The 45-kDa subunit of DmPBP makes extensive protein-DNA contacts downstream of the U1 PSEA (but not downstream of the U6 PSEA).
In earlier work, a high-resolution site-specific protein-DNA photo-cross-linking assay was used to map the interaction of the DmPBP subunits with the phosphate backbone of the U1 and U6 PSEAs (33
). In that assay, double-stranded DNA probes that had individual phosphate positions derivatized with a photo-cross-linking agent adjacent to a 32
P radiolabel were used. This method has been used by others to map the position and orientation of the general transcription factors along the DNA in the RNA polymerase II preinitiation complex (14
). In earlier work (33
), Wang and Stumph used 50 individually labeled probes to scan through a 25-bp region of the U1 and U6 PSEAs at every second phosphate position on both the template and nontemplate strands extending to a position 4 bp beyond the 3′ end of the conserved PSEAs (Fig. ). It was noted that DmPBP45 and DmPBP95 cross-linked to the phosphate in the most 3′ position examined in those studies (position 25 on the nontemplate strand). Thus, despite the fact that the 21-bp PSEAs provided the sequence specificity for DNA binding, there was the possibility that one or more subunits of DmPBP might remain in close proximity to the DNA for a substantial distance downstream of the U1 or U6 PSEA.
To investigate DmPBP-DNA interactions downstream of the PSEA, we generated 36 new probes, each containing a cross-linking agent at a unique phosphate position 3′ of the U1 or U6 PSEA (Fig. ). In addition, a number of probes (phosphate positions 17, 23, and 25 on the nontemplate strand and positions 20, 22, and 24 on the template strand) (Fig. ) were used as standards of a known cross-linking pattern and intensity based upon the results of the previous study (33
). Following the incubation of each probe individually with protein, reaction mixtures were irradiated with UV light to activate the cross-linking agent, subjected to extensive nuclease digestion, and run on SDS gels. Protein bands that contained a cross-linked radiolabel were detected by autoradiography.
Figure shows that DmPBP49 cross-linked to these probes only at the positions previously reported: U1 phosphate positions 17 (very strongly), 20 (moderately), and 22 (very strongly) (Fig. , upper panels, lanes 1, 13, and 14) and U6 phosphate positions 17 (very strongly) and 22 (weakly) (Fig. , lower panels, lanes 1 and 14). DmPBP49 did not cross-link to any of the new U1 or U6 probes that scanned for interactions further in the 3′ direction (Fig. , lanes 4 to 12 and 16 to 24). In other results, DmPBP95, the largest subunit, likewise did not cross-link to any positions further in the 3′ direction beyond those previously reported, except for one minor cross-link at phosphate position 27 with both the U1 and U6 probes (data not shown).
Notably, however, the DmPBP45 subunit cross-linked extensively and strongly to many of the new probes used in this study. When DmPBP was bound to a U1 PSEA, DmPBP45 cross-linked as far downstream as phosphate position 35 on the nontemplate strand and as far as position 40 on the template strand (Fig. , upper panels, lanes 4 to 8 and 16 to 23). Because position 20 was the site of the strongest DmPBP45 cross-linking to a U1 PSEA observed in the previous study (33
), the comparable intensities of the bands obtained with the downstream probes suggest that DmPBP45 resides in close proximity to the DNA for 20 bp beyond the end of the conserved U1 PSEA sequence. In contrast, when DmPBP was bound to a U6 PSEA, only minimal cross-linking of DmPBP45 was observed downstream of phosphate position 25 (Fig. , lower panels, lanes 4 to 12 and 16 to 24). Only site 30 on the template strand of the U6 PSEA cross-linked weakly. (Figure , lower right panel, is an overexposure to reveal the bands in lanes 13 and 14, which indicate weak cross-links [33
].) Since all the probes contained identical DNA sequences flanking the U1 and U6 PSEAs, the ability of the DmPBP45 subunit to cross-link to the downstream DNA was determined by whether DmPBP was bound to a U1 or a U6 PSEA.
Figure illustrates and summarizes the cross-linking pattern of DmPBP45 projected onto B-form DNA based upon data combined from Fig. and the previous study (33
). Phosphate positions that cross-linked strongly to DmPBP45 are indicated by colored spheres on the DNA backbone in Fig. . Positions with weak or nondetectable cross-linking are not indicated. For both PSEAs, DmPBP45 cross-linked primarily to the upper face of the duplex when the DNA is oriented as shown in the figure. When DmPBP was bound to a U1 PSEA, DmPBP45 contacted the DNA as far 3′ as position 40. In contrast, on a U6 PSEA, no strong cross-linking was observed beyond position 25. On a U1 PSEA, the very strongest cross-links of DmPBP45 occurred downstream of the PSEA at positions 28 and 30 (upper DNA duplex), but with a U6 PSEA, the very strongest cross-links occurred at positions 11 and 16 within the PSEA (lower DNA duplex).
It is interesting that the binding of DmPBP to a U1 PSEA results in the cross-linking of DmPBP45 strongly to phosphate positions within and near to PSEB, an element that contributes to the promoter strength of the RNA polymerase II-transcribed snRNA genes of fruit flies (38
). At the present time, we cannot be certain whether the cross-linking of DmPBP45 observed in the vicinity of PSEB is determined solely by the activity of the upstream U1 PSEA regardless of the downstream sequence or whether the sequence of PSEB itself may contribute to the interactions of the DNA with DmPBP45. Nonetheless, it is clear that the U6 PSEA, unlike the U1 PSEA, does not allow these close interactions to take place between DmPBP45 and the PSEB region. Thus, the interaction of DmPBP45 with the DNA is substantially different depending upon whether DmPBP is bound to a U1 or a U6 PSEA.
Identification of D. melanogaster genes that code for proteins homologous to three of the subunits of human SNAPc.
Human SNAPc consists of five distinct subunits (SNAP19, SNAP43, SNAP45, SNAP50, and SNAP190) whose genes have been cloned and characterized. We searched the D. melanogaster nucleic acid database for genes that encode proteins homologous to the human polypeptides. Highly significant matches were found for the SNAP43, SNAP50, and SNAP190 polypeptides, but no matches were found for SNAP19 and SNAP45.
Figure illustrates the structural relationships of the human SNAPs and the related sequences coded in the fly genome. For convenience, we designate the D. melanogaster
homologs as DmSNAP43, DmSNAP50, and DmSNAP190. Well-conserved regions of the proteins where the sequences from the two species are greater than 26% identical and 42% similar are shown in Fig. . Outside of these conserved regions, there is little or no significant sequence similarity. The SNAP43 homologs are well conserved within their amino-terminal halves, whereas the carboxyl-terminal two-thirds of the SNAP50 homologs share substantial identity. The SNAP190 homologs are similar in a region that contains an unusual signature motif that consists of 4.5 Myb repeats. This region of human SNAP190 has DNA binding activity (12
The primary sequences of the three putative DmSNAP proteins are shown in Fig. . The calculated molecular masses of the three polypeptides are 42, 43, and 84 kDa for DmSNAP43, DmSNAP50, and DmSNAP190, respectively. For purposes of comparison, Fig. also shows sequences from the three homologous human proteins in those regions where they exhibit significant sequence similarity to the respective fly proteins.
The predicted DmSNAP proteins are components of native DmPBP.
To obtain evidence that the proteins whose sequences are depicted in Fig. are components of the D. melanogaster
PSEA-binding protein, antibodies were raised against peptide sequences found within each of the DmSNAPs. The antibodies were then tested for their ability to supershift DmPBP-DNA complexes in a mobility shift assay. Mobility shifts were performed by using the U1 PSEA sequence and native DmPBP from fly embryos partially purified from the soluble nuclear fraction as previously described (29
). Antibodies against peptides from each of the predicted DmSNAP subunits supershifted the native DmPBP-DNA complex (Fig. , lanes 4, 9, and 14). Supershifts were not observed when preimmune serum was used instead of the specific antibodies (Fig. , lanes 3, 8, and 13). Moreover, the supershifted complexes were outcompeted by excess specific peptide (Fig. , lanes 5, 10, and 15) but not by nonspecific peptide (lanes 6, 11, and 16). These data suggest strongly that the DmSNAP43, DmSNAP50, and DmSNAP190 genes identified in the D. melanogaster
genome code for polypeptide subunits of DmPBP.
FIG. 3. Antibodies against peptide sequences from the predicted DmSNAP proteins react with native DmPBP. Electrophoretic mobility shift assays were carried out by using DmPBP prepared from fruit fly embryos and a probe that contains U1 PSEA. Additional components (more ...) Overexpression of DmSNAPs in D. melanogaster S2 cells.
We placed the DmSNAP43, DmSNAP50, and DmSNAP190 genes into insect expression vectors under the control of the inducible metallothionein promoter. Individual clones were prepared such that each protein could be expressed with or without the V5 and His6 C-terminal tags provided by the pMT/V5-His vector. These clones were cotransfected into D. melanogaster S2 cells together with a plasmid to provide blasticidin resistance. Blasticidin-resistant cell lines that could be induced to express the DmSNAP genes upon the addition of copper sulfate were selected.
The upper panels of Fig. show immunoblots in which the untagged versions of the proteins were detected with antibodies specific for each protein. In normal S2 cells, the expression levels of the endogenous proteins were too low to be detectable (lanes 1, 2, 6, 7, 11, and 12). However, each of the stably transfected cell lines expressed an increased level of a protein of an appropriate molecular weight following induction with copper sulfate (compare lanes 3 and 4, 8 and 9, and 13 and 14). (The anti-DmSNAP50 antibodies were of lower quality than the anti-DmSNAP43 and anti-DmSNAP190 antibodies, resulting in a weaker signal in the immunoblots of Fig. and a weaker supershift in the mobility shift assays of Fig. .)
FIG. 4. Inducible overexpression of DmSNAP proteins in S2 cells. We generated S2 cell lines that were stably transfected with one of the DmSNAP genes under the control of the metallothionein promoter. Lysates from control S2 cells, or from stably transfected (more ...)
The lower panels of Fig. show similar immunoblots but with protein extracts from cell lines expressing V5/His6 epitope-tagged versions of the proteins. The addition of the tags is expected to increase the molecular mass of each protein by about 5,000 Da. In each case, anti-V5 antibody detected copper-induced bands that had appropriately reduced mobilities relative to those of the respective untagged proteins (compare the mobilities relative to the markers in the upper and lower panels of Fig. ).
Tagged DmSNAPs are incorporated into functional DmPBP.
Stably transfected cell lines that overexpressed all three of the DmSNAPs within the same cells were next prepared. In each case, one of the DmSNAPs was expressed as the V5/His6-tagged version of the polypeptide, but the remaining two DmSNAPs remained untagged. However, all DmSNAPs were under the control of the metallothionein promoter. Following induction, His6-tagged proteins and protein complexes were subjected to nickel-chelating chromatography. The partially purified protein fractions were then used in mobility shift assays with a DNA fragment containing a U1 PSEA (Fig. ).
FIG. 5. Epitope-tagged DmSNAP proteins can be incorporated into DmPBP that functionally binds to DNA. Electrophoretic mobility shift assays were performed with DmPBP obtained from three different stably transfected S2 cell lines that each overexpressed all three (more ...)
The nickel affinity-purified fraction from each of the cell lines, whether expressing tagged DmSNAP43, tagged DmSNAP50, or tagged DmSNAP190, formed protein-DNA complexes that were of similar mobilities (Fig. , lanes 5, 8, and 11). Each tagged complex had a mobility just slightly less than that of the complex formed with natural DmPBP isolated from embryos (Fig. , lanes 2 and 15), probably due to the extra amino acids at the C termini of the tagged subunits. An addition of anti-V5 antibody to the reaction mixtures had no effect on the mobilities of complexes that contained untagged DmPBP from embryos (Fig. , lanes 3 and 16). On the other hand, the anti-V5 antibodies supershifted the complexes that formed with S2 cell DmPBP that contained individually tagged subunits (Fig. , lanes 6, 9, and 12). Because the DmPBP-PSEA complexes were either quantitatively supershifted (Fig. , lanes 6 and 12) or sometimes partially disrupted (lane 9) by the anti-V5 antibodies, it can be inferred that nearly all of the DmPBP present in the fractions isolated by nickel chromatography contain a tagged subunit. These data indicate, first, that the DmSNAP43, DmSNAP50, and DmSNAP190 genes each code for an integral subunit of DmPBP, and second, that the V5/His6-tagged DmSNAP proteins can be incorporated into DmPBP capable of binding to DNA that contains PSEA.
Correlating the DmSNAP gene products with the three proteins previously identified in the photo-cross-linking assay.
Data presented above indicate that we have identified and expressed fruit fly genes that code for three distinct polypeptide components of DmPBP. Moreover, the molecular masses of the proteins encoded by the cloned genes are in reasonable agreement with those of the three polypeptides detected in the photo-cross-linking assay. Those results by themselves, however, could not prove a one-to-one correspondence. We therefore further examined the relationship between the cloned genes and the proteins detected by photo-cross-linking.
The DmSNAP43 and DmSNAP50 genes code for proteins of very similar predicted sizes (42 and 43 kDa, respectively). Thus, it was not known whether the DmSNAP43 and DmSNAP50 genes code for DmPBP45 and DmPBP49, respectively, or vice versa. It was even a possibility that one or both of the genes potentially could encode additional components of DmPBP not detected by the photo-cross-linking assay. On the other hand, it seemed very likely that the DmSNAP190 gene, which codes for a protein with a calculated molecular mass of 84 kDa, was the gene for DmPBP95. DmPBP95 cross-links over the entire length of the PSEA, and the DmSNAP190 gene product was expected to cross-link to DNA because it contains 4.5 Myb repeats, similar to the region that constitutes the DNA-binding domain of human SNAP190 (12
). Moreover, human SNAP190/PTFα, like DmPBP95, has been experimentally cross-linked to DNA (36
To delineate the one-to-one relationships of the cloned D. melanogaster genes with the three polypeptides previously detected in the photo-cross-linking assay, we carried out photo-cross-linking experiments with DmPBP that contained tagged subunits. Because the extra amino acids at the C terminus of the tagged proteins increase their molecular mass by about 5,000 Da, tagged proteins exhibit slower mobilities on SDS gels than those of the corresponding untagged proteins (Fig. ). We took advantage of this property to identify the gene products that cross-link to a particular phosphate position.
We prepared S2 cell lines that stably coexpressed V5/His6
-tagged versions of DmSNAP43 and DmSNAP190 together with untagged DmSNAP50 or, alternatively, coexpressed tagged DmSNAP50 and tagged DmSNAP190 together with untagged DmSNAP43. Extracts were prepared from the two cell lines, and the tagged complexes were isolated by nickel affinity chromatography. These tagged DmSNAP complexes were then used for photo-cross-linking to U1 probes that contained the cross-linking agent at either phosphate position 17 of the upper strand or at phosphate position 28 of the lower strand. Position 17 was selected because previous data (33
) indicated that position 17 cross-links strongly to DmPBP95 and very strongly to DmPBP49, but it does not cross-link to DmPBP45. In contrast, phosphate position 28 of the U1 probe cross-links exclusively and specifically to DmPBP45 (Fig. ).
The left panel of Fig. shows results obtained with the DNA probe that contained a cross-linker at position 17 of the nontemplate strand. When the reactions were carried out with untagged DmPBP (Fig. , lanes 1 and 4), two bands that represent cross-linking to the DmPBP95 and DmPBP49 subunits were observed as expected. Lane 2 of Fig. shows results obtained when we used protein isolated from S2 cells expressing tagged DmSNAP43 and tagged DmSNAP190 but untagged DmSNAP50. In this case, the DmPBP95 band shifted to a higher position, suggesting that the DmSNAP190 gene indeed codes for the polypeptide termed DmPBP95. On the other hand, the mobility of the DmPBP49 band was unchanged, strongly suggesting that the DmSNAP43 gene does not code for DmPBP49. The results shown in Fig. , lane 3, support these conclusions and provide evidence that DmPBP49 is encoded by the DmSNAP50 gene. When the protein complex contained tagged DmSNAP50 and tagged DmSNAP190 but untagged DmSNAP43, the DmPBP49 band (as well as the DmPBP95 band) demonstrated a reduced mobility (Fig. , lane 3). This is the result expected if the DmSNAP50 gene codes for DmPBP49 and the DmSNAP190 gene codes for DmPBP95.
FIG. 6. Correlation of the DmSNAP gene products with the DmPBP subunits detected by photo-cross-linking. Photo-cross-linking was carried out with probes derivatized with a cross-linker at phosphate position 17 (probe reacts with DmPBP49 and DmPBP95 [lanes 1 to (more ...)
The identities of the DmSNAP43 and DmSNAP50 gene products were confirmed by the complementary experiment results shown in the right panel of Fig. . In this case, the U1 DNA probe contained a cross-linker at position 28 of the template strand, a position that cross-links exclusively to the DmPBP45 subunit (Fig. ). When the DmSNAP43 gene carried the tag, the band corresponding to DmPBP45 ran with a slower mobility (Fig. , lane 6). However, when DmSNAP50 was tagged, the migration of the band was unchanged (Fig. , lane 7). Taken together, the experimental results shown in Fig. provide convincing evidence that (i) the DmSNAP43 gene codes for DmPBP45, (ii) the DmSNAP50 gene codes for DmPBP49, and (iii) the DmSNAP190 gene codes for DmPBP95.