The presence of two tightly bound Zn2+ ions is essential for the structural and functional integrity of yeast RNA polymerase II.
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 (gamma-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.
Mayalagu
S
S
Centre for Cellular and Molecular Biology, Hyderabad, (A.P.), India.
Patturajan
M
M
Chatterji
D
D
D001665:Binding Sites; D015153:Blotting, Western; D002413:Cations, Divalent; D002942:Circular Dichroism; D011487:Protein Conformation; D011489:Protein Denaturation; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014544:Uridine Triphosphate / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D002413:Cations, Divalent; D012319:RNA Polymerase II; D015032:Zinc; D014544:Uridine Triphosphate