Plant DNA-dependent RNA polymerases: subunit structures and enzymic properties of the class II enzymes from quiescent and proliferating tissues

Abstract

Class II DNA-dependent RNA polymerases were purified from soybean tissues of different physiological states: (1) from seed embryo tissue, representative of a quiescent, low metabolic state and (2) from auxin-treated hypocotyl tissue, representative of a highly proliferative and metabolically active state. Dodecyl sulfate, polyacrylamide gel electrophoresis indicates that RNA polymerase II from embryonic tissue consists largely (90-95%) of the form IIA enzyme, the largest subunit having a molecular weight of 215 000. RNA polymerase II from hypocotyl tissue is exclusively a form IIB enzyme, the largest subunit having a molecular weight of 180 000. Polypeptides common to RNA polymerases IIA and IIB have the following molecular weights: 138 000; 42 000; 27 000; 22 000; 19 000; 17 600; 17 000; 16 200; 16 100; and 14 000. Peptide mapping in the presence of dodecyl sulfate suggests that the 215 000 and 180 000 subunits possess similar peptide fragments. Plant embryo tissues do not contain protease activity capable of cleaving the 215 000 subunit to the 180 000 subunit, but proliferating plant tissues do contain such an activity. Mixing experiments indicate that appreciable amounts of RNA polymerase IIB are not being artifactually produced during protein purification.

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

“…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.…”

“…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.…”

“…This communication describes an extension of our earlier studies (6,8,12) nation medium, easily accommodate 100-g quantities of embryos evenly sown as a monolayer. At the start of imbibition, the trays were covered with a layer of Saran Wrap permeated with many needle-sized holes for aeration.…”

“…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.…”

The Effect of α-Amanitin on the Arabidopsis Seed Proteome Highlights the Distinct Roles of Stored and Neosynthesized mRNAs during Germination

“…In the present work we analyzed the effect of a-amanitin on Arabidopsis seed germination. This amatoxin, which is produced by the toadstool Amanita phalloides, specifically inhibits RNA synthesis catalyzed by DNA-dependent RNA polymerase II both in vivo (Jendrisak, 1980) and in vitro (Guilfoyle and Jendrisak, 1978;de Mercoyrol et al, 1989;Bushnell et al, 2002). However a-amanitin is a relatively large molecule (M r 919.0), and it might therefore have restricted access to the embryos that are covered by a seed coat composed of five cell layers forming the outer and inner integuments .…”