The presence of two tightly bound Zn2+ ions is essential for the structural and functional integrity of yeast RNA polymerase II1

doi:10.1016/S0378-1119(96)00710-X

Gene

Volume 190, Issue 1, 1997, Pages 77-85

https://doi.org/10.1016/S0378-1119(96)00710-X Get rights and content

Abstract

DNA-dependent RNA polymerases (RNApol) are Zn²⁺ metalloproteins where the Zn²⁺ ion plays both catalytic and structural roles. Although the ubiquitous presence of Zn²⁺ with the RNApol from eukaryotes had already been established, the exact stoichiometry of Zn²⁺ ion(s) per mole enzyme is not well documented, and its role in enzymatic function remains elusive. We show here that RNApolII from Saccharomyces cerevisiae has two Zn²⁺ ions tightly associated with it which are necessary for its transcriptional activity. Upon prolonged dialysis against 10 mM EDTA for 4–5 h, the enzyme loses one Zn²⁺, as well as partial activity. However, Zn²⁺ can be added back to the enzyme, but without recovering its total activity. 5 mM orthophenanthroline (OP) removes one Zn²⁺ within 2 h; the enzyme, however, cannot be reconstituted back with Zn²⁺. Circular dichroism (CD) studies showed that the conformation of the native enzyme is unique and cannot be reproduced with Zn²⁺-reconstituted RNApolII. Similarly, the rate of abortive synthesis of a dinucleotide product over a non-specific template is faster when catalyzed by two Zn²⁺-native enzymes. Zn²⁺-reconstituted RNApolII or one Zn²⁺-RNApolII showed a slower abortive synthesis rate. ⁶⁵Zn²⁺-blotting experiments indicated that the removal of one Zn²⁺ from the enzyme destroys the Zn²⁺-binding ability of the larger subunits of yeast RNApolII. In order to check whether the presence of Zn²⁺ ions has any effect on substrate recognition, we followed the binding of (γ-AmNS)UTP, a fluorescent substrate analog to RNApolII. It was observed that OP-treated enzyme showed non-specific substrate recognition, whereas two Zn²⁺-native RNApol binds substrate at a single site.

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Citation Excerpt :
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^☆: Presented at the International Conference on ‘Eukaryotic Expression Vector Systems: Biology and Applications’, National Institute of Immunology, New Delhi, India; 4–8 February 1996.

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The presence of two tightly bound Zn2+ ions is essential for the structural and functional integrity of yeast RNA polymerase II1☆

Abstract

Cell

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

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J. Biol. Chem.

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FEBS Lett.

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Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Methods Cell Biol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

The presence of two tightly bound Zn²⁺ ions is essential for the structural and functional integrity of yeast RNA polymerase II¹☆