The prevailing view of the RNA polymerase II (RNAP II) transcription cycle is that RNAP II is recruited to the promoter, transcribes a linear DNA template, then terminates transcription and dissociates from the template. Subsequent rounds of transcription are thought to require de novo recruitment of RNAP II to the promoter. Several recent findings, including physical interaction of 3-end processing factors with both promoter and terminator regions, challenge this concept. Here we report a physical association of promoter and terminator regions of the yeast BUD3 and SEN1 genes. These interactions are transcription-dependent, require the Ssu72 and Pta1 components of the CPF 3-end processing complex, and require the phosphatase activity of Ssu72. We propose a model for RNAP II transcription in which promoter and terminator regions are juxtaposed, and that the resulting gene loops facilitate transcription reinitiation by the same molecule of RNAP II in a manner dependent upon Ssu72-mediated CTD dephosphorylation. The RNA polymerase II (RNAP II) transcription cycle involves distinct steps that include assembly of a preinitiation complex (PIC), initiation, elongation, termination, and reinitiation. Whereas initiation requires recruitment of RNAP II and a complete set of initiation factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) (Woychik and Hampsey 2002;Hahn 2004), reinitiation has been proposed to occur via a different pathway (Yudkovsky et al. 2000). Following initiation, a subset of the initiation factors and Mediator, which facilitates the interaction between gene-specific regulatory proteins and the general transcription factors, is retained at the promoter, forming a "scaffold" that functions as an intermediate for subsequent rounds of transcription in vitro. Accordingly, reinitiation can facilitate higher levels of transcription by bypassing recruitment of factors retained in the scaffold, requiring de novo recruitment of only TFIIB, TFIIF, and RNAP II (Yudkovsky et al. 2000). Whether transcription reinitiation involves terminationdependent recycling of RNAP II from the terminator to the promoter without release from template DNA is not known, although such facilitated recycling has been reported for yeast RNAP III (Dieci andSentenac 1996, 2003).Progression of RNAP II through the transcription cycle is accompanied by changes in the phosphorylation status of the CTD, a reiterated heptapeptide sequence (Y 1 -S 2 -P 3 -T 4 -S 5 -P 6 -S 7 ) present at the C terminus of the Rpb1 subunit of RNAP II (Kobor and Greenblatt 2002). RNAP II is recruited to the promoter in an unphosphorylated form (RNAP IIA) that becomes extensively phosphorylated (RNAP IIO) during transcription. In yeast, phosphorylation of Ser5 of the CTD is catalyzed by the Kin28 subunit of TFIIH, whereas Ser2 is phosphorylated by the Ctk1 subunit of the CTDK-I elongation complex (Cho et al. 2001). Dephosphorylation of Ser5-P and Ser2-P are, in turn, catalyzed by the Ssu72 and Fcp1 phosphatases, respectively (Cho et al. 2001;Krishnamurthy et al. ...

The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH.

A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in ⌬esc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in ⌬esc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.

Channel-kinase TRPM7/ChaK1 is a member of a recently discovered family of protein kinases called ␣-kinases that display no sequence homology to conventional protein kinases. It is an unusual bifunctional protein that contains an ␣-kinase domain fused to an ion channel. The TRPM7/ChaK1 channel has been characterized using electrophysiological techniques, and recent evidence suggests that it may play a key role in the regulation of magnesium homeostasis. However, little is known about its protein kinase activity. To characterize the kinase activity of TRPM7/ChaK1, we expressed the kinase catalytic domain in bacteria. ChaK1-cat is able to undergo autophosphorylation and to phosphorylate myelin basic protein and histone H3 on serine and threonine residues. The kinase is specific for ATP and cannot use GTP as a substrate. ChaK1-cat is insensitive to staurosporine (up to 0.1 mM) but can be inhibited by rottlerin. Because the kinase domain is physically linked to an ion channel, we investigated the effect of ions on ChaK1-cat activity. The kinase requires Mg 2؉ (optimum at 4 -10 mM) or Mn 2؉ (optimum at 3-5 mM), with activity in the presence of Mn 2؉ being 2 orders of magnitude higher than in the presence of Mg 2؉ . Zn 2؉ and Co 2؉ inhibited ChaK1-cat kinase activity. Ca 2؉ at concentrations up to 1 mM did not affect kinase activity. Considering intracellular ion concentrations, our results suggest that, among divalent metal ions, only Mg 2؉ can directly modulate TRPM7/ChaK1 kinase activity in vivo.A new family of protein kinases that do not display sequence homology to conventional eukaryotic protein kinases has been recently identified (1, 2). When mammalian and Caenorhabditis elegans elongation factor-2 kinases (eEF-2 1 kinases) were cloned, it was found that they do not display sequence homology to any conventional eukaryotic protein kinase (1). However, their catalytic domains appeared to be homologous to the catalytic domain of myosin heavy chain kinase A from Dictyostelium (3-5). Two more protein kinases with the same type of catalytic domain have been subsequently identified in Dictyostelium and have been called myosin heavy chain kinases B and C (6, 7). This new family of protein kinases was named ␣-kinase, because the existing evidence suggests that these protein kinases phosphorylate amino acids located within ␣-helices (2). This is different from conventional protein kinases that phosphorylate amino acids located within loops, turns, or regions with irregular structure (8). The ␣-kinase catalytic domain is characterized by several conserved motifs, which are different from the distinguishing sequence motifs that are found in conventional protein kinases (2). Surprisingly, the recently determined three-dimensional structure of the ␣-kinase catalytic domain revealed that, despite the lack of sequence homology, ␣-kinases have a fold that is very similar to conventional eukaryotic protein kinases (9).Five more human proteins with the ␣-kinase domain have been identified and cloned (2, 10, 11). Unexpectedly, it ...

Understanding the mechanism of pre‐mRNA splicing requires the characterization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes containing lariat intermediate and exon 1 from free proteins present in the whole‐cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second‐step splicing factors. We show that SLU7 protein and a novel activity that we named SSF1 (second‐step factor 1) were required in concert with PRP16 to promote progression through the second catalytic step of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.