Pa. Dezoysa et al., CONTRASTING EXPRESSION OF KAL IN CELL-FREE SYSTEMS - 5'-UTR AND CODING REGION STRUCTURAL EFFECTS ON TRANSLATION, Protein expression and purification (Print), 13(2), 1998, pp. 235-242
Citations number
32
Categorie Soggetti
Biochemical Research Methods",Biology,"Biothechnology & Applied Migrobiology
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.