Assaying CTD kinases in vitro and phosphorylation-modulated properties of RNA polymerase II in vivo.
The functional properties of RNA polymerase II are modulated by hyperphosphorylation of its unique C-terminal repeat domain (CTD). A number of enzymes with CTD kinase activity have been identified, and correlations between CTD phosphorylation and RNA polymerase II function have been made. Here we describe methods for assaying CTD kinases and for characterizing them enzymologically. In addition we present approaches for studying phosphorylation-mediated behavior of chromosome-associated RNA polymerase II by using CTD-directed, phosphorylation state-sensitive antibodies and in situ localization techniques. The methods described here should, in conjunction with genetic approaches, contribute to elucidating the physiological roles of CTD kinases.
Morris
D P
DP
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Lee
J M
JM
Sterner
D E
DE
Brickey
W J
WJ
Greenleaf
A L
AL
D000818:Animals; D004331:Drosophila melanogaster / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; D012319:RNA Polymerase II