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Molecular and Cellular Biology, February 2007, p. 926-936, Vol. 27, No. 3
0270-7306/07/$08.00+0     doi:10.1128/MCB.01361-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Role for the Ssu72 C-Terminal Domain Phosphatase in RNA Polymerase II Transcription Elongation

Mariela Reyes-Reyes and Michael Hampsey^*

Department of Biochemistry, Division of Nucleic Acids Enzymology, UMDNJ-Robert Wood Johnson Medical School, 683 Hoes Lane West, Piscataway, New Jersey 08854

Received 25 July 2006/ Returned for modification 18 August 2006/ Accepted 1 November 2006

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.

Published ahead of print on 13 November 2006.

Present address: Advaxis, Inc., Princeton, NJ 08540.

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