Fig. 6.
Mod5 is physically associated with tRNA gene complexes and pre-tRNAs. (A) ChIPs were performed using TAP-tagged versions of Mod5 and a positive control (the Brf1p subunit of the continuously bound TFIIIB transcription factor). PCR detection used primers flanking tRNAIle and tRNAGln genes as previously determined (20), with the coding region of ATG22 as an internal negative control, because the nearest tRNA gene is >5,000 bp away. Triplicate reactions from duplicate experiments are expressed as ratios of linear range PCR signal at the tRNA gene/ATG22 control. Ratios are normalized to parallel controls using an untagged strain. (B) Mod5-myc is coimmunoprecipitated with proteins bound at tRNA genes. The chromosomal MOD5 ORF was C-terminally tagged with 13xmyc in strains where the other indicated proteins are TAP-tagged as well as a strain with no secondary TAP tag. Cell lysates from 1 L midlog cultures were divided, and one-half of the samples were treated with excess DNase I (as assessed by PCR) to eliminate interactions that are mediated by co-occupation at a distance on the same DNA fragments. TAP-tagged proteins were isolated in single affinity steps, and Western blots were probed for coisolation of Myc-tagged Mod5. These results indicate that Mod5-myc is reproducibly present in affinity pull-downs of TAP-tagged Brf1p, Bdp1, Smc4, Rpc53, Rpc82, and Tfc1 but not when a TAP tag is absent. Although formation of pol III transcription complexes, including condensin, is likely to be DNA-mediated on the tRNA genes, the DNase insensitivity indicates that Mod5 is likely in close contact with the pol III transcription complex. (C) Mod5-myc is associated with pre-tRNAs. After cross-linking in culture, Mod5-myc was affinity isolated, cross-linking was reversed, and the associated RNA was analyzed by Northern blot with probes to either tRNALeu3 (not an Mod5 substrate) or tRNATyr (only the spliced form is an Mod5 substrate). In both cases, antibody to the myc tag isolated both the nonsubstrate precursors and the mature forms, with substantial enrichment for precursors (ratios given below). RNA coisolations and precursor enrichments were not sensitive to DNase treatment.