literature report literature report Yeast Pol II start-site selection : the long and the short

the formation of a pre-initiation complex (pic) is the first step that takes place during transcription by rNa polymerase ii (pol ii). the tata-binding protein binds to the tata box, thereby positioning the polymerase in the pic in such a way that it can unwind downstream DNa and lock the template strand in the active site (Bushnell et al, 2004). in metazoans, initiation takes place 25–30 base pairs (bp) downstream of the tata box (Smale & Kadonaga, 2003), whereas in Saccharomyces cerevisiae, start sites are spread across 40–120 bp downstream of the tata box (Hampsey, 1998). the rules that control selection of the pol ii start point are not well understood. in metazoans, start sites often occur at a conserved initiator sequence (Smale & Kadonaga, 2003), whereas yeast initiators are more divergent (zhang & Dietrich, 2005) and the polymerase has been proposed to ‘scan’ the downstream sequences for optimal start sites (giardina & Lis, 1993; Kuehner & Brow, 2008). Several articles recently published by the Brow, Lacroute, Libri and reines laboratories show that start-site selection has an important role in regulating the expression of genes of the S. cerevisiae nucleotidebiosynthetic pathways ( Jenks et al, 2008; Kuehner & Brow, 2008; Kwapisz et al, 2008; thiebaut et al, 2008). IMD2 and URA2 encode the initial enzymes in the pathways that lead to the synthesis of gtp and utp, respectively. the transcription of these genes is repressed by the end product Ntp (EscobarHenriques & Daignan-Fornier, 2001; Losson & Lacroute, 1981); however, despite years of searching, no repressors or activators have been identified. Studies on IMD2 identified a guanine-responsive tata box and a repressive element spanning the messenger rNa (mrNa) start site, yet how these cis-elements regulate IMD2 expression was unclear (Escobar-Henriques et al, 2003; Shaw et al, 2001). transcription profiling of strains disabled for nuclear exosome function revealed the existence of transcripts that originate upstream of the IMD2 and URA2 mrNa start point (Davis & ares, 2006; thiebaut et al, 2008), and additional work has shown that these transcripts terminate before reaching the coding region (Fig 1; Jenks et al, 2008; Kuehner & Brow, 2008). termination is directed by components of the non-poly(a) termination pathway, and include the rNabinding proteins Nrd1 and Nab3, the helicase Sen1 and components of the nuclear exosome (conrad et al, 2000; Steinmetz et al, 2001; Vasiljeva & Buratowski, 2006). this termination complex recognizes specific sequences in the nascent transcript, and directs its termination and degradation. Deletions or point mutations in the IMD2 or URA2 terminators—located upstream of the coding region—result in gene expression even under repressing conditions such as high Ntp concentration, showing that the upstream non-coding transcripts are essential for complete repression (Kuehner & Brow, 2008; Kwapisz et al, 2008; thiebaut et al, 2008). What factors determine whether pol ii initiates at the upstream or downstream sites? presumably, as pol ii scans the sequences downstream of the tata box, potential start sites pass through the active site (Fig 2). under gtp-replete or utp-replete conditions, upstream start sites predominate, whereas depletion of the specific Ntp leads to initiation at the downstream site. the upstream IMD2 transcripts begin with the dinucleotide sequence gg, which led Steinmetz and colleagues to propose that the concentration of initiating nucleotide is an important determinant of start-site selection (Steinmetz et al, 2006). recent results from the Brow laboratory show that mutations from g to a in position +1 result in the production of upstream transcripts in low-guanine conditions (Kuehner & Brow, 2008), which supports this hypothesis. When there is insufficient gtp to form the first phosphodiester bond, as would be necessary in the case of IMD2, the polymerase continues to scan downstream, eventually finding the mrNa start site. in the case of URA2, the situation is more complex: the upstream transcripts start with an a, which would argue against its regulation by the initiating Ntp (Kwapisz et al, 2008; thiebaut et al, 2008). However, the first few nucleotides of these transcripts are u-rich, which might trigger pausing and allow time for short abortive transcripts to be released from the pic during scanning. regulatory non-coding rNas have been described previously in yeast. For example, the non-coding SRG1 transcript negatively regulates expression from the downstream SER3 gene by destabilizing the assembly of a pic on the downstream SER3 promoter (Martens et al, 2004). this is not the case for URA2, however, as expression levels of both upstream and downstream transcripts are reduced by the mutation of an upstream tata box (Kwapisz et al, 2008). in addition, pol ii is present at the URA2 promoter even under repressing conditions (Kwapisz et al, 2008). a single tata box has been identified for IMD2; however, mutations of this element reveal the presence of downstream cryptic tata-like sequences (Kuehner & Brow, 2008). the increase in downstream start sites seen when the regulatory Ntp is depleted does not seem to come at the expense of upstream start sites, indicating that either pol ii is always scanning downstream and initiates there only when Ntp levels drop or Attenuated transcript ( NTP) TATA t