The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription complexes formed with TFB2 at the strong gdh promoter are similar to the orientation and occupancy of transcription complexes formed with TFB1. Initiation complexes formed by TFB2 display a promoter opening defect that can be bypassed with a preformed transcription bubble, suggesting a mechanism to explain the low TFB2 transcription activity. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening.Transcription in archaea is catalyzed by a single RNA polymerase (RNAP) that is very similar to eukaryotic RNAP II at the level of subunit identity and sequence homology (9, 21). Initiation of transcription by archaeal RNAP is guided by at least three extrinsic factors, TATA binding protein (TBP), transcription factor B (TFB), and transcription factor E (TFE), which display high levels of structural and functional conservation with their eukaryotic counterparts, TBP, TFIIB, and the TFIIE alpha subunit (4,14,15,23,29,33). Archaea apparently lack homologs of other RNAP II transcription initiation factors.Transcription in archaea initiates at simple promoters, usually containing an AT-rich TATA box about 25 bp upstream of the transcription start site, with an adjacent TFB recognition element (BRE) (26,28,39). During transcription initiation, complex formation begins when TBP binds the TATA box, followed by TFB, which binds the TBP-promoter complex and interacts with the BRE in a sequence-specific manner (6,15,22). The TBP-TFB-DNA complex recruits RNAP to the promoter, and transcription initiates. TFE facilitates transcription in cases where the TBP or TFB function is not optimal, at least in part by stabilizing the open complex, in which the DNA strands surrounding the transcription start site are separated (4,14,25,41).TFB in archaea and TFIIB in eukaryotes play a central role in recruiting RNAP and may also be involved in facilitating the structural rearrangements in the transcription complex that lead to initiation, but a detailed mechanism of action has not been determined for this transcription factor family. Like TFIIB, TFB contains a structurally complex, conserved Nterminal region that is connected by a linker to a globular...

Cerebellar adaptive plasticity regulates posture and movement in response to changing conditions of sensory stimulation. Study of adaptive plasticity of cerebellar circuitry in vitro confines experimental interest to mechanisms with a time scale of minutes. However, cerebellar plasticity, measured behaviorally or electrophysiologically in vivo, occurs over a time scale of tens of minutes and hours. Here we investigate how optokinetically-evoked increases in climbing fiber activity influence expression of key subcellular signaling proteins that regulate the accumulation of GABAA receptors (GABAARs) in the cytoplasm of Purkinje cells and their insertion into the plasma membrane. We used long-term horizontal optokinetic stimulation (HOKS) to activate climbing fibers that project to the flocculus of mice. While long-term increases in climbing fiber activity in vivo do not alter the expression of any of the subunits of GABAARs expressed by Purkinje cells, they do influence other subcellular events such as transcription and interaction of signaling proteins. Specifically, increased climbing fiber activity evoked decreased expression of 14-3-3-θ, reduced serine phosphorylation of GABAAγ2 and reduced the interaction of 14-3-3-θ with Protein Kinase C-γ (PKC-γ). Knockdown of 14-3-3-θ in vivo reduced the serine phosphorylation of GABAAγ2. Conversely, treatment of cerebellar lysates with phorbol 12-myristate-13-acetate (PMA), a PKC activator, increased serine phosphorylation of GABAAγ2. Knockdown of 14-3-3-θ or PKC-γ in N2a cells in vitro reduced serine phosphorylation of GABAAγ2 and reduced its cell-surface expression. We interpret these data to mean that a prolonged increase in climbing fiber activity decreases the cell surface expression of GABAARs in Purkinje cells and thereby reduces their sensitivity to GABAergic inhibition. This provides a homeostatic mechanism by which Purkinje cells become less sensitive to stellate cell inhibition also evoked by climbing fiber activity.

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