Subunit Structure Differences in RNA Polymerase II Purified from Ungerminated versus Germinated Wheat Embryos

Abstract

DNA-dependent RNA polymerase II (RNAP II) was purified from wheat embryos germinated for 0, 12, 24, and 36 hours and examined with several polyacrylamide gel electrophoretic systems. A changing electrophoretic pattern of RNAP II was observed on nondenaturing polyacrylamide gels. Subunit structure analysis by sodium dodecyl sulfate-polyacrylamide gel electropLoresis (SDS-PAGE) indicated that from ungerminated embryos, RNAP IA was almost exclusively obtained which has a subunit structure identical to that established for wheat germ RNAP II previously (Jendrisak, Burgess 1977 Biochemistry 16: 1959. Twelve polypeptides with molecular weights x 10-3 of 220, 140, 42, 40, 27, 25, 21, 20, 17.8, 17.0, 16.3, and 16.0 11 (14, 15, 24). The subunit structures of these intraclass variants have been investigated, and differ only in the mol wt, and in some cases, the charge of the largest enzyme subunit. Although little information exists on changes in the relative amounts of the various enzyme forms during physiological transitions in animal systems, different forms of RNAP II have been purified from plant tissues of different physiological states (6,7,12). RNAP IIA, which has a 220,000 mol wt subunit, has been purified from quiescent embryos of both monocotyledonous and dicotyledonous plant species. In metabolically active plant tissue, RNAP IIB is purified almost exclusively, which has a 180,000 mol wt largest subunit. RNA IIB appears to be proteolytically derived from the IIA enzyme form. Recently, Guilfoyle and Malcolm (7) investigated the subunit structures of RNAP II in germinating soybean embryonic axes. They found a 25-fold increase in nuclear RNAP activity during 36 h of germination, although the amount of purifiable RNAP protein remained unchanged. They also observed the apparent conversion of the highest mol wt subunit (215,00 mol wt) in the enzyme to a new mol wt of 180,000, apparently by limited proteolysis, but no changes in other subunits as determined by SDS-PAGE.This communication describes an extension of our earlier studies (6,8,12) with regard to the existence of multiple forms of RNAP II in wheat embryos. Viable wheat embryos were prepared so that RNAP II subunit structure in quiescent and germinated embryos could be compared. Results are similar to those found with germinating soybean embryonic axes (9) with regard to the proteolysis of the highest mol wt subunit. We also present evidence for a change in the electrophoretic pattern of two wheat embryo RNAP II low mol wt subunits which has not been detected in the soybean system. MATERIALS AND METHODSPreparation and Growth of Embryos. Embryos were prepared from the seed of Triticum aestivum L. var Wared by the mass isolation technique of Johnston and Stern (13) and germinated at 22°C on Whatman No. 1 filter-paper-lined cafeteria trays in 5% (w/v) sucrose containing 10 ,tg/ml chloramphenicol (5 ml/g tissue). Two 51-x 38-cm trays, each wetted with 250 ml of germi-1068 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

Enzymatic properties of plant RNA polymerases

“…These result may suggest that this limited proteolysis process could be linked to germination. Nevertheless, as stressed by Jendrisak & Skuzeski (35), before drawing any conclusions as to a physiological significance, it will be necessary to correlate the observed structural alterations with altered enzymatic properties. In fact, RNA polymerases II A and liB from wheat germ do exhibit some different catalytic properties (37).…”