RESUMEN
Studying the function or structure of proteins usually requires the generation of many protein-truncation constructs for recombinant expression, which is a tedious and error-prone job. CCD2 is a software tool designed to facilitate and automate this task. CCD2 helps scientists by aggregating the information necessary to design protein-expression constructs. This information includes sequence conservation, secondary structure prediction, domain(s) and disorder detection, post-translational modifications and information on similar (domain) structures that are available in the Protein Data Bank. CCD2 then allows users to easily choose the boundaries for protein constructs and automatically generates the primers necessary for construct amplification by polymerase chain reaction. Finally, CCD2 provides a quick analysis of the properties of the chosen constructs, together with their DNA vector maps for bookkeeping. The features of CCD2 are discussed step by step, showing that it can be a useful tool for laboratories that engage in recombinant protein production for any type of experiment, and in particular for structural biology studies.
Asunto(s)
Proteínas/metabolismo , Programas Informáticos , Estructura Secundaria de Proteína , Proteínas/químicaRESUMEN
The ASAP complex interacts with the exon-junction complex (EJC), a messenger ribonucleoprotein complex involved in post-transcriptional regulation. The three ASAP subunits (Acinus, RNPS1 and SAP18) have been individually implicated in transcriptional regulation, pre-mRNA splicing and mRNA quality control. To shed light on the basis for and consequences of ASAP's interaction with the EJC, we have determined the 1.9-Å resolution structure of a eukaryotic ASAP core complex. The RNA-recognition motif of RNPS1 binds to a conserved motif of Acinus with a recognition mode similar to that observed in splicing U2AF proteins. The Acinus-RNPS1 platform recruits the ubiquitin-like domain of SAP18, forming a ternary complex that has both RNA- and protein-binding properties. Unexpectedly, our structural analysis identified an Acinus-like motif in Pinin, another EJC-associated splicing factor. We show that Pinin physically interacts with RNPS1 and SAP18, forming an alternative ternary complex, PSAP.