A dual-luciferase reporter system for studying recoding signals.
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.
Grentzmann
G
G
Howard Hughes Medical Institute, University of Utah, Salt Lake City 84112, USA.
Ingram
J A
JA
Kelly
P J
PJ
Gesteland
R F
RF
Atkins
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003063:Cnidaria / Q000201:enzymology; D001517:Coleoptera / Q000201:enzymology; D015685:Fusion Proteins, gag-pol / Q000235:genetics; D017930:Genes, Reporter; D005815:Genetic Code; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D008969:Molecular Sequence Data; D065108:Ornithine Decarboxylase Inhibitors; D014176:Protein Biosynthesis; D016364:Reading Frames; D013539:Simian virus 40 / Q000235:genetics
D015685:Fusion Proteins, gag-pol; D065108:Ornithine Decarboxylase Inhibitors; D008156:Luciferases