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

GCN5 Is a Positive Regulator of Origins of DNA Replication in Saccharomyces cerevisiae

GCN5 encodes one of the non-essential Histone Acetyl Transferases in Saccharomyces cerevisiae. Extensive evidence has indicated that GCN5 is a key regulator of gene expression and could also be involved in transcriptional elongation, DNA repair and centromere maintenance. Here we show that the deletion of GCN5 decreases the stability of mini-chromosomes; that the tethering of Gcn5p to a crippled origin of replication stimulates its activity; that high dosage of GCN5 suppresses conditional phenotypes caused by mutant alleles of bona fide replication factors, orc2-1, orc5-1 and mcm5-461. Furthermore, Gcn5p physically associates with origins of DNA replication, while its deletion leads to localized condensation of chromatin at origins. Finally, Δgcn5 cells display a deficiency in the assembly of pre-replicative complexes. We propose that GCN5 acts as a positive regulator of DNA replication by counteracting the inhibitory effect of Histone Deacetylases

A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His(6)-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni(2+)-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs

A large-scale examination of the effectiveness of anonymous marking in reducing group performance differences in higher education assessment

The present research aims to more fully explore the issues of performance differences in higher education assessment, particularly in the context of a common measure taken to address them. The rationale for the study is that, while performance differences in written examinations are relatively well researched, few studies have examined the efficacy of anonymous marking in reducing these performance differences, particularly in modern student populations. By examining a large archive (N = 30674) of assessment data spanning a twelve-year period, the relationship between assessment marks and factors such as ethnic group, gender and socio-environmental background was investigated. In particular, analysis focused on the impact that the implementation of anonymous marking for assessment of written examinations and coursework has had on the magnitude of mean score differences between demographic groups of students. While group differences were found to be pervasive in higher education assessment, these differences were observed to be relatively small in practical terms. Further, it appears that the introduction of anonymous marking has had a negligible effect in reducing them. The implications of these results are discussed, focusing on two issues, firstly a defence of examinations as a fair and legitimate form of assessment in Higher Education, and, secondly, a call for the re-examination of the efficacy of anonymous marking in reducing group performance differences

Sub-Telomeric core X and Y' Elements in S.cerevisiae Suppress Extreme Variations in Gene Silencing

Telomere Position Effect (TPE) is governed by strong repression signals emitted by telomeres via the Sir2/3/4 Histone Deacetylase complex. These signals are then relayed by weak proto-silencers residing in the subtelomeric core X and Y' elements. Subtelomeres also contain Sub-Telomeric Anti-silencing Regions (STARs). In this study we have prepared telomeres built of different combinations of core X, Y' and STARs and have analyzed them in strains lacking Histone-Acetyltransferase genes as well as in cdc6-1 and Δrif1 strains. We show that core X and Y' dramatically reduce both positive and negative variations in TPE, that are caused by these mutations. We also show that the deletion of Histone-Acetyltransferase genes reduce the silencing activity of an ACS proto-silencer, but also reduce the anti-silencing activity of a STAR. We postulate that core X and Y' act as epigenetic “cushioning” cis-elements

Forks on the Run: Can the Stalling of DNA Replication Promote Epigenetic Changes?

Built of DNA polymerases and multiple associated factors, the replication fork steadily progresses along the DNA template and faithfully replicates DNA. This model can be found in practically every textbook of genetics, with the more complex situation of chromatinized DNA in eukaryotes often viewed as a variation. However, the replication-coupled disassembly/reassembly of chromatin adds significant complexity to the whole replication process. During the course of eukaryotic DNA replication the forks encounter various conditions and numerous impediments. These include nucleosomes with a variety of post-translational modifications, euchromatin and heterochromatin, differentially methylated DNA, tightly bound proteins, active gene promoters and DNA loops. At such positions the forks slow down or even stall. Dedicated factors stabilize the fork and prevent its rotation or collapse, while other factors resolve the replication block and facilitate the resumption of elongation. The fate of histones during replication stalling and resumption is not well understood. In this review we briefly describe recent advances in our understanding of histone turnover during DNA replication and focus on the possible mechanisms of nucleosome disassembly/reassembly at paused replication forks. We propose that replication pausing provides opportunities for an epigenetic change of the associated locus

Evidence for a role of MCM (mini-chromosome maintenance)5 in transcriptional repression of sub-telomeric and Ty-proximal genes in Saccharomyces cerevisiae

The MCM (mini-chromosome maintenance) genes have a well established role in the initiation of DNA replication and in the elongation of replication forks in Saccharomyces cerevisiae. In this study we demonstrate elevated expression of sub-telomeric and Ty retrotransposon-proximal genes in two mcm5 strains. This pattern of up-regulated genes resembles the genome-wide association of MCM proteins to chromatin that was reported earlier. We link the altered gene expression in mcm5 strains to a reversal of telomere position effect (TPE) and to remodeling of sub-telomeric and Ty chromatin. We also show a suppression of the Ts phenotype of a mcm5 strain by the high copy expression of the TRA1 component of the chromatin-remodeling SAGA/ADA (SPT-ADA-GCN5 acetylase/ADAptor). We propose that MCM proteins mediate the establishment of silent chromatin domains around telomeres and Ty retrotransposons

The Transcriptional Elongation Inhibitor 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole Inhibits Transcription Factor IIH-associated Protein Kinase

International audienceRegulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759); however the mechanisms that control this step in transcription are not well understood. The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) has long been used as an inhibitor of RNA polymerase II elongation, but its target is not known. We show that DRB is a potent inhibitor of Cdk-activating kinase, associated with the general transcription factor TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited transcriptional elongation. Furthermore, TFIIH kinase bound specifically to the herpes simplex virus VP16 activation domain which stimulates polymerase II elongation in addition to initiation (Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759). Our results suggest that DRB affects transcription by inhibiting the TFIIH-associated kinase and that this kinase functions in the control of elongation by RNA polymerase II