Functional and Structural Analysis of the Schizosaccharomyces pombe EAF protein

Abstract

newline Eukaryotic gene expression is regulated by RNA polymerase II in association with an newlinearray of other proteins. For a long time, research was focused on initiation of transcription newlineas the major regulatory step of gene expression. However, research over the past decade newlineor so has demonstrated that transcription elongation is also another major step at which newlinethe expression of a gene is controlled. ELL (Eleven Nineteen Lysine Rich Leukemia) newlineand EAF (ELL associated factor) family of transcription elongation factors s have been newlineshown to prevent transient pausing of RNA Pol II along the DNA template in vitro. Both newlineELL and EAF have been identified in higher eukaryotic organisms and shown to be newlineimportant for their survival. Interestingly, no homolog of these proteins has been newlineidentified in Saccharomyces cerevisiae, while a single homolog each of ELL and EAF newlinehas been identified in Schizosaccharomyces pombe. However, our knowledge and newlineunderstanding about their in vivo roles in various organisms is still limited. In this work, newlinewe have used S. pombe as model organism to elucidate the in vivo role(s) and structural newlinefeatures of EAF in S. pombe (SpEAF). We have shown that deletion of eaf+ or ell+ newlineresulted in slow growth of S. pombe cells under optimum growth conditions as well as newlinerendered S. pombe cells sensitive towards various DNA damaging conditions. Functional newlinemapping of SpEAF domains revealed that its carboxyl terminus region comprising of newline158-251 amino acids was sufficient to rescue these phenotypes that were associated with newlineeaf deletion. However, its amino terminus domain spanning 1-157 amino acids was newlineresponsible for interaction with SpELL. SpEAF also displayed transactivation potential, newlineand this activity resided in both its amino as well as carboxyl terminus regions. Our work newlinealso suggests that SpEAF may play a role in repair of DNA lesions caused by MMS via newlinedouble strand break repair pathway and physical association with Rhp51. Furthermore, newlinegenome-wide transcriptional response to absenc