A new reporter system has been developed for measuring
translation coupling efficiency of recoding mechanisms
such as frameshifting or readthrough. A recoding test sequence
is cloned in between the renilla and firefly luciferase
reporter genes and the two luciferase activities are subsequently
measured in the same tube. The normalized ratio of the
two activities is proportional to the efficiency with which
the ribosome “reads” the recoding signal making
the transition from one open reading frame to the next.
The internal control from measuring both activities provides
a convenient and reliable assay of efficiency. This is
the first enzymatic dual reporter assay suitable for in
vitro translation. Translation signals can be tested in
vivo and in vitro from a single construct, which allows
an intimate comparison between the two systems. The assay
is applicable for high throughput screening procedures.
The dual-luciferase reporter system has been applied to
in vivo and in vitro recoding of HIV-1 gag-pol,
MMTV gag-pro, MuLV gag-pol, and human
antizyme.