Multiple mechanisms confining RNA polymerase II ubiquitylation to polymerases undergoing transcriptional arrest.
In order to study mechanisms and regulation of RNA polymerase II (RNAPII) ubiquitylation and degradation, highly purified factors were used to reconstitute RNAPII ubiquitylation in vitro. We show that arrested RNAPII elongation complexes are the preferred substrates for ubiquitylation. Accordingly, not only DNA-damage-dependent but also DNA-damage-independent transcriptional arrest results in RNAPII ubiquitylation in vivo. Def1, known to be required for damage-induced degradation of RNAPII, stimulates ubiquitylation of RNAPII only in an elongation complex. Ubiquitylation of RNAPII is dependent on its C-terminal repeat domain (CTD). Moreover, CTD phosphorylation at serine 5, a hallmark of the initiating polymerase, but not at serine 2, a hallmark of the elongating polymerase, completely inhibits ubiquitylation. In agreement with this, ubiquitylated RNAPII is hypophosphorylated at serine 5 in vivo, and mutation of the serine 5 phosphatase SSU72 inhibits RNAPII degradation. These results identify several mechanisms that confine ubiquitylation of RNAPII to the forms of the enzyme that arrest during elongation.
Somesh
Baggavalli P
BP
Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts EN6 3LD, United Kingdom.
Reid
James
J
Liu
Wei-Feng
WF
Søgaard
T Max M
TM
Erdjument-Bromage
Hediye
H
Tempst
Paul
P
Svejstrup
Jesper Q
JQ
D002474:Cell-Free System; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000187:drug effects / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000494:pharmacology; D012694:Serine / Q000737:chemistry; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000187:drug effects / Q000378:metabolism*
D002868:Chromosomal Proteins, Non-Histone; C451742:DEF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D012694:Serine; D012319:RNA Polymerase II; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase