Abstract
The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y1S2P3T4S5P6S7) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.
We are grateful to Claire Moore (Tufts University Medical School) for critical comments on the manuscript, to Zach Burton (Michigan State University) for communicating results prior to publication, and to the members of our laboratory for many insightful discussions during the course of this work. We also thank Krishnamurthy Shankarling (RWJMS) for GST-Ssu72 expression plasmids, Grant Hartzog (UC—Santa Cruz) for strains, and Steve Brill (Rutgers University) for Rpa1 antiserum.
This work was supported by the NIH Bridge to Doctoral Degree Program (GM58389), by the NIH IMSD Award-UMDNJ/Rutgers University Pipeline Program (GM55145), by NIH Graduate Training in Cellular and Molecular Biology (GM008360), and by NIH RO1 grants GM39484 and GM68887.