CONTRASTING EXPRESSION OF KAL IN CELL-FREE SYSTEMS - 5'-UTR AND CODING REGION STRUCTURAL EFFECTS ON TRANSLATION

We investigated the expression of two different X-linked Kallmann (KAL ) gene cDNAs in two different cell-free systems using rabbit reticuloc yte lysate: (system A) transcription/translation coupled and (system B ) noncoupled. System A yielded a single band of 76 kDa corresponding t o anosmin-1, the expected full-length gene product, and upon addition of canine microsomal membranes produced a 85-kDa glycosylated form. Sy stem B did not produce any detectable protein band despite the express ion of a beta-galactosidase-positive control gene. The first 179 bases of the coding sequence are 74% GC-rich and showed the potential to fo rm imperfect hairpin structures, which in part may explain the transla tion inhibition of KAL in system B. This has further led us to specula te that coupling transcription to translation may either be preventing translating-inhibiting hairpin formation or be compensating for the l ack of certain tissue-specific proteins in reticulocyte lysate that ar e essential in overcoming inhibitory hairpins during translation. Subs titution of the 5'-UTR with an encephalomyocarditis virus internal rib osomal entry site (EMCV IRES) sequence resulted paradoxically in a low er yield of anosmin 1, suggesting that elements in the 5'UTR may be ne cessary for maintaining a ''normal'' level of expression. The use of K AL and luciferase reporters (containing different 5'UTRs) demonstrated that the native KAL 5' UTR is not involved in translational efficienc y. However, this sequence may influence faithful translation initiatio n. Theoretical RNA conformation data imply that effective EMCV IRES us age with KAL may require favorable pairing between the IRES and uniden tified sequences within the 5' coding region of the gene. This work pr ovides a foundation both for the investigation of KAL regulation and f or the characterization of its function, (C) 1998 Academic Press.