In vitro translation of mRNAs into proteins is frequently used to stud
y the coding capacity of RNAs or cDNAs and the functional effects of m
utations. In vitro translation assays have traditionally been monitore
d by following the incorporation of a radiolabeled amino acid into new
ly synthesized protein. We have optimized an alternative nonradioactiv
e biotin-labeling method. rRNA(Lys) is first aminoacylated with lysine
, which is then chemically labeled with biotin. When biotin-lysine-tRN
A(Lys) is added to translation systems, the biotinylated lysine is inc
orporated into the growing polypeptide chain. After electrophoresis an
d transfer to a blotting membrane, the biotin-labeled translation prod
ucts are detected by a chemiluminescent reaction of luminol/iodophenol
with streptavidin-coupled horseradish peroxidase. This nonradioactive
method yields results equivalent to those obtained using the radioact
ive method. Biotin-labeled translation products are also biologically
functional: (i) biotinylated precursor proteins are transported and pr
ocessed correctly by dog pancreas microsomes; (ii) transcription facto
rs synthesized by biotin in vitro translation bind specifically to the
ir DNA recognition sequence; and (iii) biotin-modified luciferase keep
s its enzymatic activity. The major advantage of the biotin in vitro t
ranslation system is that no radioactivity is required and the method
is easy, economical, reproducible and fast-the whole nonradioactive pr
ocedure, from translation to detection, can be completed within six ho
urs.