organelles, such as mitochondria and chloroplasts  <cit> .
thus, eukaryotes, such as yeast, commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized in the cytoplasm and the other in the mitochondria.
however, in some cases, the cytoplasmic and mitochondrial forms of a trna synthetase with a given amino acid specificity are encoded by the same nuclear gene through alternative initiation of translation, examples of which include ala <dig>   <cit> , grs <dig>   <cit> , hts <dig>   <cit> , and vas <dig> )  <cit> .
because the two isoforms are essentially generated from the same open reading frame, they have the same polypeptide sequence, except for a short sequence attached to the amino-terminus of the mitochondrial precursor that functions as a mitochondrial targeting signal.
as a consequence, the two isoforms cannot be substituted for each other in vivo.
many eukaryotic cytoplasmic trna synthetases contain an amino- or carboxyl-terminal polypeptide extension, which is absent from their bacterial counterparts  <cit> .
these extensions are generally rich in lysine residues and capable of non-specifically binding to rna.
a well-studied example is the appended domain  of yeast glutaminyl-trna synthetase , which binds unfractionated yeast trnas, single-stranded rna, and pseudoknot rna with comparable affinities, with kd values of around  <dig>  μm  <cit> .
many other examples were also identified, such as the carboxyl-terminal domain of rice methionyl-trna synthetase  <cit>  and the amino-terminal domain of mammalian lysyl-trna synthetase  <cit> .
in addition to rna binding, the ads of some trna synthetases were found to participate in protein-protein interactions, such as those of yeast glutamyl- and methionyl-trna synthetases   <cit>  and mammalian valrs  <cit> .
however, more generally, the exact role of the ad in the biological functions of this family of enzymes remains elusive.
recently, it was found that many of these ads contain one or several classical nuclear localization signals  <cit>  which are believed to play a role in the nuclear importation of these otherwise "cytoplasmic" proteins.
even more exciting are the findings that several trna synthetases from both prokaryotes and eukaryotes take part in functions unrelated to aminoacylation, including roles in mitochondrial rna splicing, transcriptional and translational regulation, cytokine-like activity, and amino acid biosynthesis  <cit> .
as with many known yeast trna synthetases, the cytoplasmic form of yeast valrs also contains an amino-terminal polypeptide extension.
while the ad of mammalian valrs was shown to interact with the four subunits of the elongation factor, ef-1h, to form a high-molecular-weight complex  <cit> , relatively little is known about the biological function of its yeast counterpart.
our earlier studies suggested that the ad of yeast valrs  possesses non-specific trna-binding activity  that significantly contributes to trna binding and aminoacylation activities of the enzyme  <cit> .
we report herein that the n-terminal domain  of yeast valrs can act as an ad when fused to a sequence-specific dbd, and this transcriptional activity is mainly attributable to a tryptophan-rich peptide  within the n domain.
using this pentapeptide as a reference structure, a much more active peptide, heptatryptophan , was consequently devised.
furthermore, w <dig> stimulated transcription initiation via interaction with tfiib, a general transcription factor.
it is our hope that information obtained in this study will advance our understanding of the biochemical properties of ads in general, and also provide new insights into the mechanisms of transcription activation in particular.
RESULTS
identification of an activation peptide
in addition to trna binding, we wondered whether the n-terminal domain of yeast valrs possesses another function.
to explore this possibility, the n-terminal domain  was cloned by fusion to the dna-binding protein, lexa, and used as bait to screen a yeast library for interacting partners.
note that the n-terminal sequence used in this assay was  <dig> residues longer than the ad  used in previous studies  <cit>  to ensure that all of the sequence elements that might be important for interactions were included.
after all, the exact length of the ad has yet to be clearly defined.
the dna sequence encoding the n-terminal domain was pcr-amplified and cloned as a lexa fusion into pgilda  as described in "materials and methods", and the resulting construct was cotransformed with the reporter plasmid, p8oplacz , into the egy <dig> yeast strain.
to rule out the possibility that the bait hybrid protein was itself an autoactivator, the cotransformants were first tested on selection medium  in the absence of any prey hybrid protein.
unexpectedly, the lexa-n domain fusion by itself could turn on the leu <dig> and lacz reporter genes; the cotransformants grew robustly on the x-gal agar plate and turned blue .
this result suggests that the bait hybrid protein per se is a transcription activator, and the n domain of yeast valrs acts as an ad.
it was noted that the operator sequences in the reporter genes leu <dig> and lacz were not identical, and only fusions that could turn on both reporter genes were considered positive in this assay.
to determine which segment of the n domain actually accounted for this transcriptional activity, various segments of the domain were individually fused to lexa, and their transcriptional activities were tested.
as shown in figure 1a, segments containing residues 98~ <dig>  101~ <dig>  104~ <dig>  107~ <dig>  and 111~ <dig> were active in the transcriptional assay .
in contrast, segments consisting of residues 113~ <dig>  98~ <dig>  and 98~ <dig> had no detectable activity .
thus, the segment containing residues 111~ <dig>  is essential and sufficient for this activity .
quantitative assays of the β-gal activity  further showed that among these active peptides, the segments containing residues 98~ <dig>  101~ <dig>  and 104~ <dig> had the highest activities ; the segment containing residues 107~ <dig> had medium activity ; and the segments containing residues 1~ <dig> and 111~ <dig> had the lowest activities  .
as expected, functionally inactive peptides had no detectable β-gal activity in the assays .
to check whether all of these lexa fusion constructs were properly expressed in the reporter yeast strain, the expression profiles of these constructs were analyzed by western blotting using an anti-lexa antibody.
as shown in figure 1c, all of the lexa fusion proteins were stably expressed by the constructs, with only minor variations in protein levels, suggesting that the negative phenotype observed on the x-gal plate for the functionally inactive peptides was not caused by severe protein degradation or insufficient protein synthesis .
plexa-pos  and pgilda respectively served as positive and negative controls in the assays.
repetition of the pentapeptide sequence enhanced activity
while the pentapeptide, wydww, can function as an ad, its activity was relatively low, only ~50% of that of the positive control and ~30% of that of the segment containing residues 98~ <dig> of the n domain .
to enhance its activity, two or three tandem repeats of the pentapeptide sequence were cloned, and the activities of the resulting constructs were tested.
as shown in figure 2a and 2b, duplication of the pentapeptide, resulting in  <dig>  strongly enhanced the activity  .
however, an additional replication of the sequence, resulting in  <dig>  did not further enhance the activity;  <dig> exhibited activity comparable to that of  <dig> .
western blotting assays showed that these constructs expressed similar levels of lexa fusion proteins.
thus, changes in the transcriptional activity of these fusion constructs did not result from different protein expression levels .
w <dig> had activity higher than that of wydww
to examine which amino acid residues of the pentapeptide are critical for its activity, the residues were mutated to alanine residues, and the activities of the resulting constructs were tested.
as shown in figure  <dig>  mutation of the first two amino acid residues  to aa, the last two residues  to aa, or the middle residue  to a or k drastically reduced the activities .
in contrast, mutation of the middle residue  to w had little effect on the activity .
moreover, mutation of the second residue  to w significantly enhanced the activity  .
thus, it appears that w is preferred in all positions of the pentapeptide for activity.
a western blot analysis showed that these mutations had only minor effects on the expression levels of the fusion proteins .
to gain further insights, both of the non-w residues in the pentapeptide were mutated to w residues , and the activity of the resulting construct was tested.
as expected, w <dig> had an activity ~ <dig> -fold higher than that of the native pentapeptide .
most amazingly, inserting two more w residues into w <dig>  yielding w <dig>  further enhanced the activity  .
that is, the activity of w <dig> was ~3-fold higher than that of the native pentapeptide .
however, the activity of w <dig> was almost equivalent to that of w <dig> .
thus, w <dig> appeared to be the strongest ad among those tested.
to investigate whether the transcriptional activities of these peptides were attributable to their hydrophobic property, w <dig> was mutated to f <dig>  and the activity of the resulting construct was tested.
as shown in figure 4a and 4b, changing w <dig> to f <dig> resulted in a functionally inactive peptide that failed to turn on the reporter genes .
with respect to protein expression, these fusion constructs expressed considerably different levels of proteins.
lexa-w <dig> and lexa-w <dig> produced the highest protein expression levels ; lexa-wydww and lexa-w <dig> had medium protein expression levels ; and lexa-f <dig> had the lowest protein expression level .
to further investigate whether the different protein expression levels observed herein were caused by different protein stabilities in vivo, a cycloheximide-chase assay  was carried out on the representative constructs, lexa-wydww, lexa-w <dig>  and lexa-f <dig>  to exclude the interference of transcription activation, the host cell used for the assay was invsc <dig>  instead of the reporter yeast strain, egy <dig>  as shown in figure 4e, these fusion proteins had conspicuously distinct turnover rates.
lexa-wydww was much more stable than lexa-w <dig>  and lexa-w <dig> was much more stable than lexa-f <dig>  lexa-f <dig> had a short half-life  and was degraded in vivo at a much faster speed than the other two fusion proteins tested.
it remains to be seen whether this attribute actually accounted for the negative phenotype of lexa-f <dig> in the transcriptional assay .
another interesting finding was the discovery that while lexa-w <dig> had the highest transcriptional activity among these three constructs, it did not have the highest protein stability or expression level .
thus, it appears that there were no direct correlations between transcriptional activity and protein expression levels in these instances.
figure 4d shows that similar levels of cdna products were generated from these fusion constructs, suggesting that the sequences encoding wydww, w <dig>  and f <dig> did not compromise the stability of the specific lexa mrnas in vivo.
w <dig> is a non-promoter-specific ad
we next tested whether the transcriptional activities of w <dig> and  <dig> were promoter-specific, and whether they were affected by the dbd used.
to this end, w <dig> and  <dig> were assayed in a gal4-based system, where the ad was fused in-frame to the gal4-dbd cloned in pgbkt <dig> , and the reporter genes used were his <dig> and mel <dig>  under the control of two completely heterologous gal4-responsive upstream activating sequences and promoter elements, gal <dig> and mel <dig>  respectively.
as shown in figure  <dig>  both gal4-dbd fusion proteins turned on the reporter genes; transformants carrying either of these two fusion constructs 2) grew robustly and turned blue on selection medium containing x-α-gal but lacking tryptophan and histidine , suggesting that both of these peptides acted as ads in gal4-dbd fusion proteins.
thus, the transcriptional activities of these two peptides were non-promoter-specific and were operational in both lexa- and gal4-dbd-responsive reporter genes.
quantitatively, these two fusion proteins had transcriptional activities ~ <dig> -fold lower than that of the positive control .
this is not surprising, considering the fact that the positive control was a wild-type gal <dig> gene.
western blot assays showed that these two gal4-dbd fusion constructs had protein expression levels much lower than that of the gal4-dbd alone .
whether the ads destabilized the gal4-dbd fusion proteins and whether the relatively poor transcriptional activity of these two fusion constructss was caused by a lower level of protein expression are yet to be determined.
however, regardless of the diverse protein expression levels, these results clearly demonstrate the ability of these two peptides to function as ads in the gal4-based system.
w <dig> acts as an ad in a two-hybrid system
so far, we have shown that w <dig> and  <dig> can act as ads when directly fused to a sequence-specific dbd such as lexa and gal4-dbd .
the question arose as to whether these two short peptides can act as efficiently when they are physically separated from the dbd.
to answer this question, dna sequences encoding these two peptides were individually cloned into pgadt7-t  to replace the sequence encoding the gal4-ad, and the transcriptional activities of the resulting constructs were tested following the protocols devised for the yeast two-hybrid system.
as shown in figure  <dig>  murine p <dig>  specifically interacted with the sv <dig> large t-antigen  and turned on the reporter genes, his <dig> and mel <dig> .
in contrast, human lamin c  did not interact with the large t-antigen and therefore failed to turn on the reporter genes .
interestingly, both w7-t and 2-t fusions turned on the reporter genes when acting in concert with the gal4-dbd- <dig> fusion , but failed to do so when acting in concert with the gal4-dbd-lam fusion .
this observation provides strong evidence that both w <dig> and  <dig> can act as ads in a traditional two-hybrid system, albeit with efficiencies poorer than that of gal4-ad .
w <dig> mediates transcription activation through interacting with tfiib
to date, only a few protein factors have unambiguously been identified as direct targets of transcriptional activators, including tbp, tfiib, tfiih, and a few others  <cit> .
we wondered whether w <dig> also stimulates transcription initiation through interaction with one of these protein factors.
pursuant to this objective, tbp, tfiib, and tfiih were individually cloned into pgilda , and then their interaction with w <dig>  was tested using a yeast two-hybrid system.
lexa and b <dig> respectively served as the dbd and ad in this assay.
as shown in figure  <dig>  all of these lexa fusion proteins were well expressed in the test yeast strain egy <dig> , with lexa-tfiih having a lower expression level.
in the absence of w <dig>  none of these fusion constructs per se could simultaneously turn on the designated reporter genes, leu <dig> and lacz .
however, in the presence of w <dig>  tfiib, but not tbp or tfiih, could turn on both reporter genes .
this result strongly suggests that w <dig> specifically interacts with tfiib.
discussion
we report herein that the n-terminal domain of yeast valrs acts as an ad when fused to a sequence-specific dbd, lexa .
however, there is no direct evidence so far showing that yeast valrs actually functions as a transcription activator in the nucleus.
more experiments are currently underway to validate this possibility.
despite this uncertain aspect, a core sequence element accounting for this activity was mapped to a region near the c-terminus of the n domain.
this core sequence element contains only five amino acid residues: wydww  .
interestingly, the core sequence element per se retained much of the transcriptional activity and could be stably expressed as a fusion to the dbd in yeast .
a mutagenesis study indicated that w residues in the pentapeptide were crucial for its activity.
even the y residue, which also possesses an aromatic ring in its side chain, was not as supportive of the activity as were the w residues .
as a result, the native pentapeptide acted less efficiently  than did wwdww and wwwww as an ad .
using this core sequence element as a reference, a much-stronger activation peptide, w <dig>  was successfully produced.
w <dig> had ~3-fold higher transcriptional activity than those of the native pentapeptide and the n domain .
while we cannot rule out the possibility that w <dig> somehow altered the affinity or specificity for the lexa operators of the lexa fusion protein, w <dig> has to be capable of interacting with one of the components of the transcription apparatus to stimulate transcription of the reporter genes.
to our knowledge, w <dig> is so far the smallest ad that retains strong transcriptional activity.
with its small size, w <dig> is unlikely to interfere with the folding of fused proteins or to occlude the normal site of interaction.
in addition, w <dig> can significantly reduce the overall size of the prey fusion construct, and therefore has the potential to enhance the efficiency of plasmid transformation, which is a long-standing issue when screening a large library for novel interacting partners.
similar to the reasons that the his <dig> tag is widely accepted and used for protein purification, w <dig> may be an excellent replacement for the gal4-ad or b <dig> in two-hybrid screening as an ad.
a preliminary study suggested that the direct target of w <dig> may be the general transcription factor, tfiib , which was previously shown to be one of the interacting partners of the acidic transcription activator, gal <dig>  <cit> .
it is believed that recruitment of tfiib and others by gal <dig> leads to assembly of the transcription apparatus at the promoter site and subsequent transcription initiation  <cit> .
however, sequence alignment between these two ads  showed little sequence homology.
thus, gal4-ad and w <dig> may interact with different sites of tfiib or through a different mechanism.
alternatively, a certain portion of gal <dig> may fold into a three-dimensional structure that displays a hydrophobic feature similar to that of w <dig>  in any case, it is interesting to find that both of these ads mediate transcription activation through interacting with tfiib.
thus, the minute ad, w <dig>  might turn out to be an interesting paradigm for further mechanistic studies of transcription activation.
CONCLUSIONS
a short peptide containing seven consecutive tryptophan residues  can function as an activation domain when fused to a dna-binding protein, lexa, and turn on reporter genes with distinct lexa-responsive promoters.
like the activation domain of gal <dig>  w <dig> mediates transcription activation through interacting with the general transcription factor, tfiib.

 eukaryotic transcriptional activators that stimulate transcription initiation of a particular set of target genes usually consist of a sequence-specific dna-binding domain and a transcription activation domain. the dbd targets these activators to a specific location in the promoter region of a gene, and the ad mediates transcription initiation by recruiting gene-specific factors, chromatin-remodeling factors, mediator complexes, and general transcription factors. dbds are classified into several distinct patterns or motifs according to their sequences and structural similarities. in contrast, ads do not share significant sequence homologies, and therefore no specific motif has been defined. despite this, several classes of ads with distinctive sequence features were identified, including acidic activators, glutamine-rich activators, and proline-rich activators. while an increasing number of transcriptional activators have been identified, the direct targets or interacting partners of most transcriptional activators and the detailed mechanisms through which these interacting partners induce transcription initiation remain largely unknown. to date, only a few protein factors have been explicitly identified as direct targets of transcriptional activators, including the tata box-binding protein, tfiib, tfiih, and a few others. one of the earliest and best-studied models for transcriptional regulation in eukaryotes is that of yeast gal which is involved in regulating galactose metabolism in response to changes in the concentration of the carbohydrate. the gal system consists of four structural and three regulatory genes. protein products of the structural genes are required for transport and metabolism of galactose, and protein products of the regulatory genes control expression of the structural genes. gal is an acidic transcriptional activator comprised of two functionally independent domains: an n-terminal sequence-specific dbd and a c-terminal ad. induction of the gal4-responsive genes by galactose is mediated through the specific binding of the gal4-dbd to an upstream activating sequence in their promoter regions and subsequent recruitment of the general transcription apparatus by the gal4-ad. under non-inducing conditions in the absence of galactose, gal activity is repressed by the interaction of gal with the gal4-ad. under inducing conditions when galactose is present, the repressor, gal is taken away by gal from the gal4-ad, which is then able to recruit the transcription machinery and initiate transcription. in prokaryotes, there are typically aminoacyl-trna synthetases, one for each amino acid. in eukaryotes, protein synthesis occurs in the cytoplasm, and also in organelles, such as mitochondria and chloroplasts. thus, eukaryotes, such as yeast, commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized in the cytoplasm and the other in the mitochondria. however, in some cases, the cytoplasmic and mitochondrial forms of a trna synthetase with a given amino acid specificity are encoded by the same nuclear gene through alternative initiation of translation, examples of which include ala, grs, hts, and vas ). because the two isoforms are essentially generated from the same open reading frame, they have the same polypeptide sequence, except for a short sequence attached to the amino-terminus of the mitochondrial precursor that functions as a mitochondrial targeting signal. as a consequence, the two isoforms cannot be substituted for each other in vivo. many eukaryotic cytoplasmic trna synthetases contain an amino- or carboxyl-terminal polypeptide extension, which is absent from their bacterial counterparts. these extensions are generally rich in lysine residues and capable of non-specifically binding to rna. a well-studied example is the appended domain of yeast glutaminyl-trna synthetase, which binds unfractionated yeast trnas, single-stranded rna, and pseudoknot rna with comparable affinities, with kd values of around μm. many other examples were also identified, such as the carboxyl-terminal domain of rice methionyl-trna synthetase and the amino-terminal domain of mammalian lysyl-trna synthetase. in addition to rna binding, the ads of some trna synthetases were found to participate in protein-protein interactions, such as those of yeast glutamyl- and methionyl-trna synthetases and mammalian valrs. however, more generally, the exact role of the ad in the biological functions of this family of enzymes remains elusive. recently, it was found that many of these ads contain one or several classical nuclear localization signals which are believed to play a role in the nuclear importation of these otherwise "cytoplasmic" proteins. even more exciting are the findings that several trna synthetases from both prokaryotes and eukaryotes take part in functions unrelated to aminoacylation, including roles in mitochondrial rna splicing, transcriptional and translational regulation, cytokine-like activity, and amino acid biosynthesis. as with many known yeast trna synthetases, the cytoplasmic form of yeast valrs also contains an amino-terminal polypeptide extension. while the ad of mammalian valrs was shown to interact with the four subunits of the elongation factor, ef-1h, to form a high-molecular-weight complex, relatively little is known about the biological function of its yeast counterpart. our earlier studies suggested that the ad of yeast valrs possesses non-specific trna-binding activity that significantly contributes to trna binding and aminoacylation activities of the enzyme. we report herein that the n-terminal domain of yeast valrs can act as an ad when fused to a sequence-specific dbd, and this transcriptional activity is mainly attributable to a tryptophan-rich peptide within the n domain. using this pentapeptide as a reference structure, a much more active peptide, heptatryptophan, was consequently devised. furthermore, w stimulated transcription initiation via interaction with tfiib, a general transcription factor. it is our hope that information obtained in this study will advance our understanding of the biochemical properties of ads in general, and also provide new insights into the mechanisms of transcription activation in particular.  identification of an activation peptide in addition to trna binding, we wondered whether the n-terminal domain of yeast valrs possesses another function. to explore this possibility, the n-terminal domain was cloned by fusion to the dna-binding protein, lexa, and used as bait to screen a yeast library for interacting partners. note that the n-terminal sequence used in this assay was residues longer than the ad used in previous studies to ensure that all of the sequence elements that might be important for interactions were included. after all, the exact length of the ad has yet to be clearly defined. the dna sequence encoding the n-terminal domain was pcr-amplified and cloned as a lexa fusion into pgilda as described in "materials and methods", and the resulting construct was cotransformed with the reporter plasmid, p8oplacz, into the egy yeast strain. to rule out the possibility that the bait hybrid protein was itself an autoactivator, the cotransformants were first tested on selection medium in the absence of any prey hybrid protein. unexpectedly, the lexa-n domain fusion by itself could turn on the leu and lacz reporter genes; the cotransformants grew robustly on the x-gal agar plate and turned blue. this result suggests that the bait hybrid protein per se is a transcription activator, and the n domain of yeast valrs acts as an ad. it was noted that the operator sequences in the reporter genes leu and lacz were not identical, and only fusions that could turn on both reporter genes were considered positive in this assay. to determine which segment of the n domain actually accounted for this transcriptional activity, various segments of the domain were individually fused to lexa, and their transcriptional activities were tested. as shown in figure 1a, segments containing residues 98~ 101~ 104~ 107~ and 111~ were active in the transcriptional assay. in contrast, segments consisting of residues 113~ 98~ and 98~ had no detectable activity. thus, the segment containing residues 111~ is essential and sufficient for this activity. quantitative assays of the β-gal activity further showed that among these active peptides, the segments containing residues 98~ 101~ and 104~ had the highest activities; the segment containing residues 107~ had medium activity; and the segments containing residues 1~ and 111~ had the lowest activities. as expected, functionally inactive peptides had no detectable β-gal activity in the assays. to check whether all of these lexa fusion constructs were properly expressed in the reporter yeast strain, the expression profiles of these constructs were analyzed by western blotting using an anti-lexa antibody. as shown in figure 1c, all of the lexa fusion proteins were stably expressed by the constructs, with only minor variations in protein levels, suggesting that the negative phenotype observed on the x-gal plate for the functionally inactive peptides was not caused by severe protein degradation or insufficient protein synthesis. plexa-pos and pgilda respectively served as positive and negative controls in the assays. repetition of the pentapeptide sequence enhanced activity while the pentapeptide, wydww, can function as an ad, its activity was relatively low, only ~50% of that of the positive control and ~30% of that of the segment containing residues 98~ of the n domain. to enhance its activity, two or three tandem repeats of the pentapeptide sequence were cloned, and the activities of the resulting constructs were tested. as shown in figure 2a and 2b, duplication of the pentapeptide, resulting in strongly enhanced the activity. however, an additional replication of the sequence, resulting in did not further enhance the activity; exhibited activity comparable to that of. western blotting assays showed that these constructs expressed similar levels of lexa fusion proteins. thus, changes in the transcriptional activity of these fusion constructs did not result from different protein expression levels. w had activity higher than that of wydww to examine which amino acid residues of the pentapeptide are critical for its activity, the residues were mutated to alanine residues, and the activities of the resulting constructs were tested. as shown in figure mutation of the first two amino acid residues to aa, the last two residues to aa, or the middle residue to a or k drastically reduced the activities. in contrast, mutation of the middle residue to w had little effect on the activity. moreover, mutation of the second residue to w significantly enhanced the activity. thus, it appears that w is preferred in all positions of the pentapeptide for activity. a western blot analysis showed that these mutations had only minor effects on the expression levels of the fusion proteins. to gain further insights, both of the non-w residues in the pentapeptide were mutated to w residues, and the activity of the resulting construct was tested. as expected, w had an activity ~ -fold higher than that of the native pentapeptide. most amazingly, inserting two more w residues into w yielding w further enhanced the activity. that is, the activity of w was ~3-fold higher than that of the native pentapeptide. however, the activity of w was almost equivalent to that of w. thus, w appeared to be the strongest ad among those tested. to investigate whether the transcriptional activities of these peptides were attributable to their hydrophobic property, w was mutated to f and the activity of the resulting construct was tested. as shown in figure 4a and 4b, changing w to f resulted in a functionally inactive peptide that failed to turn on the reporter genes. with respect to protein expression, these fusion constructs expressed considerably different levels of proteins. lexa-w and lexa-w produced the highest protein expression levels; lexa-wydww and lexa-w had medium protein expression levels; and lexa-f had the lowest protein expression level. to further investigate whether the different protein expression levels observed herein were caused by different protein stabilities in vivo, a cycloheximide-chase assay was carried out on the representative constructs, lexa-wydww, lexa-w and lexa-f to exclude the interference of transcription activation, the host cell used for the assay was invsc instead of the reporter yeast strain, egy as shown in figure 4e, these fusion proteins had conspicuously distinct turnover rates. lexa-wydww was much more stable than lexa-w and lexa-w was much more stable than lexa-f lexa-f had a short half-life and was degraded in vivo at a much faster speed than the other two fusion proteins tested. it remains to be seen whether this attribute actually accounted for the negative phenotype of lexa-f in the transcriptional assay. another interesting finding was the discovery that while lexa-w had the highest transcriptional activity among these three constructs, it did not have the highest protein stability or expression level. thus, it appears that there were no direct correlations between transcriptional activity and protein expression levels in these instances. figure 4d shows that similar levels of cdna products were generated from these fusion constructs, suggesting that the sequences encoding wydww, w and f did not compromise the stability of the specific lexa mrnas in vivo. w is a non-promoter-specific ad we next tested whether the transcriptional activities of w and were promoter-specific, and whether they were affected by the dbd used. to this end, w and were assayed in a gal4-based system, where the ad was fused in-frame to the gal4-dbd cloned in pgbkt, and the reporter genes used were his and mel under the control of two completely heterologous gal4-responsive upstream activating sequences and promoter elements, gal and mel respectively. as shown in figure both gal4-dbd fusion proteins turned on the reporter genes; transformants carrying either of these two fusion constructs 2) grew robustly and turned blue on selection medium containing x-α-gal but lacking tryptophan and histidine, suggesting that both of these peptides acted as ads in gal4-dbd fusion proteins. thus, the transcriptional activities of these two peptides were non-promoter-specific and were operational in both lexa- and gal4-dbd-responsive reporter genes. quantitatively, these two fusion proteins had transcriptional activities ~ -fold lower than that of the positive control. this is not surprising, considering the fact that the positive control was a wild-type gal gene. western blot assays showed that these two gal4-dbd fusion constructs had protein expression levels much lower than that of the gal4-dbd alone. whether the ads destabilized the gal4-dbd fusion proteins and whether the relatively poor transcriptional activity of these two fusion constructss was caused by a lower level of protein expression are yet to be determined. however, regardless of the diverse protein expression levels, these results clearly demonstrate the ability of these two peptides to function as ads in the gal4-based system. w acts as an ad in a two-hybrid system so far, we have shown that w and can act as ads when directly fused to a sequence-specific dbd such as lexa and gal4-dbd. the question arose as to whether these two short peptides can act as efficiently when they are physically separated from the dbd. to answer this question, dna sequences encoding these two peptides were individually cloned into pgadt7-t to replace the sequence encoding the gal4-ad, and the transcriptional activities of the resulting constructs were tested following the protocols devised for the yeast two-hybrid system. as shown in figure murine p specifically interacted with the sv large t-antigen and turned on the reporter genes, his and mel. in contrast, human lamin c did not interact with the large t-antigen and therefore failed to turn on the reporter genes. interestingly, both w7-t and 2-t fusions turned on the reporter genes when acting in concert with the gal4-dbd- fusion, but failed to do so when acting in concert with the gal4-dbd-lam fusion. this observation provides strong evidence that both w and can act as ads in a traditional two-hybrid system, albeit with efficiencies poorer than that of gal4-ad. w mediates transcription activation through interacting with tfiib to date, only a few protein factors have unambiguously been identified as direct targets of transcriptional activators, including tbp, tfiib, tfiih, and a few others. we wondered whether w also stimulates transcription initiation through interaction with one of these protein factors. pursuant to this objective, tbp, tfiib, and tfiih were individually cloned into pgilda, and then their interaction with w was tested using a yeast two-hybrid system. lexa and b respectively served as the dbd and ad in this assay. as shown in figure all of these lexa fusion proteins were well expressed in the test yeast strain egy, with lexa-tfiih having a lower expression level. in the absence of w none of these fusion constructs per se could simultaneously turn on the designated reporter genes, leu and lacz. however, in the presence of w tfiib, but not tbp or tfiih, could turn on both reporter genes. this result strongly suggests that w specifically interacts with tfiib. discussion we report herein that the n-terminal domain of yeast valrs acts as an ad when fused to a sequence-specific dbd, lexa. however, there is no direct evidence so far showing that yeast valrs actually functions as a transcription activator in the nucleus. more experiments are currently underway to validate this possibility. despite this uncertain aspect, a core sequence element accounting for this activity was mapped to a region near the c-terminus of the n domain. this core sequence element contains only five amino acid residues: wydww. interestingly, the core sequence element per se retained much of the transcriptional activity and could be stably expressed as a fusion to the dbd in yeast. a mutagenesis study indicated that w residues in the pentapeptide were crucial for its activity. even the y residue, which also possesses an aromatic ring in its side chain, was not as supportive of the activity as were the w residues. as a result, the native pentapeptide acted less efficiently than did wwdww and wwwww as an ad. using this core sequence element as a reference, a much-stronger activation peptide, w was successfully produced. w had ~3-fold higher transcriptional activity than those of the native pentapeptide and the n domain. while we cannot rule out the possibility that w somehow altered the affinity or specificity for the lexa operators of the lexa fusion protein, w has to be capable of interacting with one of the components of the transcription apparatus to stimulate transcription of the reporter genes. to our knowledge, w is so far the smallest ad that retains strong transcriptional activity. with its small size, w is unlikely to interfere with the folding of fused proteins or to occlude the normal site of interaction. in addition, w can significantly reduce the overall size of the prey fusion construct, and therefore has the potential to enhance the efficiency of plasmid transformation, which is a long-standing issue when screening a large library for novel interacting partners. similar to the reasons that the his tag is widely accepted and used for protein purification, w may be an excellent replacement for the gal4-ad or b in two-hybrid screening as an ad. a preliminary study suggested that the direct target of w may be the general transcription factor, tfiib, which was previously shown to be one of the interacting partners of the acidic transcription activator, gal. it is believed that recruitment of tfiib and others by gal leads to assembly of the transcription apparatus at the promoter site and subsequent transcription initiation. however, sequence alignment between these two ads showed little sequence homology. thus, gal4-ad and w may interact with different sites of tfiib or through a different mechanism. alternatively, a certain portion of gal may fold into a three-dimensional structure that displays a hydrophobic feature similar to that of w in any case, it is interesting to find that both of these ads mediate transcription activation through interacting with tfiib. thus, the minute ad, w might turn out to be an interesting paradigm for further mechanistic studies of transcription activation.  a short peptide containing seven consecutive tryptophan residues can function as an activation domain when fused to a dna-binding protein, lexa, and turn on reporter genes with distinct lexa-responsive promoters. like the activation domain of gal w mediates transcription activation through interacting with the general transcription factor, tfiib. 
we report herein that the n-terminal domain of yeast valyl-trna synthetase can function as an ad when fused to a dna-binding protein, lexa, and turn on reporter genes with distinct lexa-responsive promoters. the transcriptional activity was mainly attributed to a five-residue peptide, wydww, near the c-terminus of the n domain. remarkably, the pentapeptide per se retained much of the transcriptional activity. eukaryotic transcription activators normally consist of a sequence-specific dna-binding domain and a transcription activation domain. further study indicated that w mediates transcription activation through interacting with the general transcription factor, tfiib. mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide significantly enhanced its activity, while mutations which substituted aromatic residues with alanine residues severely impaired its activity. accordingly, a much more active peptide, pentatryptophan, was produced, which elicited ~3-fold higher activity than that of the native pentapeptide and the n domain. since w shares no sequence homology or features with any known transcription activators, it may represent a novel class of ad.
 eukaryotic transcription activators normally consist of a sequence-specific dna-binding domain and a transcription activation domain. while many sequence patterns and motifs have been defined for dbds, ads do not share easily recognizable motifs or structures.  we report herein that the n-terminal domain of yeast valyl-trna synthetase can function as an ad when fused to a dna-binding protein, lexa, and turn on reporter genes with distinct lexa-responsive promoters. the transcriptional activity was mainly attributed to a five-residue peptide, wydww, near the c-terminus of the n domain. remarkably, the pentapeptide per se retained much of the transcriptional activity. mutations which substituted tryptophan residues for both of the non-tryptophan residues in the pentapeptide significantly enhanced its activity, while mutations which substituted aromatic residues with alanine residues severely impaired its activity. accordingly, a much more active peptide, pentatryptophan, was produced, which elicited ~3-fold higher activity than that of the native pentapeptide and the n domain. further study indicated that w mediates transcription activation through interacting with the general transcription factor, tfiib.  since w shares no sequence homology or features with any known transcription activators, it may represent a novel class of ad. 