Differential Ability of Various Plasmid DNAs to Sequester Inhibitors of RNA Polymerase III Transcription

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Abstract
Deletion mutants of the Drosophila tRNAArg gene that lack A-box promoter sequences are not transcribed in several cell-free systems; however, they are actively expressed in vivo in Xenopus oocytes (Sharp et al., 1983a). We show that two A-box deletion mutants of the tRNAArg gene can be transcribed by a HeLa cell-free transcription system if it is preincubated with various DNAs, indicating that an inhibitor is responsible for the lack of mutant tRNA gene transcription. Optimal mutant transcription rescue, and presumably optimal binding of inhibitor, is facilitated by the presence of an active RNA polymerase II promoter in the preincubating DNA. Plasmid DNAs containing RNA polymerase III or weak RNA polymerase II promoters are of intermediate rescue efficiency, and pBR322 DNA is least efficient. Competition studies indicate that the stability of the inhibitor-DNA complex formed initially is apparently increased if the preincubating DNA contains an active RNA polymerase II promoter. Thus, HeLa whole-cell lysates contain a specific inhibitor(s) of RNA polymerase III transcription that primarily affects weakened RNA polymerase III promoters (e.g., A-box deletion mutants) and binds preferentially to DNAs containing an active RNA polymerase II promoter. Yet this apparent sequestration of inhibitor by Class II templates does not appear to inhibit their subsequent transcription by RNA polymerase II. These data raise the possibility that there may be interactions between the RNA polymerase II and III transcription machinery.