Binding of the Termination Factor Nsi1 to Its Cognate DNA Site Is Sufficient To Terminate RNA Polymerase I Transcription In Vitro and To Induce Termination In Vivo

Abstract

Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination of rDNA transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like DNA binding domains and associates in vivo near the 3= end of rRNA genes to rDNA, but information about which and how DNA sequences might influence Nsi1-dependent termination is lacking. Here, we show that binding of Nsi1 to a stretch of 11 nucleotides in the correct orientation was sufficient to pause elongating Pol I shortly upstream of the Nsi1 binding site and to release the transcripts in vitro. The same minimal DNA element triggered Nsi1-dependent termination of pre-rRNA synthesis using an in vivo reporter assay. Termination efficiency in the in vivo system could be enhanced by inclusion of specific DNA sequences downstream of the Nsi1 binding site. These data and the finding that Nsi1 blocks efficiently only Pol I-dependent RNA synthesis in an in vitro transcription system improve our understanding of a unique mechanism of transcription termination. C ellular multisubunit RNA polymerases of all three domains of life share common structural and functional features. After initiation of transcription at their cognate gene promoters, they extend the transcripts until they reach specific DNA cis elements, at which transcription is terminated. Termination occurs when the contacts of the RNA-DNA hybrid within the elongating RNA polymerase are destabilized. Termination finally results in the stopping of RNA synthesis, release of the transcript, and dissociation of the RNA polymerase from the DNA template (reviewed in reference 1). A first step in the termination pathway can be the pausing of the elongating polymerase, which is then followed by the disruption of the elongation complex.Dissociation of the ternary elongation complex formed by RNA polymerase, RNA, and DNA can be caused either by auxiliary protein factors or solely by interactions of DNA and RNA with the transcribing RNA polymerase. Both principles, factordependent and intrinsic termination, have been described for bacterial transcription termination (reviewed in reference 1). In the case of eukaryotic RNA polymerase II (Pol II), either 3=-end processing factors of the mRNA or the yeast RNA-DNA helicase Sen1 (human senataxin) was suggested to disrupt the ternary complex (reviewed in references 2 and 3). This might resemble Rho-dependent termination in bacteria in which the ATP-dependent RNA-DNA helicase Rho destabilizes the elongation complex (4, 5; for a review, see reference 1). RNA polymerase III (Pol III) termination resembles the other bacteria-like termination mechanism, the intrinsic termination, in which the RNA-DNA hybrid is destabilized by a stem-loop in the nascent RNA (6, 7) (8).In contrast to these mechanisms of transcription termination, Pol I requires a termination factor which binds to a specific DNA sequence t...

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

“…This conclusion is also consistent with recent data showing that binding of S. cerevisiae terminator Nsi1 (also known as Ytt1) to its DNA binding site is sufficient to terminate RNA pol I transcription (28). However, at this time, we do not know the details of exactly how the TTD-Rpa12 interaction causes transcription termination.…”

“…In S. cerevisiae, an in vitro system consisting of purified pol I, Reb1 (ScReb1), the Ter site, and an upstream AT-rich release element were necessary and sufficient for transcription termination (7). However, recent work has shown that Nsi1 (Ytt1) is also responsible for transcription termination in S. cerevisiae (28).…”

Transcription Termination: Variations on Common Themes

“…Although early work suggested that Reb1 is the roadblocking factor [46,47], later studies have shown that this role is carried out by another protein containing a Myb-like DNA binding domain, Nsi1 (NTS1 silencing protein 1), which recognizes a sequence that is similar to the Reb1 binding site present at the rDNA terminator [48,49]. The specific implication of Nsi1 instead of Reb1 might be due to some subtle differences in DNA binding of the two proteins [48].…”

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases

“…In vitro transcription using tailed templates (10 nM) was performed as described 51 . In brief, THL or control reactants were preincubated with the respective polymerase.…”