We have shown previously that an internal ribosome entry segment (IRES) directs the synthesis of the p36 isoform of Bag-1 and that polypyrimidine tract binding protein 1 (PTB-1) and poly(rC) binding protein 1 (PCBP1) stimulate IRES-mediated translation initiation in vitro and in vivo. Here, a secondary structural model of the Bag-1 IRES has been derived by using chemical and enzymatic probing data as constraints on the RNA folding algorithm Mfold. The ribosome entry window has been identified within this structural model and is located in a region in which many residues are involved in base-pairing interactions. The interactions of PTB-1 and PCBP1 with their cognate binding sites on the IRES disrupt many of the RNA-RNA interactions, and this creates a largely unstructured region of approximately 40 nucleotides that could permit ribosome binding. Mutational analysis of the PTB-1 and PCBP1 binding sites suggests that PCBP1 acts as an RNA chaperone to open the RNA in the vicinity of the ribosome entry window while PTB-1 is probably an essential part of the preinitiation complex.
