Fig. 5. An SII-induced oligomer resulting from reiterative RNA synthesis at a promoter-proximal location.

A. Standard in vitro transcription reactions with a complete set of nucleotides using naked pA_4XMLΔ53 (lanes 1,2) or pKS (lanes 3,4) templates. Reactions were reconstituted from basic components plus HNF4α. SII was added as indicated. Analysis on 25% PAG.

B. In vitro transcription reactions using naked pA_4XMLΔ53 template contained only subsets of nucleotides. All reactions were primed with CpA; those in lanes 1–3 additionally contained α³²P-CTP whereas those in lanes 4–7 contained α³²P-CTP and UTP. dATP was used as energy source. Reactions were reconstituted from basic components plus HNF4α and SII and Mediator were added as indicated. Analysis on 25% PAG.

C. In vitro transcription reactions using naked “G_n-STOP” templates to measure n-mer length. Reactions were primed with CpA and additionally contained α³²P-CTP, UTP, 3′O-methyl-GTP and dATP. Reactions were reconstituted from basic components plus HNF4α and SII was added to reactions with the G₁₅-STOP template (lane 5). Indicated products (6-mer, 8-mer, 10-mer, 16-mer and the n-mer) were analyzed on a 25% PAG.

D. In vitro transcription reactions using naked “G₁₅-STOP” template, as described in (C). Both reactions contained SII but dATP was omitted in the reaction in lane 2. The data in lane 1 was quantified to gauge the relative stoichiometries of the n- and 16-mers (see main text).

E. Nucleotide sequence around the transcription start site of the pA_4XMLΔ53 template. The expected RNA products when transcription is carried out with the indicated subsets of nucleotides are also shown.