The number of reactive cysteine residues of yeast RNA polymerase I was determined and their function was studied using parachloromercury benzoate(pCMB), dithiobisnitrobenzoate (DTNB) and N-ethyl-maleimide (NEM) as modifying agents. By treatment with DTNB about 45 sulfhydryl groups react in the presence of 8M urea. Under non-denaturing conditions only 20 sulfhydryl groups are reactive with pCMB and DTNB. Both reagents completely inactivate the enzyme and this effect can be reversed by reducing agents. The sedimentation coefficient and the subunit composition are not affected when the enzyme is inactivated. Two of the most reactive sulfhydryl groups are necessary for activity. The modification of these groups is partially protected by substrates and DNA, suggesting that they are involved either in catalysis or in the maintenance of the conformation of the active site. Experiments with 14C-NEM indicate that the most reactive groups are located in subunits of 185,000, 137,000 and 41,000 daltons.
Bibliographical noteFunding Information:
We thank Or. Graeme Bell for helpful discussions during the preparation of this manuscript and to Rebeca Espinoza and Leslie Spector for typing 1t. This research was funded by grants from NIH (GM24157) and from DIUC of Catholic University.