Structural characterisation of the Saccharomyces cerevisiae transcription elongation factor Spt4/5 through RNA interactions

[Truncated] The transcription elongation factor Spt4/5 (Spt4/5) tightly associates with RNA polymerase II (RNAPII) regulating elongation and co-transcriptional pre-mRNA processing events; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro and that this function may be key in understanding the ability of Spt4/5 to exert its control over transcription elongation.

Spt5 is an essential protein and the only universally conserved RNAP-associated transcription elongation factor. A unique feature of eukaryotic Spt5 is the presence of multiple Kyrpides, Ouzounis and Woese (KOW) domains. The KOW domains are thought to be responsible for the RNA binding function of Spt4/5 although there is little evidence in the literature to support this theory. Therefore, the main focus of this study was to characterise the RNA binding specificity of Saccharomyces cerevisiae Spt4/5.

When expressed in Escherichia coli, the Spt4/5 complex is innately insoluble, possibly due to the large unstructured regions connecting the KOW domains to each other and to the Spt5 core. Therefore, the first section of this thesis presents the expression and purification of milligram quantities of three different multi-KOW domain complexes of yeast Spt4/5 through the use of ubiquitin fusion constructs. The multi-KOW domain Spt4/5 complexes were characterised by various biophysical techniques showing that the proteins are folded with an Spt4:Spt5 hetero-dimeric stoichiometry of 1:1.