An insight into structural plasticity and conformational transitions of transcriptional co-activator Sus1

Abstract

RNA biogenesis and mRNA transport are an intricate process for every eukaryotic cell. SAGA, a transcriptional coactivator and TREX-2 are the two major complexes participate in this process. Sus1 is a transcription export factor and part of both the SAGA and the TREX-2 complex. The competitive exchange of Sus1 molecule between SAGA and TREX-2 complex modulates their function which is credited to structural plasticity of Sus1. Here, we portray the biophysical characterization of Sus1 from S. cerevisiae. The recombinant Sus1 is a α-helical structure which is stable at various pH conditions. We reported the α-helix to βsheet transition at the low pH as well as at high pH. Sus1 showed 50% reduction in the fluorescence intensity at pH-2 as compared to native protein. The fluorescence studies demonstrated the unfolding of tertiary structure of the protein with variation in pH as compared to neutral pH. The same results were obtained in the ANS binding and acrylamide quenching studies. Similarly, the secondary structure of the Sus1 was found to be stable till 55% alcohol concentration while tertiary structure was stable up to 20% alcohol concentration. Further increase in the alcohol concentration destabilizes the secondary as well as tertiary structure. The 300 mM concentration of ammonium sulfate also stabilizes the secondary structure of the protein. The structural characterization of this protein is expected to unfold the process of the transportation of the mRNA with cooperation of different proteins.

Proteome analysis of Saccharomyces cerevisiae after methyl methane sulfonate (MMS) treatment

“…The expression of the protein is directly linked to the transcription of their respective genes which is regulated by the RNAPII and epigenetic modification (phospho/dephosphorylaltion) on the CTD [ [12] , [13] , [14] , [15] ]. In order to further investigate the effect of MMS treatment on the phosphorylation status of RNAPII-CTD the western blotting was done using phosphospecific antibodies against CTD [ 28 , 29 ].…”

“…Although many of the proteins that comprise the initial response to MMS have been identified but, the complete repertoire of downstream DDR events induced by MMS especially with respect to the RNAPII remains poorly understood [ [12] , [13] , [14] , [15] , 54 ]. Comparison of fractions associated with RNAP-II in MMS treated sample indicate proteins that are differentially regulated and have a greater degree of association with RNAP-II in response to the MMS during active transcription.…”

“…There are some proteins which regulate the transcription by the phosphorylation of the carboxy terminal domain (CTD) of RNA Polymerase-II (RNAP-II). The CTD acts as a platform for the recruitment of transcription regulatory proteins [ [12] , [13] , [14] , [15] ]. The phosphorylation of the CTD exists in multiple forms [ 16 ].…”

Recombinant expression and preliminary characterization of Peptidyl-prolyl cis/trans-isomerase Rrd1 from Saccharomyces cerevisiae

“…Dialyzing the Rrd1 protein in the preferred buffer pH 7.0. With flow (0.4 ml/min) and with detection at 280 nm, the columns were washed and pre-equilibrated suitable buffer at 25°C [ 23 ].…”

Recombinant expression and biophysical characterization of Mrt4 protein that involved in mRNA turnover and ribosome assembly from Saccharomyces cerevisiae

“…Furthermore, Mrt4 was purified using Ni–NTA (Qiagen, USA) following manual instructions. The concentration of recombinant proteins was calculated through the Bradford method using BSA as a standard [ 22 , 23 ].…”