Sir2 Silences Gene Transcription by Targeting the Transition Between RNA Polymerase II Initiation and Elongation

Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932

^* To whom correspondence should be addressed. Email: dgross{at}lsuhsc.edu.

	Abstract

It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here we show that recruitment of Pol II, as well as the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMRa1 and HML1/2 mating promoters. This, together with the fact that Pol II is Ser5-phosphorylated, implies that SIR-mediated silencing is permissive to both PIC assembly and transcription initiation. By contrast, occupancy of factors critical to both mRNA capping and Pol II elongation – including Cet1, Abd1, Spt5, Paf1C and TFIIS – is virtually abolished. In agreement, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2, and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing with a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II and TFIIH recruitment to the HML1 promoter, thereby abrogating PIC formation.