Phosphorylation causes a conformational change in the carboxyl-terminal domain of the mouse RNA polymerase II largest subunit

J Zhang,J L Corden

doi:10.1016/s0021-9258(18)52243-2

Abstract

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II can be phosphorylated by a p34cdc2/CDC28-containing CTD kinase. Phosphorylated serine (or threonine) is located at positions 2 and 5 in the repetitive heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show here that phosphorylation of the mouse CTD retards its electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels in a way similar to that observed for the II0 form of the largest subunit of RNA polymerase II phosphorylated in vivo. At the maximum level of phosphorylation by CTD kinase in vitro, there are 15-20 phosphates evenly distributed among the 52 heptapeptide repeats that comprise the mouse CTD. Gel filtration chromatography and sucrose gradient ultracentrifugation analyses indicate that phosphorylation induces a dramatic conformational change in the CTD with the phosphorylated form adopting a far more extended structure than the unphosphorylated CTD.

Highlights

From the Howard Hughes Medical Institute and Departmentof Molecular Biology and Genetics, The JohnsHopkins Uniuersity School of Medicine, Baltimore, Maryland 21205
Phosphorylated serine is located at po- heptapeptidesubstrates
The extinction coefficient for ATP at 259 nm was taken as 1.54 X lo4 cm” M”. An aliquot of this ATP fraction was Phosphorylation of CTDa Causes a Shift of Its Electrophoretic Mobility-The use of a complete mouse carboxyl-terminal domain (CTD) substrate has allowed us to carefully examine the phosphorylation remixed with 10 mlof high flash point mixture, counted in a liquid action catalyzed by mouse CTD kinase E2

Summary

RESULTS

Phosphorylation of CTDa Causes a Shift of Its Electrophoretic Mobility-The use of a complete mouse CTD substrate has allowed us to carefully examine the phosphorylation remixed with 10 mlof high flash point mixture, counted in a liquid action catalyzed by mouse CTD kinase E2. During this stage the more phosphates added, the more the mobility is retarded. The 32P-CTDaphosphorylated by casein kinase I1 and its protease fragments were analyzed as described above for CTD kinase-labeled 3ZP-CTDo. Urea-PAGE Separation of Phosphorylated CTD Intermediates-20 charge to mass ratio and the electrophoretic mobility. Kinase E2 is added, and theresulting reaction products, taken at various times, are resolved by urea-PAGE electrophoresis (see “Experimental Procedures”) Under these conditions, we see that the addition of phosphates to the CTD substrate increases the charge to mass ratio of the CTD andthus.

Are the phosphorylated residues randomly distributed among a

TABLIE Relative phosphate contents of CTDofragments

DISCUSSION

OS a