Analysis of TAF II Function in the Yeast Saccharomyces Cerevisiae

Transcription by RNA polymerase II is a highly regulated process requiring a number of general and promoter specific transcription factors. Although many of the factors involved in the transcription reaction are known, exactly how they function to stimulate or repress transcription is not well understood. Central to understanding gene regulation is understanding the mechanism by which promoter specific transcription activators (activators) stimulate transcription. A group of factors called coactivators have been shown to be required for activator function in vitro. The best characterized coactivators to date are members of the TFIID complex. TFIID is a multisubunit complex composed of the TATA box binding protein (TBP) and 8-12 TBP associated factors (TAFIIs). Results from numerous in vitro experiments indicate that TAFIIs function by binding to activators and forming a bridge between the activator and the basal transcription machinery. In order to gain insight into the mechanism by which activators stimulate transcription, we chose to analyze the in vivo function of TAFIIs, their proposed targets. Results from the genetic disruption of a number of TAFIIs in the yeast Saccharomyces cerevisiae showed that most are encoded by essential genes. In order to study their function, temperature-sensitive and conditional alleles were constructed. Cells depleted of individual TAFIIs by either of these two methods displayed no defect in global transcription activation. Inactivation of yTAFII17, however, resulted in a promoter specific defect. In addition, inactivation of yTAFII145, yTAFII90, or TSM1, resulted in an inability of cells to progress through the cell-cycle. In an attempt to identify genes whose expression required yTAFII90, we performed subtractive hybridization on strains containing wild-type and temperature-sensitive alleles. Although this technique successfully identified genes differentially expressed in the two strains, it failed to identify genes whose expression required yTAFII90. These results indicate that TAFIIs are not the obligatory targets of activators, and that other factors must provide this role in vivo. Furthermore, that many of TAFIIs are required for cell-cycle progression

RGG-boxes of the EWS oncoprotein repress a range of transcriptional activation domains

The Ewings Sarcoma Oncoprotein (EWS) interacts with several components of the mammalian transcriptional and pre-mRNA splicing machinery and is also found in the cytoplasm and even on the cell surface. The apparently diverse cellular functions of EWS are, however, not well characterized. EWS harbours a potent N-terminal transcriptional activation domain (the EAD) that is revealed in the context of oncogenic EWS-fusion proteins (EFPs) and a C-terminal RNA-binding domain (RBD) that recruits pre-mRNA splicing factors and may couple transcription and splicing. In contrast to EFPs, the presumed transcriptional role of normal EWS remains enigmatic. Here, we report that multiple RGG-boxes within the RBD are necessary and sufficient for cis-repression of the EAD and that RGG-boxes can also repress in-trans, within dimeric partners. Lys can functionally substitute for Arg, indicating that the basic nature of the Arg side chain is the critical determinant of RGG-box-mediated repression. In addition to the EAD, RGG-boxes can repress a broad range of activation domains (including those of VP16, E1a and CREB), but repression can be alleviated by the simultaneous presence of more than one activation domain. We therefore propose that a key function of RGG boxes within native EWS is to restrict promiscuous activation by the EAD while still allowing EWS to enter functional transcription complexes and participate in other transactions involving pre-mRNAs

Construction and utilisation of a bidirectional reporter vector in the analysis of two nod-boxes in of Rhizobium loti : a thesis presented in partial fulfilment of the requirements for the Degree of Master of Science in Molecular Genetics at Massey University

The nod-box is a 47bp cis-acting regulatory region which has been conserved amongst every species of Rhizobium studied to date. In species such as R. meliloti and R. leguminosarum, the nod-box has been shown to promote constitutive activity towards the regulatory nodD gene, and flavonoid-inducible expression towards the divergently-transcribed nodABCIJ operon. This bidirectional regulation of the so-called common nod genes was not observed in R. loti. A previous analysis of this species had shown that its nod-box promoted inducible activity towards the truncated 'nodD' gene, as well as the nodACIJ operon. It was the unusual arrangement of these R. loti nod genes that had initially aroused interest in this bacteria. To further investigate the role of the nod-box in the regulation of the R. loti common nod genes, a bidirectional reporter vector (pSPV4) was constructed. This novel vector allowed the promoter activity of a cloned nod-box-containing fragment to be concurrently measured in either direction using the same culture of cells. To achieve this construct, the gusA gene from pRAJ260 was blunt-end ligated into pUC21. An in-frame ribosome binding site (rbs) was cloned upstream of the gusA coding sequence to facilitate transcriptional fusions. The rbs and gusA gene were later excised as a functional unit and blunt-end ligated into pMP220 alongside the B-galactosidase reporter gene but in the opposite orientation. Hence, both reporter genes could be divergently transcribed from a common regulatory region cloned into the multiple cloning site that separated the genes. The fragments of DNA that were eventually cloned into the bidirectional vector were generated through the polymerase chain reaction. Each DNA insert contained the nod-box bracketed by differing lengths of flanking region. Once these PCR-generated fragments had been sequenced in pUC118 and subcloned into pSPV4, the resulting constructs were transformed into R. loti cells by electroporation. As the electroporation of these cells had not previously been reported, the conditions for this procedure were established and optimised. The results obtained from the bidirectional reporter assays disagreed with those observed in the earlier assays by Teo (1990). Neither the nodACIJ nod-box of NZP2037 nor the nodB nod-box of NZP2213, showed bidirectional inducible expression. In fact, both nod-boxes showed constitutive expression in the 'nodD' direction and inducible expression in the opposite direction. This indicates that the control of the nod genes in R. loti is fundamentally the same as that seen in other fast-growing Rhizobium species. Three regulatory elements affecting the levels of nod gene expression have tentatively been identified outside the nod-box sequence, though the results indicating their presence may simply be·due to spacing differences between the nod-box and the reporter gene

Nucleosome Distortion as a Possible Mechanism of Transcription Activation Domain Function

After more than three decades since the discovery of transcription activation domains (ADs) in gene-specific activators, the mechanism of their function remains enigmatic. The widely accepted model of direct recruitment by ADs of co-activators and basal transcriptional machinery components, however, is not always compatible with the short size yet very high degree of sequence randomness and intrinsic structural disorder of natural and synthetic ADs. In this review, we formulate the basis for an alternative and complementary model, whereby sequence randomness and intrinsic structural disorder of ADs are necessary for transient distorting interactions with promoter nucleosomes, triggering promoter nucleosome translocation and subsequently gene activation

The pause-initiation limit restricts transcription activation in human cells.

Eukaryotic gene transcription is often controlled at the level of RNA polymerase II (Pol II) pausing in the promoter-proximal region. Pausing Pol II limits the frequency of transcription initiation ('pause-initiation limit'), predicting that the pause duration must be decreased for transcriptional activation. To test this prediction, we conduct a genome-wide kinetic analysis of the heat shock response in human cells. We show that the pause-initiation limit restricts transcriptional activation at most genes. Gene activation generally requires the activity of the P-TEFb kinase CDK9, which decreases the duration of Pol II pausing and thereby enables an increase in the productive initiation frequency. The transcription of enhancer elements is generally not pause limited and can be activated without CDK9 activity. Our results define the kinetics of Pol II transcriptional regulation in human cells at all gene classes during a natural transcription response

Transcription Regulation and Membrane Stress Management in Enterobacterial Pathogens

Transcription regulation in a temporal and conditional manner underpins the lifecycle of enterobacterial pathogens. Upon exposure to a wide array of environmental cues, these pathogens modulate their gene expression via the RNA polymerase and associated sigma factors. Different sigma factors, either involved in general 'house-keeping' or specific responses, guide the RNA polymerase to their cognate promoter DNAs. The major alternative sigma54 factor when activated helps pathogens manage stresses and proliferate in their ecological niches. In this chapter, we review the function and regulation of the sigma54-dependent Phage shock protein (Psp) system-a major stress response when Gram-negative pathogens encounter damages to their inner membranes. We discuss the recent development on mechanisms of gene regulation, signal transduction and stress mitigation in light of different biophysical and biochemical approaches.This is the peer reviewed version of the paper: Zhang, N., Jovanovic, G., McDonald, C., Ces, O., Zhang, X., & Buck, M. (2016). Transcription Regulation and Membrane Stress Management in Enterobacterial Pathogens. У M. C. Leake (Ур.), Biophysics of Infection (стр. 207–230). Springer International Publishing. [https://doi.org/10.1007/978-3-319-32189-9_13

Functional interplay between PPM1G and the transcription elongation machinery

Transcription elongation is a critical regulatory step in the gene expression cycle. One key regulator of the switch between transcription initiation and elongation is the P-TEFb kinase, which phosphorylates RNA polymerase II (Pol II) and several negative elongation factors to relieve the elongation block at paused promoters to facilitate productive elongation. Here, we highlight recent findings signifying the role of the PPM1G/PP2Cg phosphatase in activating and maintaining the active transcription elongation state by regulating the availability of P-TEFb and blocking its assembly into the catalytic inactive 7SK small nuclear ribonucleoprotein (snRNP) complex

Regulation of Transcriptional Elongation by RNA Polymerase II

Transcriptional attenuation by RNA polymerase II (pol II) has been shown to regulate the expression of many genes (Spencer and Groudine, 1990) but the mechanism of this control has been poorly understood. In this thesis I present data which indicate that in X.laevis oocytes transactivator proteins stimulate transcriptional elongation by pol II. Transcription complexes activated by recombinant factors bound to promoter elements in synthetic genes have high competence to elongate meaning they are able to read through pausing and termination sites efficiently. Furthermore, activation domains differ in the processivity of the transcription they stimulate from a given promoter. In contrast, non-activated transcription and transcription "squelched" by a non-binding factor mostly terminates prematurely. A general transcription factor, TBP, is found to stimulate initiation, but not elongation of pol II transcription. These results suggest that programming the competence of RNApolymerase II to elongate is an integral part of the initiation step which is controlled by activators co-operating with the basal transcriptional machinery. The positive effect of transcriptional activators on pol II processivity is counteracted by the suppressor of transcriptional elongation DRB (Dichlororibofuranosyl-benzimidazole) and by protein kinase inhibitors such as H-8 and H-7. Here I characterise a transactivator binding CTD-protein kinase which is highly sensitive to DRB, H-7 and H-8. This protein kinase co-purifies with the general transcription factor TFIIH on affinity chromatography resins and has properties indistinguishable from the TFIIH associated kinase. I suggest that the effect of DRB on transcriptional elongation is mediated by inhibition of the TFIIH associated kinase activity. The human protein BM28, which is analogous to the yeast MCM2 and MCM3 proteins, has been proposed to participate in DNA replication. In this thesis I include experiments which indicate that BM28 is also essential for pol II transcription in X.laevis oocytes

The Host-Cell Architectural Protein HMG I(Y) Modulates Binding of Herpes Simplex Virus Type 1 ICP4 to Its Cognate Promoter

AbstractThe productive infection cycle of herpes simplex virus is controlled in part by the action of ICP4, an immediate-early gene product that acts as both an activator and repressor of transcription. ICP4 is autoregulatory, and IE-3, the gene that encodes it, contains a high-affinity binding site for the protein at its cap site. Previously, we had demonstrated that this site could be occupied by proteins found in nuclear extracts from uninfected cells. A HeLa cell cDNA expression library was screened with a DNA probe containing the IE-3 gene cap site, and clones expressing the architectural chromatin proteins HMG I and HMG Y were identified by this technique. HMG I is shown to augment binding of ICP4 to its cognate site inin vitroassays and to enhance the activity of this protein in short-term transient expression assays