Recombinant Schizosaccharomyces pombe RNA polymerase II subunit A C-terminal domain phosphatase ssu72 (ssu72)
Description
Enzymatic Activity and Substrate Specificity
Ssu72 exhibits strict specificity for Ser5-P and Ser7-P dephosphorylation, dependent on the cis-conformation of the Pro6 residue . Its activity is modulated by phosphorylation of adjacent residues:
Kinetic studies show that Thr4 phosphorylation decreases Ssu72’s activity by ~50%, highlighting its sensitivity to CTD modification states .
Initiation and Promoter Clearance
-
Ssu72 associates with the preinitiation complex (PIC), maintaining the CTD in a hypo-phosphorylated state to facilitate RNAP II recruitment .
-
Post-initiation, Ser5 phosphorylation by Kin28/CDK7 enables promoter escape, followed by incremental Ser5-P dephosphorylation by Ssu72 to transition RNAP II into elongation .
Elongation and Termination
-
Ssu72 ensures efficient elongation by resolving Pol II pausing through Ser5/Ser7 dephosphorylation, which promotes P-TEFb recruitment and Thr4/Ser2 phosphorylation .
-
Near gene termini, Ssu72 collaborates with the CPF complex to terminate transcription and recycle RNAP II via gene looping .
Regulatory Functions in Phosphate Homeostasis
In S. pombe, Ssu72 links transcription termination to phosphate metabolism:
| Function | Mechanism | Consequence |
|---|---|---|
| Phosphate sensing | Ssu72 activity modulates CTD phosphorylation, altering Pho1 expression | Pho1 regulates intracellular phosphate levels by degrading phosphate stores |
| Feedback loop | Phosphate scarcity increases CTD Ser5-P, enhancing Ssu72 activity to upregulate Pho1 | Maintains cellular phosphate equilibrium |
In Vitro Transcription Assays
-
Recombinant Ssu72 rescues transcription defects in ssu72-2 mutant extracts, confirming its role in elongation efficiency .
-
Depletion of Ssu72 increases Pol II occupancy at promoters while reducing gene body density, indicating elongation defects .
Phospho-CTD Dynamics
-
Pol II Phosphorylation States:
Condition pSer5-Pol II pSer7-Pol II pSer2-Pol II pThr4-Pol II Ssu72 depletion ↑ at promoter and gene body ↑ at promoter and gene body ↓ in gene body ↓ in gene body -
Gene-Specific Effects:
Applications in Biochemical Research
Recombinant Ssu72 is utilized to:
Product Specs
Target Background
KEGG: spo:SPAC3G9.04
STRING: 4896.SPAC3G9.04.1
Q&A
Basic Research Questions
-
What is Ssu72 and what is its primary biochemical function?
Ssu72 is a highly conserved phosphatase that specifically targets the C-terminal domain (CTD) of RNA polymerase II (RNAPII). It functions primarily as a CTD phosphatase with specificity for Serine-5 phosphorylation (Ser5-P), playing a crucial role in the transcription cycle. Initially identified as a component of the yeast cleavage/polyadenylation factor (CPF) complex, Ssu72 has dual functions: it acts as a CTD phosphatase that regenerates the initiation-competent, hypophosphorylated form of RNAPII, and also serves as a factor necessary for pre-mRNA cleavage and efficient transcription termination . Current research conclusively demonstrates that Ssu72 dephosphorylates Ser5-P specifically at the initiation-elongation transition, correcting previous misconceptions that it acted only during termination .
-
Which specific phosphorylation sites does Ssu72 target on the RNA Polymerase II CTD?
The CTD of RNAPII's largest subunit (Rpb1) consists of multiple repeats of a heptapeptide sequence (Y₁S₂P₃T₄S₅P₆S₇) that undergoes dynamic phosphorylation and dephosphorylation during the transcription cycle. Ssu72 exhibits catalytic specificity for both Ser5-P and Ser7-P residues within this heptapeptide repeat . A unique property of Ssu72 is that it recognizes Ser5-P in one orientation of the CTD and Ser7-P when bound in the opposite orientation. While Ssu72 does not directly dephosphorylate Ser2-P, research shows it indirectly affects Ser2 phosphorylation status during the elongation stage of transcription . This dual specificity and indirect regulation of other phosphorylation sites highlights Ssu72's complex role in modulating the CTD phosphorylation code during transcription.
-
How can researchers generate experimental systems to study Ssu72 function?
Several experimental approaches are commonly employed to study Ssu72 functions:
-
Mutant strain generation: Researchers typically create three types of mutants:
-
Chromatin Immunoprecipitation (ChIP): ChIP experiments using specific antibodies against different phosphorylated forms of the CTD (such as 3E10 antibody against Ser2-P) or against RNAPII subunits (like Rpb3) allow monitoring of recruitment and phosphorylation status across genes .
-
Biochemical assays: Western blotting to assess phosphorylation status of bulk RNAPII in whole cell extracts and in vitro phosphatase assays with recombinant proteins .
-
Cell cycle studies: Synchronization methods like cdc25-22 block-release can be employed to study temporal recruitment of Ssu72 to specific genomic regions during cell cycle progression .
-
Mass spectrometry: This technique can identify phosphorylation sites on proteins regulated by Ssu72, as demonstrated in the identification of serine-74 phosphorylation on Stn1 .
-
-
What phenotypes are observed in different Ssu72 mutants?
Different Ssu72 mutants display distinct molecular and cellular phenotypes:
-
Depletion mutants (ssu72-td): Show depletion of hypophosphorylated RNAPII with increased Ser5-P in whole cell extracts. Despite this, RNAPII elongation continues in vivo, suggesting Ssu72 is not essential for RNAPII progression along genes .
-
Catalytically inactive mutants (ssu72-4, ssu72-C13S): Display accumulation of Ser5-P signal uniformly high across genes rather than the normal pattern of Ser5-P diminishing with increasing distance from the promoter . These mutants also exhibit longer telomeres, indicating Ssu72's phosphatase activity is required for normal telomere length regulation .
-
All mutant types: Show deregulation of Ser2 phosphorylation, with increased Ser2-P observed from the promoter region rather than the gradual increase typically seen in wild-type strains .
-
Human cell SSU72 knockdowns: Result in telomere elongation (inhibitable by telomerase inhibitor BIBR1532) and single-stranded DNA accumulation at telomere termini, suggesting conserved functions across species .
-
-
How does Ssu72 differ functionally between yeast and human systems?
Ssu72 is evolutionarily conserved from yeast to humans, with research demonstrating functional conservation particularly in telomere regulation. In both fission yeast (S. pombe) and human cells (HT1080), reduction of Ssu72 leads to telomere elongation . The phosphorylation site on Stn1 (serine-74) that is regulated by Ssu72 in fission yeast is conserved throughout higher eukaryotes, including humans and mice .
In human cells, SSU72 knockdown using specific shRNAs results in telomere elongation from a median length of 3.4 Kb to 3.7-3.8 Kb, an effect that can be prevented by telomerase inhibitor treatment. This indicates that the telomere elongation occurs through a telomerase-dependent mechanism . Additionally, as in yeast, human SSU72 knockdown leads to single-stranded DNA accumulation at telomere termini, further supporting functional conservation .
While the core functions appear conserved, the complexity of human transcription regulation likely means additional roles or regulatory mechanisms exist in higher eukaryotes that have yet to be fully characterized.
