Diagrams showing the overall primary and secondary structures of the 5'-untranslated region (5'-UTR) (-575) of human p27 mRNA. (a) Overall primary structure of human 5'-UTR of p27 gene showing the location of upstream open reading frame (uORF) (-521 to -425) and the polypyrimidine tract (-66 to -41) in the internal ribosome entry site (IRES) (i.e., IRES region of Hengst: -417 to -1) [11, 34]. (b) The most stable secondary stem and loop structure of the wild-type IRES region of Hengst (-417 to -1) [11, 34–36] at several lowest Gibbs free energy values. This structure was generated using Zucker's RNA mfold software version 2.3 [56, 57]. (c) Same as in Figure 8b, except that UU at -57 and -56 in the polypyrimidine tract (-66 to -41) were mutated to GG. This mutation completely destroyed the overall secondary stem and loop structure of IRES. Various other mutations introduced into the polypyrimidine tract also significantly modified the overall stem and loop structure of IRES.