Protocol to reconstitute translationally arrested heat shock mRNPs and condensates in vitro

Abstract

Summary: Heat shock (HS) coincides with the assembly of translationally arrested heat shock messenger ribonucleoprotein particles (HS-mRNPs) and condensates. Here, we present a protocol to reconstitute HS-mRNPs and HS condensates with eIF4G, eIF4E, Pab1p, and mRNA in vitro. In addition, we describe the necessary steps to measure the effect of HS-mRNPs and HS condensates on translation in yeast extracts. The protocol can be modified to study mRNPs and condensates assembled with other proteins and to study translation in extracts prepared from different cells.For complete details on the use and execution of this protocol, please refer to Desroches Altamirano et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Protein Biochemistry
• Protein expression and purification