Sequestration analysis for RNA polymerase I transcription factors with various deletion and point mutations reveals different functional regions of the mouse rRNA gene promoter

Abstract

We compared the ability of various deletion and substitution mutants of the mouse rRNA gene promoter to bind essential factors required for accurate transcription initiation by RNA polymerase I. Different amounts of a competitor template were first incubated with a mouse cell extract containing the whole complement of factors and RNA polymerase I, and then a tester template was added for the second incubation. Transcription was started by adding nucleoside triphosphates (one labeled), and the accurate transcripts were determined on a gel. The results indicated that the ability of 5' deletion mutants to sequester essential factors decreased almost concurrently with the impairment of in vitro transcription activity, whereas when the promoter sequence was removed from the 3' side, the transcription activity decreased earlier and more drastically than the sequestration ability. Similar, though not identical, results were obtained by preincubation with fraction D separated on a phosphocellulose column, indicating that the major factor which was sequestered was TFID, the species-dependent transcription initiation factor that binds first to the promoter in the initiation reaction (H. Kato, M. Nagamine, R. Kominami, and M. Muramatsu, Mol. Cell. Biol. 6:3418-3427, 1986). Compilation of the data suggests that a region inside the 5' half of the core promoter (-40 to -1) is essential for the binding of TFID. The 3' half of the promoter (-1 to downstream) is not essential for the binding of TFID but is highly important for an efficient transcription initiation. A strong down-mutant with a one-base substitution at -16 (G to A) had a reduced ability to bind to TFID, whereas a null mutant with a single base substitution at -7 (G to A) showed a binding ability similar to that of the wild-type promoter when tested with whole-cell extract. This null mutant, however, could not sequester the TFID well when incubated with fraction D alone, suggesting that the binding of TFID with this mutant is unstable in the absence of another factor(s) present in cell extract. The factor is not TFIA, which binds after TFID, because the addition of fraction A containing TFIA did not cause TFID to bind to the mutant. The availability of different mutants having lesions at different steps of transcription initiation will provide a powerful tool for the dissection of the initiation reaction of the RNA gene.