FIGURE 6.

The C terminus of Mtf1p contacts the template strand near positions −3 and −4. A, the DNA template contained 4-thio-dTMP residues at positions −3 and −4 (bold) and [³²P]dCMP at position −2 (italics). The consensus sequence is underlined. B, NTCB-induced cleavage of Mtf1p-DNA photocross-linking products. The template shown in panel A was incubated with Rpo41p and Mtf1p (WT or mutant as indicated), and the resulting complexes were irradiated with UV. The cleavage patterns generated in the presence of either 150 (+) or 300 (++) mm NaOH were resolved by gel electrophoresis; the apparent molecular weights of the fragments (including attached DNA) are indicated. The diagram to the right of the autoradiogram indicates the positions of NTCB cleavage sites in WT Mtf1p (left side of the bar) and the molecular masses of the expected protein fragments (without contribution of the attached DNA; in kDa, right side). The Mtf1p protein used in these experiments contained an N-terminal His tag (MAHHHHHH), indicated in light gray. The results of this experiment indicate that the region that is involved in cross-linking extends from residues 220 to 341 (dark gray). C, this panel is the same as panel B except using Mtf1p mutant D253C/C302S, in which the NTCB cleavage site at position 302 was eliminated, and a new site was introduced at position 253. The region that is involved in cross-linking is indicated in dark gray. D, hydroxylamine-induced cleavage of Mtf1p-DNA photocross-linking products. Mtf1p mutants having hydroxylamine cleavage sites (NG) at the positions indicated were constructed and used to prepare complexes as above. The cross-linking products were subjected to hydroxylamine-induced cleavage (NH₂OH) and resolved by gel electrophoresis. As the NG sites were moved incrementally toward the C terminus of the protein, the sizes of the labeled products became correspondingly smaller. The results indicate that the site of cross-linking lies in the interval from 320 to 341 (dark gray).