Mot1 is an essential yeast Snf2/Swi2-related ATPase that exerts both positive and negative effects on gene expression. In vitro, Mot1 can disrupt TATA-binding protein–DNA complexes in an ATP-dependent reaction. This activity can explain Mot1-mediated transcriptional repression, but how Mot1 activates transcription is unknown. We demonstrate that, remarkably, Mot1 is localized in vivo to promoters for both Mot1-repressed and Mot1-activated genes. Moreover, Mot1 ATPase activity is required for both activation and repression of gene activity. These findings suggest a novel function for the Mot1 ATPase at activated genes, perhaps involving ATP-driven reorganization of the preinitiation complex. Mot1 regulates the expression of ≈3% of yeast genes in cells grown in rich medium. Most of these genes are repressed by Mot1, consistent with Mot1's ATP-dependent TATA-binding protein–DNA dissociating activity. Additionally, ≈77% of the Mot1-repressed genes are involved in the diauxic shift, stress response, mating, or sporulation. The gene sets controlled by NC2 and Srb10 are strongly correlated with the Mot1-controlled set, suggesting that these factors cooperate in transcriptional control on a global scale

Single-molecule surface-enhanced Raman scattering (SM-SERS) is one of the vital applications of plasmonic nanoparticles. The SM-SERS sensitivity critically depends on plasmonic hot-spots created at the vicinity of such nanoparticles. In conventional fluid-phase SM-SERS experiments, plasmonic hot-spots are facilitated by chemical aggregation of nanoparticles. Such aggregation is usually irreversible, and hence, nanoparticles cannot be re-dispersed in the fluid for further use. Here, we show how to combine SM-SERS with plasmon polariton-assisted, reversible assembly of plasmonic nanoparticles at an unstructured metal-fluid interface. One of the unique features of our method is that we use a single evanescent-wave optical excitation for nanoparticle assembly, manipulation and SM-SERS measurements. Furthermore, by utilizing dual excitation of plasmons at metal-fluid interface, we create interacting assemblies of metal nanoparticles, which may be further harnessed in dynamic lithography of dispersed nanostructures. Our work will have implications in realizing optically addressable, plasmofluidic, single-molecule detection platforms.

Mot1 is an essential Snf2/Swi2-related ATPase and TATAbinding protein (TBP)-associated factor (TAF). In vitro, Mot1 utilizes ATP hydrolysis to disrupt TBP-DNA complexes, but the relationship of this activity to Mot1's in vivo function is unclear. Chromatin immunoprecipitation was used to determine how Mot1 affects the assembly of preinitiation complexes (PICs) at Mot1-controlled promoters in vivo. We find that the Mot1-repressed HSP26 and INO1 promoters are both regulated by TBP recruitment; inactivation of Mot1 leads to increased PIC formation coincident with derepression of transcription. For the Mot1-activated genes BNA1 and URA1, inactivation of Mot1 also leads, remarkably, to increased TBP binding to the promoters, despite the fact that transcription of these genes is obliterated in mot1 cells. In contrast, levels of Taf1, TFIIB, and RNA polymerase II are reduced at Mot1-activated promoters in mot1 cells. These results suggest that Mot1-mediated displacement of TBP underlies its mechanism of repression and activation at these genes. We suggest that at activated promoters, Mot1 disassembles transcriptionally inactive TBP, thereby facilitating the formation of a TBP complex that supports functional PIC assembly.

Mot1 is an essential

Although pathways for assembly of RNA polymerase (Pol) II transcription preinitiation complexes (PICs) have been well established in vitro, relatively little is known about the dynamic behavior of Pol II general transcription factors in vivo. In vitro, a subset of Pol II factors facilitates reinitiation by remaining very stably bound to the promoter. This behavior contrasts markedly with the highly dynamic behavior of RNA Pol I transcription complexes in vivo, which undergo cycles of disassembly/reassembly at the promoter for each round of transcription. To determine whether the dynamic behavior of the Pol II machinery in vivo is fundamentally different from that of Pol I and whether the static behavior of Pol II factors in vitro fully recapitulates their behavior in vivo, we used fluorescence recovery after photobleaching (FRAP). Surprisingly, we found that all or nearly all of the TATA-binding protein (TBP) population is highly mobile in vivo, displaying FRAP recovery rates of <15 s. These high rates require the activity of the TBP-associated factor Mot1, suggesting that TBP/chromatin interactions are destabilized by active cellular processes. Furthermore, the distinguishable FRAP behavior of TBP and TBP-associated factor 1 indicates that there are populations of these molecules that are independent of one another. The distinct FRAP behavior of most Pol II factors that we tested suggests that transcription complexes assemble via stochastic multistep pathways. Our data indicate that active Pol II PICs can be much more dynamic than previously considered.fluorescence recovery after photobleaching ͉ Mot1 ͉ TFIID T ranscription preinitiation complex (PIC) formation involves the assembly of general transcription factors (GTFs) at appropriately remodeled chromatin templates. Regulation of RNA polymerase (Pol) II transcription initiation can occur by many different mechanisms involving facilitated recruitment of PIC components or stimulation of their activities at the promoter. Although the diversity of such regulatory mechanisms is widely appreciated, comparatively little is known about the dynamic behavior of GTFs in vivo vis a vis their interactions with each other and with chromatin. Most nuclear proteins studied to date, including a number of gene-specific transcription factors and chromatin-modifying enzymes, display highly dynamic behavior in vivo (1-3). In many cases, this dynamic behavior is energy-independent (4). Even chromatin structural proteins once thought to be stably bound to DNA display dynamic behavior in vivo (3-9). However, the dynamic behavior of GTFs is largely unexplored. In vitro, a PIC scaffold nucleated by TATA binding protein (TBP) is very stable, a property that facilitates multiple rounds of transcription at an activated promoter (10). A stable TBP association with chromatin is supported by analysis in human cells, which yields a slow fluorescence recovery time for TBP of 20 min (11). In striking contrast, in vivo analysis of RNA Pol I PICs yields a much more dynamic picture, with fluores...

The phosphatidylinositol-3-kinase (PI3K) and AKT (protein kinase B) signaling pathways play an important role in regulating cell cycle progression and cell survival. In previous studies, we demonstrated that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to p53 inhibition and cell survival. In the present study, we extend these observations to identify regulatory pathways affected by AKT in HTLV-1-transformed cells. We demonstrate that inhibition of AKT reduces the level of phosphorylated Bad, an important member of the pro-apoptotic family of proteins. Consistent with the decrease of phosphorylated Bad, cytochrome c is released from the mitochondria and caspase-9 is activated. Pretreatment of the cells with caspase-9 specific inhibitor z-LEHD-FMK or pan caspase inhibitor Ac-DEVD-CHO prevented LY294002-induced apoptosis. Of interest, p53 siRNA prevents LY294002-induced apoptosis in HTLV-1-transformed cells, suggesting that p53 reactivation is linked to apoptosis. In conclusion, the AKT pathway is involved in targeting multiple proteins which regulate caspase- and p53-dependent apoptosis in HTLV-1-transformed cells. Since AKT inhibitors simultaneously inhibit NF-kappaB and activate p53, these drugs should be promising candidates for HTLV-1-associated cancer therapy.

Nucleotide excision repair factor 4 (NEF4) is required for repair of nontranscribed DNA in Saccharomyces cerevisiae. Rad7 and the Snf2/Swi2-related ATPase Rad16 are NEF4 subunits. We report previously unrecognized similarity between Rad7 and F-box proteins. Rad16 contains a RING domain embedded within its ATPase domain, and the presence of these motifs in NEF4 suggested that NEF4 functions as both an ATPase and an E3 ubiquitin ligase. Mutational analysis provides strong support for this model. The Rad16 ATPase is important for NEF4 function in vivo, and genetic analysis uncovered new interactions between NEF4 and Rad23, a repair factor that links repair to proteasome function. Elc1 is the yeast homologue of a mammalian E3 subunit, and it is a novel component of NEF4. Moreover, the E2s Ubc9 and Ubc13 were linked to the NEF4 repair pathway by genetic criteria. Mutations in NEF4 or Ubc13 result in elevated levels of the DNA damage recognition protein Rad4 and an increase in ubiquitylated species of Rad23. As Rad23 also controls Rad4 levels, these results suggest a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4.

Transcription consists of a series of highly regulated steps: assembly of the preinitiation complex (PIC) at the promoter, initiation, elongation, and termination. PIC assembly is nucleated by TFIID, a complex composed of the TATA-binding protein (TBP) and a series of TBP-associated factors (TAFs). One component, TAF7, is incorporated in the PIC through its interaction with TFIID but is released from TFIID upon transcription initiation. We now report that TAF7 interacts with the transcription factors, TFIIH and P-TEFb, resulting in the inhibition of their Pol II CTD kinase activities. Importantly, in in vitro transcription reactions, TAF7 inhibits steps after PIC assembly and formation of the first phosphodiester bonds. Further, in vivo TAF7 coelongates with P-TEFb and Pol II downstream of the promoter. We propose a model in which TAF7 contributes to the regulation of the transition from PIC assembly to initiation and elongation.MHC class I genes ͉ regulation ͉ transcription initiation I n eukaryotic cells, expression of protein-encoding genes depends on the ordered recruitment of the general transcription factors (GTF) TFIID, TFIIB, and TFIIA to the promoter, followed by association of Pol II, the mediator, and the remaining GTFs, TFIIF, TFIIE, and TFIIH, to form a preinitiation complex (PIC) (1). Once PIC assembly is complete, transcription initiation ensues; Pol II with the elongation complex dissociate from the PIC (2, 3). A required step during transcription initiation is the phosphorylation of serine 5 in the carboxy terminal domain (CTD) heptad repeat of Pol II by the kinase subunit of TFIIH, CDK7 (4, 5), after which Pol II pauses to ensure proper pre-mRNA capping (6-9). The transition from pausing to elongation is facilitated by the P-TEFb elongation complex, which also mediates efficient elongation (10). P-TEFb consists of two subunits, cyclin T1 and the kinase CDK9, which phosphorylates serine 2 of the CTD, required for productive elongation and the recruitment of complexes involved in mRNA processing (splicing and polyadenylation) (10-15).Although the general mechanics of transcription have been characterized, relatively little is known about how the transitions from PIC assembly to initiation/pausing to elongation are regulated. Promoter recognition is largely mediated by TFIID, which is composed of the TATA binding protein (TBP) and over a dozen TBP associated factors (TAFs) (16,17,18). The largest TFIID component, TAF1, has both acetyltransferase (AT) and kinase activities (19,20). We demonstrated that TAF1 and its intrinsic acetyltransferase activity are essential for transcription of an MHC class I gene (21). Importantly, MHC class I transcription is inhibited both in vitro and in vivo by the viral transactivator, HIV Tat, which binds to the TAF1 AT domain, inhibiting its enzymatic activity (22, 23). TAF7, a cellular 55-kDa TFIID component (24,25), also binds to TAF1 inhibiting its AT activity and repressing MHC class I transcription (26) Significantly, we have demonstrated that TAF7 remains boun...