Distinct effects of ATP on transcription complex formation and initiation in a yeast in vitro transcription system

Abstract

The stages and kinetics of transcription complex formation in a Saccharomyces cerevisiae in vitro transcription system were analysed using the anionic detergent sarkosyl. In contrast to findings from other systems, we were not able to distinguish between a fully formed pre-initiation complex and a ‘rapid start’ complex to which nucleotides were added. A further increase in resistance of transcription to sarkosyl was only observed 12 min after transcription initiation, by which time elongation was underway. Low concentrations of ATP, dATP or, suprisingly, the non-hydrolysable analogue ATPγS selectively stimulated transcription when present during assembly of transcription complexes, although the level of stimulation dropped when ATP was added progressively later. The effect of ATP did not correlate with the kinetics of template commitment, signifying that it functioned at a later stage than this, but prior to the full assembly of sarkosyl-resistant pre-initiation complexes. ATP also altered the sarkosyl resistance of initiating transcription complexes possibly by affecting a rate-limiting step leading to earlier appearance of elongated transcripts. This effect was antagonised by ATPγS, thus providing evidence that the stimulatory effect of ATP on pre-initiation complex formation and its effect on the lag between initiation and elongation phases are distinct.