Preferential protein-protein binding of HB6, RDM1 and DMS3 to BPM1

BPM proteini vrste A. thaliana usmjeravaju nekoliko transkripcijskih faktora u 26S proteasomsku razgradnju ovisnu o ubikvitinu. U preliminarnom istraživanju, nakon kopurifikacije proteina TAP-tag metodom iz klijanaca uročnjaka, pokazana je interakcija između proteina BPM1 i DMS3, odnosno RDM1, članova puta DNA metilacije usmjerene RNA molekulom (RdDM). U ovom diplomskom radu, interakcije između BPM1, transkripcijskog faktora HB6 te DMS3, odnosno RDM1 proteina, analizirane su metodom kopurifikacije (pull-down), jedan na jedan i jedan na dva. U tu su svrhu prikladni ekspresijski plazmidi konstruirani pomoću ligacije ili In-Fusion metode. Plazmidi su kemijski transformirani u E. coli soj RosettaTM. Nakon indukcije ekspresije gena, fuzijski su proteini pročišćeni afinitetnom kromatografijom i vizualizirani SDS-PAGE metodom, bojanjem proteina u gelu i imunodetekcijom. U ovom diplomskom radu, potvrđena je interakcija između RDM1 i BPM1, te DMS3 i BPM1. Interakcija između HB6 i BPM1-GST nije potvrđena, budući da se protein HB6 vezao i na GST (negativna kontrola). Ovi eksperimenti predstavljaju osnovu za daljnja istraživanja koja bi mogla razriješiti potencijalnu ulogu proteina BPM1 u putu RdDM.BPM proteins in A. thaliana are known to direct several transcription factors to ubiquitinmediated 26S proteasome degradation. Preliminary investigation, after protein copurification by TAP-tag from Arabidopsis seedlings, has shown that BPM1 also potentially interacts with members of the RNA-directed DNA methylation (RdDM) pathway, DMS3 and RDM1. Here, interactions of BPM1 with transcription factor HB6 and proteins DMS3 and RDM1 were analyzed in one to one and one to two pull-down assays. For this purpose, suitable expression plasmids were generated using ligation or In-Fusion cloning methods. RosettaTM E. coli strain was chemically transformed with generated constructs. Protein expression was induced, and fusion proteins were purified. Finally, pull-down assays were performed and proteins visualized by SDS-PAGE, staining and immunodetection. In the conditions carried out here, RDM1 and DMS3 interacted with BPM1. Since HB6 interacted with both BPM1-GST and GST alone (negative control), a direct HB6 interaction could not be shown. These experiments provide a basis for future analyses which could elucidate the potential function of BPM1 in the RdDM pathway