We have isolated the Fcp1/TFIIF/pol IIA complex from S. pombe
for the first time. This type of complex has not been reported from other organisms. The successful isolation resulted from the use of low-salt buffer throughout the purification and the simple method for complex isolation. Fcp1, however, was not contained in the S. cerevisiae
pol II complex isolated with the anti-CTD antibody (71
). This discrepancy might have originated from the use of different antibodies. Otherwise, the S. cerevisiae
Fcp1 does not form a complex with pol II.
We used the fission yeast S. pombe
because the transcriptional mechanism is more similar to that of higher eukaryotes (40
), all the pol II subunit genes have been cloned (4
), and the subunit-subunit interactions have been well characterized (31
). A large complex of pol II holoenzyme has been isolated from S. pombe
). A similar holoenzyme could not be purified, at least by the method employed. It might not be extracted, or the FLAG tag on the N terminus of Rpb3 or C terminus of Fcp1 might be masked by other components in the holoenzyme. In fact, a large amount of pol II remained unextracted in the final precipitates (see Table ). Large amounts of Fcp1 were also left unsolved in the final precipitates, suggesting the existence of other Fcp1-containing complexes.
The stoichiometry of Fcp1 in the Rpb3-tagged complex was less than those of pol II subunits, indicating that Fcp1 is associated with a portion of pol II molecules. The total number of Fcp1 molecules in strain JY741, which contains 10,000 molecules/cell of pol II at exponential phase in a rich medium (33
), was measured as 3,500 ± 300 molecules/cell by quantitative Western blotting (data not shown). The amount of Fcp1-asociated pol II complex herein determined is consistent with the intracellular level of Fcp1. This ratio supports the binding of Fcp1 to pol II in a certain stage of the transcription cycle for the processive reaction of CTD dephosphorylation.
The association of Fcp1 with pol II that is not engaged in transcription may prevent phosphorylation of the CTD by CTD-kinases. The pol II isolated from the nuclease-treated extract was also associated with a small amount of Fcp1. Most of the nuclease-treated pol II was the pol IIO form, but Fcp1 was found to be associated only with pol IIA. This agrees with the notion that elongation control includes phosphorylation and dephosphorylation of the CTD (45
Although the phosphatase activity of the recombinant Fcp1-H was reasonably high with the artificial substrate p
-nitrophenyl phosphate (see Results), the CTD-phosphatase activity in vitro was rather low as measured using purified pol II substrate. Efficient dephosphorylation of CTD may require an additional factor or condition. In the case of S. cerevisiae
and humans, TFIIFα interacts directly with Fcp1 and stimulates its CTD-phosphatase activity (1
). As expected from the function of Fcp1, the IIa form of Rpb1 increased in the Fcp1-overexpressing cells. When the cells were disrupted by the glass beads method, Rpb1 degraded into the IIb form. However, the degradation was less in the Fcp1-overexpressing cells, suggesting that formation of the Fcp1/pol II complex protects the CTD from degradation by proteinases. Since fcp1
disruptants are inviable, there seems to be no other phosphatase that can substitute for Fcp1.
Several lines of evidence indicated direct binding between pol II and Fcp1. From the previous results (1
), however, the possibility cannot be excluded that a factor such as TFIIF bridges pol II and Fcp1 in the absence of Fcp1-pol II direct binding. We concluded that Rpb4, which is a pol II-specific subunit, is one of the Fcp1-interacting subunits of pol II. This is consistent with the observation that neither recombinant CTD nor purified Rpb1 is the substrate of CTD-phosphatase (10
). The C-terminal region of S. cerevisiae
Fcp1 was shown to interact with TFIIFα (35
), and human TFIIFβ was reported to interact with Rpb5 (72
). Taken together, the whole protein-protein interactions among Fcp1, TFIIF, and pol II have been elucidated.
In S. pombe
, Rpb4 is a stably associated subunit of pol II, and the rpb4
gene is essential for cell growth (60
). In contrast, it is one of the dissociable subunits in S. cerevisiae
), and the rpb4
gene is nonessential for cell viability (74
). Taking advantage of its nonessential nature, the functions of Rpb4 were characterized in detail using S. cerevisiae
. Altogether, the results indicate the importance of Rpb4 in transcription initiation (16
), especially at high temperature (44
). Here we propose another function of Rpb4: it plays a role in assembly of the Fcp1-pol II complex and thereby promotes CTD dephosphorylation for the reutilization of pol II in a new cycle of transcription. This conclusion was strongly supported by the result of the rpb4
shut-off experiment. After the shut-off of Rpb4 synthesis, apparent defects were clearly observed in both Fcp1 binding to pol II and CTD dephosphorylation, long before the cells showed any notable growth defect.
Another notable point in this work is the identification of S. pombe
Tfg3 in the same complex with TFIIFα and -β. This suggested that S. pombe
TFIIF, like S. cerevisiae
) but different from human TFIIF, is composed of three species of subunit, although a functional assay is needed to define the essentiality of the Tfg3 subunit in TFIIF function. This finding is rather unexpected, because the transcription mechanism of S. pombe
has been reported to be more similar in several aspects to that of higher eukaryotes than to that of S. cerevisiae
; for example, the start site position (40
), the functionality of different classes of activator (55
), and the structure and stoichiometry of Rpb4 (60
). The S. pombe
transcription mechanism might have in-between characteristics useful to connect the knowledge of S. cerevisiae
and higher eukaryotes.