Zinc-binding subunits of yeast RNA polymerases.

I Treich,M Riva,A Sentenac

doi:10.1016/s0021-9258(18)54732-3

I Treich, M Riva + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)54732-3

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Abstract

The zinc-binding subunits of yeast RNA polymerase A(I) and B(II) have been identified by a zinc-blotting technique. The two largest subunits of each enzyme (A190, A135, B220, and B150), as well as A12.2, A10, B44.5, B12.6, and B10, bind 65Zn(II). Predicted zinc-binding motifs have been noted in the NH2-terminal part of B220 and the COOH-terminal region of B150 subunits. Subdomains encompassing these motifs have been overproduced as MalE-fusion proteins and shown to retain zinc binding activity. Site-directed mutagenesis in the predicted metal-binding domain of B150 demonstrated its role in zinc binding. Mutations of cysteine residues C1163, C1166, C1182, and C1185 affected 65Zn2+ binding in vitro and caused a lethal or thermosensitive phenotype for growth. The ability to bind zinc is not sufficient for function since mutations in vicinal residues not affecting zinc binding were either lethal or thermosensitive. The role of zinc in RNA polymerase structure and function is discussed in the light of the present results.

Highlights

From the Service de Biochimie et Genetioue Moleculaire,Centre d’Etudes Nuckaires de Saclny, F-91191 Gif-Sur- Yvette Cedex, France
A(1) and B(I1) have been identified by a zinc-blotting prised of two large subunits, structurally homologous to b’
The two largest subunits of each enzyme and p, and of a collection of smaller polypeptides, some of (A190, A135, B220an, d B150), aws ell as A12.2, A10,which areshared betweentwo or allthreeforms of the

Summary

RESULTS

Zinc-binding Subunits inRNA Polymerases A and B-Zinc were investigated using the 6sZn(II) blotting assay (Fig. 3). The las1t 60 amino acids of B150 zinc ions bound/subunit as several experimental factors were were fused to MalE, and thepurified fusion was probed with likely to affect metal binding efficiency. The filter was probed w“itZhnC12 (10 pg) (lane b ) were subjected to a 10% polyacrylamide gel electroas described under “Experimental Procedures” and zinc-binding sub-phoresis with SDS,transferred to a PVDFmembraneandprobed units were revealed by autoradiography for 15 h (6sZn). The mutant fusion proteins expressed in E. coli cells were purified on amylose columns and analyzed for zinc binding activity using the“Zn blot assay (Fig. 6). Purified MalE-B150 fusion protein (5 pg) (lane b), ADH Comparable amounts of wild-type and mutantMalE-B150 fusion proteins (about 2.5 pg) were subjected to SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and probed with “ZnCI2 in different competitor cations.

Plamia CIKWBODB

DISCUSSION