Free luciferase may acquire a more favorable conformation than ribosome-associated luciferase for its activity expression.
A variant of firefly luciferase in which the C-terminal end was extended with 44 amino acid residues served as a model protein in this study. After transcription and translation in vitro, the enzyme activity was measured when still attached to the ribosome and when released from the ribosome by incubation with RNase A or puromycin. It was found that the C-terminally extended luciferase already had activity when linked to the ribosome, but its activity was greatly increased when released from the ribosome. These results indicate that the luciferase is folded during synthesis on the ribosome; however, some conformational adjustments occur after its release from the ribosome which are required for the full expression of its enzymatic activity.
Yang
F
F
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Jing
G Z
GZ
Zhou
J M
JM
Zheng
Y Z
YZ
D019943:Amino Acid Substitution; D000818:Animals; D003001:Cloning, Molecular; D001517:Coleoptera; D004926:Escherichia coli; D014644:Genetic Variation; D008156:Luciferases / Q000096:biosynthesis* / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D008156:Luciferases