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While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

We have analyzed transcription initiation by RNA polymerase II (pol II) in a highly efficient in vitro transcription system composed of essentially homogeneous protein preparations. The pol II complex was stalled on adenovirus major late promoter templates at defined positions, and the open region and RNA products of these complexes were examined. The first transition is formation of the open complex, which can be reversed by addition of ATPgammaS. The open region is no longer sensitive to ATPgammaS after formation of a four-nucleotide RNA, which constitutes the second transition. This indicates that the ATP-dependent DNA helicase activity of TFIIH is required to maintain the open region only during formation of the first three phosphodiester bonds. The downstream part of the transcription bubble expands in a continuous motion, but the initially opened region (-9/-2 on the non-template strand) recloses abruptly when transcription reaches register 11. This third transition is accompanied by a switch from abortive to productive RNA synthesis, which implies promoter clearance. Our findings provide a framework to analyze regulation of these specific transitions during transcription initiation by pol II.

The role of the basal transcription factor TFIIE was investigated in RNA polymerase II transcription reactions reconstituted with purified proteins. Using negatively supercoiled templates, which circumvent the requirement for TFIIH, we observed that transcription from the adenovirus major-late (ML) core promoter is more dependent on TFIIE than transcription from the adenovirus E4 (E4) or mouse mammary tumor virus (MMTV) promoters. For all three promoters, an increase in the ionic strength of the reaction mixtures led to an increased dependence on TFIIE. Analysis of hybrid ML/MMTV promoters showed that the region encompassing the start site, from -10 to +10, dictates this dependence. Transcription from a relaxed E4 template with a pre-melted -8 to +2 region was completely independent of both TFIIE and TFIIH. We propose that on negatively supercoiled templates TFIIE can facilitate promoter melting.