A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5′-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.

In eukaryotic cells, RNA polymerase II (RNA Pol II) 5 and its associated factors carry out transcription of pre-mRNAs, snRNAs, telomerase RNA, and other noncoding RNAs. Pol II also couples transcription to nuclear processes including pre-mRNA modifications (1)(2)(3)(4), mRNA export, and chromatin reconfiguration (5)(6)(7). Coupling RNA processing with synthesis is presumed to be critical in restricting the temporal window during which unmodified transcripts are vulnerable to degradation by endogenous ribonuclease activities (8 -10) and directing RNA processing factors to sites of Pol II transcription at specific steps during Pol II progression through a gene. The Pol II elongation complex coordinates these transactions to help orchestrate control over gene expression (for review, see Ref. 11). Such coordination is mediated by specific and reversible interactions among Pol II and the factors involved in elongation.
The formation of RNA 5Ј cap structure, m 7 GpppN, is the first transcription-coordinated RNA modification event, and it occurs as soon as the transcript attains ϳ25 nucleotides (12)(13)(14)(15). The RNA cap is formed in three enzymatic steps (16,17); (i) removal of the 5Ј ␥-phosphate catalyzed by the RNA triphosphatase; (ii) attachment of a GMP to the 5Ј diphosphate by the guanylyltransferase; (iii) methylation of the 5Ј guanine by the cap methyltransferase. The first two of these steps are closely linked and coupled to Pol II transcription in most organisms. Mammals combine their triphosphatase and guanylyltransferase into a bi-functional capping enzyme (CE) encoded by a single gene. Fungal CEs comprise the two enzymes in a tightly associated complex, as exemplified by the Saccharomyces cerevisiae CE, a heterotetrameric complex consisting of the guanylyltransferase Ceg1 and triphosphatase Cet1 subunits (18) (for review, see Ref. 19). Emerging evidence has also suggested non-catalytic roles of the CE in transcription regulation. For instance, stimulation of CE by HIV Tat promotes read-through of the viral genome (20,21). CE has been implicated in the formation of an early elongation checkpoint, as has been observed in 5Ј-paused Pol II complexes (15,(22)(23)(24)(25). Cotranscriptional capping is effectuated by the specific interaction between a CE and its cognate RNA polymerase that supports a stoichiometric CE-polymerase complex (13,26), as exemplified in certain viral transcription/processing systems in each of which the transcription of viral genes is mediated by a single virus-encoded RNA polymerase (27).
The issue of class specificity in CE recruitment arises in eukaryotes because of the existence of three related RNA poly-merases, Pol I, Pol II, and Pol III, that share five subunits (Rpb5, -6, -8, -10, and -12) but synthesize different classes of RNAs (28). Capping is specifically targeted to Pol II transcription complexes (1,3) after the phosphorylation of Pol II by TFIIH during transcription initiation (29). Importantly, mRNAs that are forced to be transcribed by Pol I, Pol III, or T7 RNA polymerase are uncapped (30 -32). The mechanism for the specific recruitment has been ascribed to the interaction between CE and the phosphorylated C-terminal repeated domain (CTD) of Rpb1, a domain that is unique to the Pol II largest subunit (1,3,33,34). The absence of CTD in Pol I and Pol III (35,36) suggests that the CTD imparts the Pol II apparatus with a unique ability to regulate interactions with various factors including the CE in a phosphorylation-dependent manner. It is known that CTD phosphorylation occurs reversibly on the serine-2, -5, or -7 residues in its heptapeptide motif, 1 YSPTSPS 7 . It is also known that CTD phosphorylation patterns correlate with discrete stages of transcription (37,38). CE binding to Pol II is stimulated by the phosphorylation at either Ser-2 or Ser-5 positions in the CTD (39,40), but Ser(P)-5 catalyzed by the Kin28 kinase of TFIIH predominates during early stages of transcription (41)(42)(43). The binding per se does not require the presence of transcript in Pol II, as has been observed either in the absence of RNA polymerization events (3) or with the purified polymerase (33). Interestingly, the elongation factor Spt5 of the fission yeast Schizosaccharomyces pombe seems to contribute a CE recruitment function in vivo that overlaps with the function provided by the CTD (45,46). Although evidence exists for a functional interaction between Spt5 and CE in the budding yeast S. cerevisiae (47), it remains unclear if this interaction is critical in targeting CE to Pol II transcription because of a lack of biochemical and structural data on this issue. Because S. cerevisiae Spt5 also participates in Pol I transcription (48), Spt5 is not likely to be a decisive factor in the specific recruitment of CE to Pol II in budding yeast, although it may contribute a salubrious effect on the proper assembly of CE into the early elongation complex.
The current model for Pol II-linked cotranscriptional RNA 5Ј-capping rests on the binding of CE to the phosphorylated CTD (CTD-P) (1,3,33,34). The CTD is present but disordered in the Pol II crystal structure and may extend from the globular core of Pol II into bulk solution (49,50). Structural studies of a complex between the Cgt1 subunit (Candida albicans homolog of S. cerevisiae Ceg1) and synthetic CTD heptads bearing Ser(P)-5 have revealed interactions with about 2.5 CTD repeats that are coordinated in a surface groove within the nucleotidyltransferase (NT) domain of Cgt1 (51). Given that there are 26 -27 heptad repeats in the CTD of S. cerevisiae and the CE structures are highly conserved between C. albicans and S. cerevisiae, one could expect at least eight copies of CE to bind, possibly distributively, along the CTD of yeast Pol II upon CTD phosphorylation. However, it is difficult to conceive that such spatially undefined interactions can provide the precise spatial and temporal information required to target CE activity to a narrow window within the transcription cycle. Furthermore, recent experiments with human cells found that the CTD alone was not sufficient for enhancing cotranscriptional processing in vivo, suggesting the involvement of additional Pol II compo-nents and/or associated factors in the process (52). It, therefore, appears that CTD-P alone does not fully account for the Pol II specificity in CE recruitment even though CTD phosphorylation is critical for RNA capping in vivo.
Here, we present evidence for a mechanism that complements the CTD-P-only recruitment model for CE function, thus shedding light on the Pol II-specificity in cotranscriptional targeting of the budding yeast CE. We demonstrate a distinctive and stoichiometric CE-Pol IIO complex that requires two interfaces on the polymerase. One interface is based on the CTD-P as expected from the common model and the Cgt1/CTD-P cocrystal structure (51). The other interface deemed important for the recognition is formed with the Pol II multihelical Foot domain (50). Mutational impairment of either interface disrupts the stability of the complex in vitro and compromises cell growth, whereas overexpression of CE suppresses the in vivo effects of the interface mutations. Mutational evidence also indicates that the Foot domain plays an essential role in imparting specificity toward Pol II as opposed to Pol I and Pol III. Our data indicate that the simultaneous formation of the dual interface is necessary and sufficient to stabilize CE binding to the polymerase. Hence, the dual interface is the physical basis for specifying RNA CE to Pol II transcription in vivo. We also show that each of the two interfaces alone is unable to support stable interactions with CE. Therefore, the post-initiation Pol II capping complex may dissociate as soon as the state of either interface is changed, a modus operandi that seems suitable for the dynamic assembly and disassembly of multiprotein complexes involved in transcription regulation.

EXPERIMENTAL PROCEDURES
RNA polymerases were purified directly from the budding yeast S. cerevisiae by applying the TAP-tagging strategy (53) with the procedures given in the supplemental Experimental Procedures. Pol IIO was generated using the in vitro phosphorylation by MAP kinase 2 (MAPK2) under a condition known to produce both Ser(P)-2 and Ser(P)-5 sites but with preference toward Ser-5 positions (supplemental Experimental Procedures). The Pol IIO was indistinguishable from that generated with the yeast TFIIK kinase, a subcomplex of TFIIH (supplemental Fig. S1B), which is responsible for in vivo phosphorylation of the CTD and the recruitment of CE (29,54). As such, the Pol IIO generated in vitro was suitable for binding studies with CE. The heterodimeric yeast CE and its individual subunits (Ceg1 and Cet1) were expressed in Escherichia coli and purified as described in the supplemental Experimental Procedures.
Glutathione S-transferase (GST) and maltose-binding protein (MBP) fusions were constructed using PCR primers corresponding to sequence regions that each encompassed a Pol II structural domain (50), i.e. the CTD of Rpb1, Rpb1-Foot, Rpb2-Lobe (residues 216 -407), Rpb5, Rpb6, and Rpb8 subunits and Rpb6 truncations. GST-CTD-P and MBP-CTD-P were generated by the same in vitro phosphorylation procedure as used for generating Pol IIO. The GST-and MBP-mediated chromatographic procedures are described in the supplemental Experimental Procedures.
Affinity capture by calmodulin-coupled beads was used in analyzing CE recognition of RNA polymerases. The calmodulin-binding peptide on the C terminus of a polymerase subunit (Rpb7 for Pol II, Rpb8 for Pol I) was derived from the TAP purification procedure. The calmodulin-binding peptide allowed the immobilization of a polymerase complex on calmodulin beads. Detailed conditions are given in the supplemental Experimental Procedures. Size exclusion chromatography was applied to assess stabilities of the various polymerase-CE complexes under the conditions given in the supplemental Experimental Procedures.
For the determination of protein stoichiometry, the gels resulting from affinity assays and size-exclusion chromatography experiments were stained with Coomassie Blue and digitized with an optical scanner (CanoScan-4400F) that proved to produce an even background. Intensities of bands (e.g. Ceg1 and Rpb2 for CE and Pol II, respectively) were quantified with ImageQuant software (GE Healthcare), normalized against their respective molecular weights, and used to determine the stoichiometry.
Western blotting was performed using antibodies directed against Ceg1 (55) and Cet1 (56) for the yeast CE and against Rpb4 (NeoClone) for yeast Pol II. The bands were quantified using the ChemiDoc system (Bio-Rad).
Plasmid shuffle assays were based on the counter-selection of URA3containing cells by 5-fluoroorotic acid (FOA) and performed with standard procedures. Details of the shuffling experiment and also the Ceg1 overexpression in the strains are described in the supplemental Experimental Procedures. The coimmunoprecipitation experiments were done with cell extracts of FOA-resistant strains, each carrying an rpb1 allele, using the procedure given in the supplemental Experimental Procedures. X-ray crystallography was employed to reveal structural aspects of the interaction between CE and Pol II. Detailed procedures and conditions are given in the supplemental Experimental Procedures.

Differential Binding Affinities
Underlie the Recognition of Pol II by Cellular CE-To uncover the biochemical determinants for Pol II-specific recruitment of CE, we measured the amount of yeast CE captured by Pol I, Pol IIA (Pol II unphosphorylated on its CTD), and Pol IIO after each had been immobilized on calmodulin beads ("Experimental Procedures"). As shown in Fig. 1, we observed weak interactions between yeast CE and either Pol IIA (Fig. 1, A and B, lanes 2) or Pol I (Fig. 1B, lane 4). In contrast, we observed stoichiometric interactions between CE and Pol IIO, the phosphorylated post-initiation isoform of Pol II (Fig. 1, A, lane 4, and B, lane 3). The stoichiometry values were 1.0 for CE/Pol IIO and 0.15 for CE/Pol IIA (Fig.  1A, inset), as measured by using the ratio of Ceg1/Rpb2; thus, the retention of CE by Pol IIO increased 7-fold when compared with Pol IIA. This result is consistent with the notion that Pol II-specific targeting of 5Ј capping is dependent on CTD phosphorylation (1,3,29,33,34). These data also demonstrate a CTD phosphorylation-dependent stoichiometric interaction between a bifunctional CE and a structurally intact cellular RNA polymerase. Elut, bound fractions. Protein subunits are marked on the sides in black for polymerases and red for CE. Note that Rpb1o is the Rpb1 with its CTD phosphorylated at serine 2 and 5 sites by MAPK2. The sphere represents CalM-coupled beads that bind the calmodulin-binding peptide (CBP) on the Rpb7 C terminus derived from TAP purification. The box shows molar ratios of CE over the requisite forms of Pol II, as gauged by band intensities of Ceg1/Rpb2. No CTD degradation was observed in the polymerase preparations. B, shown is a comparison of CE stoichiometry in binding to Pol IIA, Pol IIO, and Pol I. The assay method was the same as used in A. Note that A# denotes a Pol I subunit, AC# are subunits shared between Pol I and III, and Rpb5, -6, and -8 are shared among all three polymerases. Molar ratios of CE/Pol I was estimated to be 0.10, based on band intensities of Ceg1 and A190. C, CE separates from Pol IIA in SEC. An incubation mixture of CE and Pol IIA (Load) was developed over a Superose-6 column. Eluted fractions (0.3 ml each, numbered on the top) were analyzed with SDS-PAGE and Coomassie-stained. Subunits of CE (red) are separated from those of Pol IIA (black). Pol IIA and CE peaks are marked by the double arrows. D, CE comigrates with Pol IIO in SEC. The interaction between CE with Pol IIO was analyzed with the same protocol as used in C. The peak of Pol IIO-CE complex migrated slightly faster than that of Pol IIA in C.
We next tested whether CTD-P alone was sufficient to support the stoichiometric interaction with CE. Previous detections for CE binding to GST-CTD-P monitored formation of the radioactive CE-*GMP intermediate (1,3) or used Western analysis (33). Due to the nature of these assays, the stoichiometry of the respective complexes remained unknown. We confirmed the interaction between CE and CTD-P using a similar assay with radioactive labeling of the CE (supplemental Fig.  S1C). However, we were unable to detect CE in the complex by staining with silver (data not shown) even when a 30-fold molar excess of CTD-P was used. We concluded that CTD-P alone is not capable of sustaining the stoichiometric Pol IIO-CE complex. Together, these results suggest that cellular CE recognizes specific features in the globular structure of the respective Pol II molecule, a mechanism reminiscent of that of the viral systems (13,26).
To assess the stability of the respective complexes, excess CE was added to both Pol IIA and Pol IIO, and interactions were characterized by size-exclusion chromatography (SEC). While CE readily separated from Pol IIA (Fig. 1C), it coeluted with Pol IIO with a stoichiometry of 1:1 (Fig. 1D). In the presence of CE, the Pol IIO (peak fractions #42-47) migrated slightly faster than Pol IIA (peak fractions #46 -50), consistent with an increase in molecular weight as a result of CE binding to Pol IIO, but in the absence of CE, the two forms of Pol II migrated indistinguishably (not shown). These data indicate the formation of a stable and stoichiometric complex between CE and Pol IIO but not Pol IIA.
Additional information can be obtained from this analysis. The relative amounts of Cet1 and Ceg1 that are bound to Pol IIO persistently approach 1:1 in repeated experiments (supplemental Table S1). That this stoichiometry is equivalent to that of CE/Pol IIO (see above) suggests that the CE-Pol IIO complex contains a 1Ceg1:1Cet1 heterodimer. However, when we consider the molecular state of the excess Ceg1 and Cet1 complex, its apparent size as indicated by the migration position is more consistent with a 2Ceg1:2Cet1 heterotetramer that has been observed in SEC and the crystal structure (18). Apparently, the heterodimer found in the complex with Pol IIO is not consistent with the previously observed heterotetramer or the 1Ceg1: 2Cet1 heterotrimer CE (18) nor does it explain the in vivo requirement of Cet1 homodimerization (57). Nonetheless, our data suggest the possibility that the heterotetrameric and heterotrimeric states may be dynamic and could change upon the interaction with Pol IIO.
Identifying Structural Determinants for Pol IIO-CE Recognition-As pointed out in the Introduction, the CTD-P-alone model for CE recruitment does not fully explain the precision and specificity of CE targeting. As described in the preceding section, CTD-P alone cannot be responsible for the stoichiometric complex either. A recruitment model solely dependent on CTD-P interactions also fails to account for the recent finding that addition of the CTD to Pol III or the T7 RNA polymerase is not sufficient to direct RNA processing to the CTD-bearing polymerases (52). It is, therefore, necessary to elucidate additional structural determinants required for the preferential binding of CE to Pol IIO as opposed to other forms of RNA polymerases.
As a first step toward this end, we mapped the interaction using the subcomplexes of CE and Pol II that are structurally stable when isolated in solution. These included the Ceg1 (guanylyltransferase) and Cet1 (triphosphatase) subunits of the yeast CE, the Rpb4-Rpb7 subcomplex (consisting of Pol II subunits Rpb4 and Rpb7), and the 10-subunit Pol II lacking Rpb4-Rpb7. In experiments described in the supplemental Data, Section 2.1, we determined that the stable and stoichiometric interaction with CE requires only the 10-subunit core of Pol IIO (supplemental Fig. S2C); the Rpb4-Rpb7 subcomplex is dispensable for this interaction. Moreover, a stable complex was observed between the 12-subunit Pol IIO and Ceg1 but not Cet1 (supplemental Fig. S2, A and B), suggesting that Cet1 is not critical for the interaction with the polymerase.
To further probe the structural features of Pol IIO that might be involved in recruiting CE, we copurified the 12-subunit Pol IIO with recombinant Ceg1-Cet1 by SEC and crystallized the complex (supplemental Fig. S3A). Similar attempts using the 10-subunit Pol IIO did not yield crystals at all. SDS-PAGE analysis revealed Ceg1, Cet1, and Pol II subunits in the crystals (supplemental Fig. S3B). The crystals diffracted weakly, and native anomalous data were eventually collected to 4.7 Å at the zinc edge (supplemental Table S2). Crystallographic phases were experimentally determined using the multi-crystal approach that proved effective for resolving the electron density of Pol II (58). The experimental map of the cocrystal showed Pol II density that matched the known atomic model (59) (Fig. 2A), validating the experimental phase values for the complex. We did not refine the Pol II model against the cocrystal data due to the low resolution. Additional protein-like density not ascribed to Pol II was observed adjacent to the multihelical Foot domain of Rpb1 (Fig. 2, A and B). As a control, diffraction data from Pol IIO crystals obtained in the absence of CE were analyzed, but no significant density was observed adjacent or near to the Rpb1 Foot domain (not shown). These data indicated that the extra density was not due to a folded portion of Pol II. The possibility that the CTD-P on Pol IIO might fold up and account for the additional density was also diminished by the fact that the CTD-P tail of Pol IIO was as susceptible to chymotrypsin digestion as the unstructured CTD of Pol IIA (supplemental Fig. S3C).
To account for the additional density, we attempted to manually dock domains within the CE structure (18) into the complex. We found that neither the NT domain of Ceg1 subunit nor the Cet1 subunit could be docked into this density, whereas several differently orientated models of the oligonucleotide binding (OB) domain of Ceg1 could be placed inside the density (supplemental Fig. S3D), indicating a local rotational disorder in the cocrystal. A difference Fourier (2F o Ϫ F 1c ) map phased by the unrefined Pol II model revealed density also at the same location (not shown), but the density was much weaker than that based on the experimental phases. This was taken as an additional indication for the disorder. This structural disorder could reflect potential different modes of CE binding to Pol IIO in the absence of a transcription bubble and other factors. Nevertheless, the location of the additional experimental density adjacent to a surface on the Foot domain (Fig. 2B) indicated that this Pol II domain might interact with CE and most likely via the OB domain.
The Rpb1 Foot is an independently folded domain in the Pol II structure (50). The Foot domain is conserved among Pol IIs from different species (supplemental Fig. S4A), but it is one of the two regions that vary the most when comparing sequences of the largest subunits from Pol I, Pol II, and Pol III (supplemental Fig. S4, B-D). The structural volume of this domain differs noticeably between Pol I and Pol II (60). These observations suggest a model wherein the Foot domain, in concert with the CTD-P, presents surface determinants for the specific recognition of Pol II by CE. We designed biochemical and functional experiments to investigate this model. We designated the crystallographic interface between the CE OB and Pol II Foot domain as PCI1 (polymerase CE Interface 1) (Fig. 2B) Fig. S5A) using GST-mediated affinity chromatography, we observed weak interactions between CE and an isolated Foot domain, in agreement with the crystallographically observed PCI1. Furthermore, we confirmed the Foot as the only non-CTD site for CE recognition (supplemental Fig. S5B; details are in supplemental Data, Section 2.2). No interactions were found between an isolated Cet1 and the Pol II domains (supplemental Fig. S5C), consistent with the result described above that Ceg1, but not Cet1, was essential for binding.
To evaluate the role of the CTDbased PCI2, we produced MBP-CTD, a construct of intact yeast CTD fused with MBP (maltosebinding protein). We phosphorylated it to produce MBP-CTD-P by the same procedure as for generating Pol IIO. SEC results showed that MBP-CTD-P did not form a stable complex with CE (Fig. 3, A and B). We reasoned that formation of the stable complex might require the presence of both PCI1 and PCI2 and, hence, produced an MBP-Foot fusion protein. This construct was combined in trans with MBP-CTD-P and incubated with CE. Each of the proteins in this mixture migrated independently through the SEC column (Fig. 3C). The GST-Foot protein was tested as well and produced similar results (not shown). Therefore, in contrast to Pol IIO, neither PCI1 nor PCI2 alone nor their cop-  OCTOBER 29, 2010 • VOLUME 285 • NUMBER 44

JOURNAL OF BIOLOGICAL CHEMISTRY 34031
resence as separate proteins could support formation of a stoichiometric complex with CE.
To combine PCI and PCI2 in cis, we expressed a soluble MBP fusion protein with the Rpb1 module consisting of tandem domains of the Foot, nucleic acid cleft, and CTD (FCC) in their natural order (Rpb1 861-1733) (Fig. 4A). This protein, named BFCC (B for Pol II) module herein, was partially purified using the MBP-amylose affinity chromatography and then phosphorylated in the same way as for Pol II (Fig. 4B). The impurities in the preparation proved to be breakdown products as they all bound to the amylose resin; they likely formed high molecular weight aggregates as indicated by their SEC elution positions, possibly via the Cleft domain, which is mostly buried in the Pol II tertiary folding but exposed in this construct. The phosphorylated (BFCCo) but not the unphosphorylated (BFCCa) module formed a stable and stoichiometric complex with CE (Fig. 4, C  and D). It is worth noting that the profile of unbound Ceg1-Cet1 complex (Fig. 4D) does not spread into the fractions that correspond to elution positions of the bound CE. This characteristic is persistent throughout the SEC runs including that shown in Fig. 1C. Because this binding was phosphorylationdependent and the breakdown products in the BFCC preparation were not responsive to the CTD phosphorylation (hence CTD-less) as determined by mobility shift in SDS-PAGE (Fig.  4B), the stable complex in Fig. 4C was deemed to have resulted from interactions with the BFCCo protein (top band). The breakdown products common to the BFCCa and BFCCo prep-

. Separated Foot and CTD-P domains do not support stable interactions with CE.
A, shown is a SEC profile of the recombinant CTD-P with the peak fraction indicated by the upward arrow. B, CE separates from the recombinant CTD-P in SEC (upward arrows). The downward arrow approximately marks a potential peak position for a stable complex. C, copresence of the CTD-P and Foot domains in trans does not support formation of a stable complex with the CE. The staggered profiles of each protein (upward arrows) indicate their separation. A complex formed among the proteins would create a comigration peak (approximated by the downward arrow) ahead of the individual peaks. Note that the CTD is unfolded and its migration is abnormally slow. A contaminating protein is marked by an asterisk. MK, an M r marker.
arations apparently did not influence the results in this assay. The positive interactions detected using only the BFCCo construct also suggests that the multihelical Foot domain is properly folded within this module. These observations demonstrate that the stable stoichiometric interactions between CE and intact Pol IIO can be recapitulated when the Foot and CTD-P domains are linked in cis as they occur in the BFCCo module. As such, the BFCC module offered a means to analyze each component of the dual interface using mutagenesis.
To assess the functional significance of PCI1, we replaced the Foot domain in BFCC with its counterparts from Pol I and Pol III to produce the AFCC and CFCC (A for Pol I; C for Pol III) modules, respectively. These domain-swapped constructs were partially purified and phosphorylated (supplemental Fig. S6A) but failed to form stable complexes with CE (Fig. 4, E and F), indicating that the Foot domains of Pol I and Pol III cannot functionally substitute for that of Pol II and that the Pol II Foot is required for stable association with CE. This biochemical result offers an explanation for the observation that a CTDtailed mutant Pol III is not able to impart RNA processing to its transcripts in vivo (52). Introduction of the analogous domainswapped mutations into yeast RPB1 conferred lethality (see below), confirming that the Rpb1 Foot domain is essential for Pol II function in vivo. The possibility that other Pol II subunits (e.g. Rpb5 and Rpb6) might be involved in the interaction with CE was ruled out through the additional experiments described in the supplemental Data, Section 2.2.
A Minimum CTD Length in Pol IIO Is Required for Stable Interaction with CE-We probed PCI2 by evaluating the minimal length of CTD that is capable of maintaining interactions with CE. Mutant BFCC proteins were produced by terminating the CTD domain at the carboxyl ends of the 16th, 14th, 12th, and 10th heptad repeats (Fig. 5A). Again, the degradation products evident in these preparations were not responsive to the phosphorylation as judged by mobility in the SDS-PAGE (supplemental Fig.  S6A). In SEC performed at 20°C, CE formed stable complexes with the construct of 16CTD in a phosphorylation-dependent manner (Fig. 5, B  and C). Characteristically, again, the profile of unbound CE did not spread into positions of the complexed CE (Fig. 5C). Complexed CE was also found with BFCCo(14CTD) in the SEC profile (Fig. 5D). However, we were not able to discern the formation of a stable complex with BFCCo(12CTD) (Fig. 5E) or the 10CTD module (not shown), as no CE was detectable by Coomassie staining. However, a slight shift (0.5-1 fraction, 0.15 ml/fraction) of the CE profile toward the peak of BFCCo(12CTD) was noticed, which might suggest a certain level of unstable interactions with the 12CTD module. Overall, these results indicate that CE requires at least 14 linear heptads on the polymerase to form a stable complex at 20°C in vitro in this assay. It should also be noted that the last 10 nonconsensus residues from the very C terminus of yeast CTD are probably not involved in CE binding, as they were present in the 10CTD construct (Fig. 5A), and no interaction were detected (data not shown). These biochemical data correlated well with phenotypes of the strains (shown below) bearing the same CTD truncations in Rpb1; namely, that the 12CTD allele could not support yeast growth at 18°C and only allow very slow growth at 30 and 37°C and the 10CTD allele is inviable at all the temperatures tested.
These observations indicate that a CTD containing 13-14 heptad repeats on the yeast Pol II is required and sufficient for

Determinants of Cotranscriptional RNA Capping
the stable interaction with CE. This notion is consistent with earlier findings that the S. pombe CE requires 12-14 CTD heptads for interaction in a two-hybrid assay (61) and that human Pol II with 15 heptads from either the N-or C-terminal half of the CTD supports capping in vivo (62). When considered together with the C. albicans structure mentioned above in which the PCI2 encompasses ϳ2.5 CTD heptads (51), our data suggest a possibility that the yeast CE might interact with 2-3 consecutive heptads between registers 11-14 on the native Pol IIO molecule; an alternative possibility is that formation of the complex might require 2-3 consecutive heptads at a certain linker distance from the body of the polymerase -a relaxed form of the former model. Such a restriction of CE interaction to a specific segment within the CTD could be a mechanism that explains why CTD di-heptads appear to be functional units of the CTD (63).
Functional Significance of the Pol IIO-CE Interface-To assess the importance of PCI1 and PCI2 for yeast growth, we used the plasmid shuffle strategy (64) to substitute a wild-type (WT) RPB1 in the yeast with a copy of rpb1 carrying mutations in PCI1 or PCI2. Both the WT and mutant alleles were under the control of the native promoter of RPB1 ("Experimental Procedures"). As shown in Fig. 6A, when the Rpb1 Foot was substituted by A-Foot and C-Foot, its counterparts from Pol I and Pol III (sequences aligned in supplemental Fig. S4B), or by ⌬-Foot, a di-glycine linkage between Rpb1 872 and 1059, cells were not viable (Fig. 6A, ϪLeu ϩFOA). These results are consistent with the observed loss of CE interactions with the corresponding AFCCo and CFCCo modules in the SEC assays (Fig. 4, E and F). The C-Foot and ⌬-Foot mutations displayed a dominant negative growth effect over WT RPB1 allele born on the URA3 plasmid that was used for shuffling (Fig. 6A, ϪLeu). This phenomenon first manifested in the persistently low frequencies of transformation of the LEU2-marked plasmids carrying these alleles (Fig. 6B). A simple explanation for the dominance is that these mutations generate defective Pol II complexes that blocked or interfered with transcription by the normal Pol II complexes that comprise a WT Rpb1. . Phenotypes of the PCI1 and PCI2 mutants. A, growth effects due to Foot swaps and deletion (A-Foot, C-Foot, and ⌬-Foot) as tested with 20-fold serially diluted cultures are shown. FOA is toxic to URA3-containing cells and, thus, allows the LEU2-linked alleles to be assayed in the absence of the WT gene. The LEU2-linked RPB1 (WT) served as the positive control, and the empty LEU2 shuffle plasmid (vector) served as the negative control. B, frequencies of the LEU2 plasmids carrying a Foot-swap, Foot deletion, or PCI2 (CTD shortening) mutant rpb1 to transform the shuffle strain that contains RPB1 on the URA3 plasmid are shown. Constant amounts of competent cells and plasmid DNA (key below) were used. C, locations of the mutated residues in Rpb1 Foot domain are shown. Mutated PCI1 residues in the FtH4, FtH2, and FtLP elements are colored red, magenta, and yellow, respectively. D, the PCI1 and PCI2 mutations impair yeast growth. The shuffle assays were performed at 37°C (not shown), 30°C (upper left), and 18°C (lower left). The former two conditions yielded similar phenotypes. The slow-growth defects of PCI1 worsened at 18°C, indicating cold sensitivity. The PCI2 mutations also slowed yeast growth (right panels). Shortening the CTD to 12 heptads severely impaired growth at 30°C and prevented growth at 18°C; the 10CTD allele could not support growth at all temperatures. E, the PCI2 mutations dominantly impair yeast growth at lower temperature (18°C). The LEU2-marked plasmids carrying rpb1 with the PCI1 and PCI2 mutations were each transformed into a WT strain derived from W303. The selection and growth of LEU ϩ strains were carried out on SCϪLeu plates, and growth rates were assessed using 20-fold serial dilution.
The PCI2 mutant with a CTD identical to that in the 10CTD module used in the SEC experiment was not able to support growth at all temperatures tested (Fig. 6D, right panels), the 12CTD mutant grew extremely slowly at 30°C and was inviable at 18°C, the 14CTD and 16CTD alleles were apparently wild type at 30°C, but the 14CTD mutant exhibited cold sensitivity. These observations are similar to those made previously by Young and co-workers (65) and Corden and co-workers (66) and support the conclusion that a minimum of 11-12 CTD repeats is required for budding yeast viability. This minimum CTD length required for viability is nearly identical to the aforementioned threshold length, 13-14 repeats, for forming a stable complex with CE in vitro. Combined with the biochemical results, our data pinpoint the impairment of PCI2 and loss of CE interactions as another detrimental consequence of CTDshortening beyond a minimum length in addition to the disruption of Mediator interactions, 3Ј-processing, and termination (2,11,38).
To test for dominant effects of the non-lethal mutations over an endogenous RPB1, we transformed the LEU2-marked PCI1 and PCI2 alleles (under the native RPB1 promoter) into a WT strain (a derivative of W303). As shown in Fig. 6E, the PCI1 surface mutants (FtH4, FtH2, and FtLP) did not affect growth in the WT background. The 14CTD and 12CTD alleles displayed dominant negative effects at the lower temperature (18°C), which reflected the low frequency and small colony size in the initial transformation (Fig. 6B). Similar dominant effects had been noticed for S5A substitutions in the CTD and for the complete removal of CTD (67). These mutational studies confirm that the PCI1 and PCI2 sites identified in vitro contribute to essential functions in vivo.
Direct Functional Interaction between CE and the PCI1 Site on Pol II-The x-ray diffraction and biochemical data described thus far indicate a direct and Pol II-specific interaction between CE and the PCI1 site on the Foot domain. To address whether the impairment of CE interaction at PCI1 is a cause for the growth defects that had been observed for the PCI1 mutants, we examined CE associations with the mutant Pol II molecules using coimmunoprecipitation via the Pol II Rpb3 subunit, which was modified with a TAP tag on its C terminus (53). As shown in a typical experiment (Fig. 7A, left) and by the averaged result from multiple experiments (right, histogram), the amounts of CE associated with Pol II bearing the FtH4, FtH2, and FtLP mutations were significantly reduced relative to that with the WT Pol II. In contrast, the levels of both CE and Pol II in cell extracts, as judged by their Ceg1 and Rpb4 subunits, respectively, remained unchanged (Fig. 7B).
If the impairment of CE interaction at PCI1 is indeed responsible for the slow growth phenotypes observed for the PCI1 mutants, overexpression of the CE might suppress this phenotype. We introduced pRS423, a HIS3-marked multicopy plasmid (68,69), into the PCI1 mutant strains for the expression of yeast CE. Consistent with the results shown in Fig. 6D, the PCI1 mutants (FtH2, FtH4, and FtLP) transformed with pRS423 alone grew more slowly at 18°C than the isogenic strain bearing the WT RPB1, as gauged by their colony sizes (Fig. 7C, CEG1Ϫ). Transformation of a plasmid carrying the CEG1 under its natural promoter (pRS423ϩCEG1) restored growths of the mutants to levels identical to that of the WT strain (Fig. 7C,  CEG1ϩ) with the suppression most apparent for the FtLP allele. Similar suppression was observed in experiments performed at 23 and 30°C (not shown). Based on the reduction of CE binding as a result of the PCI1 mutations and the suppression of the PCI1 alleles by CE overexpression, we conclude that the PCI1 surface on the Foot domain contributes to CE recruitment in vivo.
Because neither PCI1 nor PCI2 is sufficient on its own to stabilize interactions with CE in vitro ( Fig. 3) but both are important for yeast growth and critical for CE binding in vivo, we conclude that a productive interaction with the CE is achieved in vivo only when the two interfaces are simultaneously formed in cis as they occur in a Pol IIO molecule. We propose that the formation of a stoichiometric Pol IIO-CE complex based on the dual interface (PCI1 plus PCI2) is the basis for CE targeting to sites of Pol II elongation.

DISCUSSION
The Dual-interface CE Recruitment Model-The biochemical, structural, and functional data presented here allow us to propose a dual-interface model that explains the physical basis for the specific targeting of RNA capping to Pol II transcripts (Fig. 8). In this model CE forms a stoichiometric complex with Pol IIO, the phosphorylated, post-initiation isoform of Pol II; the specificity of this interaction is achieved by combining the two determinants, PCI1 and PCI2, in cis as they occur in a native Pol IIO molecule. PCI1 is unique to Pol II and, thus, contributes to the isozyme specificity during recruitment of CE. PCI2 is also unique to Pol II and allows further differentiation between the phosphorylated and unphosphorylated forms of Pol II. Our functional data suggest that the dual interface may be metastable in nature, as a mutated interface of either PCI1 or PCI2 is prone to inactivation by cold (Fig. 6, D and E), and impairing either PCI1 or PCI2 is detrimental to the recognition by CE in vitro (Figs. 4 and 5) and yeast survival in vivo (Fig. 6A). In summary, CE targeting depends upon the combined PCI1 and PCI2, which is a characteristic of elongating Pol II molecules.
A Specific CTD Segment Involved in the Assembly of Pol II Cotranscriptional Capping Complex-Our data suggest a possibility that CE might interact with a particular segment of 2-3 haptads within the CTD-P when forming a specific complex with Pol IIO. This proposal is consistent with the binding of ϳ2.5 heptads by C. albicans CE in the cocrystal structure (51); it could also offer a physical explanation for the observation that CTD functions are confined within di-heptads (63). Factors involved in CTD-linked RNA processing and other nuclear events could use similar modes to interact with the phosphor-ylated CTD. With the data available, however, it remains a distinct possibility that a longer CTD would afford CE with many alternative binding sites beyond register 11. Resolution of this issue will require further experimentation. It is tempting to suggest that PCI1 might spatially coordinate productive CE binding on the CTD, raising the possibility that higher order complexes between factors and CTD may have some organization along the length of CTD in the absence of primary sequence differences in CTD repeats. A spatial constraint of CTD by bridging the interactions between PCI1 and PCI2 via CE might in turn be important for the productive binding or regulation of other CTD-targeting factors and would represent a role for CE beyond its enzymatic functions.
Definition of Dynamic Regulatory Complexes by Combining Weak Interactions-The relatively weak interactions observed for isolated PCI1 and PCI2 may reflect a dynamic property of the system that may be important for cotranscriptional capping. Conceivably, this process is highly specific and temporally sensitive to ensure that CE actions are minimized toward other classes of RNAs abundant in the nucleus. In this light, a stable complex formed based on combining the individually weak PCI1 and PCI2 seems well suited for this task, as dissolution of this complex would occur rapidly upon perturbation of either interface. For instance, PCI1 and/or PCI2 may separate as a result of the CE conformational change that accompanies the capping reaction; likely, the action by the Ser-5specific CTD phosphatase Ssu72 may weaken the PCI2 interactions at a post-capping step (70), although the mechanism that determines the order of these events is unknown. It also appears that this principle, namely combining weak interactions alongside post-translational modifications such as phosphorylation, may universally govern the dynamic assembly and disassembly of multiprotein regulatory complexes that function in transcription and possibly other pathways.
Relationship of the Dual Interface to Other Aspects of CE Recruitment-Our model is consistent with the majority of published data that implicate CTD phosphorylation in CE recruitment. In addition, the interaction based on PCI1 offers an alternative explanation for the residual coupling activities for capping (a 4-fold enhancement) by the CTD-less Pol IIA (71) and for the inability of a CTD-tailed human Pol III to instigate processing to its transcripts (52). However, our data do not address the reported inhibition of Pol II due to direct interactions with the triphosphatase Cet1 (56). FIGURE 7. PCI1 contributes to the specific recognition of Pol IIO by CE in vivo. A, mutation of PCI1 reduces the association of CE with Pol II in the cell. Whole-cell extracts prepared from FOA-resistant strains bearing the PCI1 mutations were assayed by coimmunoprecipitation (co-IP) via the polymerases (using Rpb3-TAP). The left panels show a typical Western blot that reveals the levels of Ceg1 bound to Pol II in the strains, with the latter detected for its Rpb4 subunit. The right histogram shows the levels of Rpb4-nomalized Ceg1 in the mutants relative to that in the WT (allele/WT). The values are the averages from four independent experiments. B, the levels of Ceg1 and Pol II remain unchanged in extracts of the various strains. A typical blotting experiment is shown on the left, and the quantification of Ceg1 and Rpb4 based on four experiments is shown on the right. C, overexpression of CE suppresses the slow-growth phenotypes of the PCI1 mutants. FOA-resistant strains bearing the PCI1 mutations were transformed with either the empty pRS423 (CEG1Ϫ) or CEG1-carrying vector (CEG1ϩ). pRS423 is a multicopied yeast 2-m plasmid bearing the HIS3 marker (69). The cultures were brought to the same cell density, serially diluted, and spotted onto the SC-His medium and incubated at 18°C.
Our model highlights the binding of CE via the OB domain of Ceg1 with certain disorder, which would leave the NT domain of Ceg1 free to interact with CTD-P to form PCI2. We believe this and additional disorders may have prevented the visualization of electron density for the NT domain and the entire Cet1 subunit, both present in the crystal (supplemental Fig. S3B). The absence of the NT domain in our structure is consistent with the known flexibility between NT and OB domains in guanylyltransferases (19). Also, the absence of Cet1 is consistent with the observation that Cet1 is flexibly attached to Ceg1 OB domain via a linker peptide (Cet1 241-268) (18).
Because our Pol IIO-CE complex does not contain a template and DNA/RNA hybrid, it represents at best a core structure of a complete elongation complex that may ordinarily include Spt4-Spt5. Given the functional involvement of Spt5 in early elongation and the salubrious contributions of Spt5 C-terminal region to CE recruitment as seen in S. pombe (46), the Spt5 C-terminal region might interact with parts of Cet1 and/or the Ceg1 NT domain to help orient and position the entire CE in a productive assembly on the polymerase.
Implications of the Dual Interface for RNA Early Elongation and Processing-The dominant negative effects on cell growth observed for some of the PCI1 and PCI2 mutants suggest that an impaired PCI1 or PCI2 surface might confer defects at Pol II post-initiation steps, pointing to a role of CE in releasing the early elongation complex from promoter regions (22) or its participation in the elongation checkpoint (23, 47) either through RNA capping or by providing additional protein interactions in these complexes.
Our disclosure of the importance of the non CTD-based PCI1 for CE function has mechanistic implications for other Pol II-linked nuclear processes including splicing, 3Ј-processing, and mRNA export. The recruitment of factors that catalyze these activities may not be precise in the chromatin matrix if the mechanism is solely based on the CTD, because the CTD does not assume a regularly folded structure and may spatially extend over 900 Å (50). Thus, it may be useful to suggest that the targeting of these events to discrete steps in the transcription cycle may also require interactions with the globular core of Pol II so as to restrict the activities to sites of active Pol II elongation. Whether the Foot domain would again be involved in any of these processes remains to be determined.