Regulation of Transcriptional Elongation by RNA Polymerase II

Yankulov, Krassimir Yankov (1994). Regulation of Transcriptional Elongation by RNA Polymerase II. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fdec

Abstract

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.