Cotranslational formation of active photoprotein obelin in a cell-free translation system: direct ultrahigh sensitive measure of the translation course.
Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotranslational formation of active photoprotein. Active obelin formation is recorded by its luminescence, either direct in the translation mixture in the presence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the synthesized product in both cases allows use of apoobelin mRNA at very low concentrations as an internal marker for immediate measure of protein biosynthesis activity of in vitro translation systems. It is shown that the simultaneous translation of any other mRNA does not affect translation of photoprotein mRNAs under standard conditions. Continuous registration of luminescence in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus increasing the numerical values of the recorded signals. Registration of photoprotein luminescence during translation can be used to obtain additional information about the translation process, for example codon reading speed, about protein folding, and about the formation of active proteins on ribosomes.
Branch of the Institute of Bioorganic Chemistry, Russian Academy of Science, Pushchino, 142292, Russia.
N G
NG
Berestovskaya
L A
LA
Shaloiko
A Y
AY
Gorokhovatsky
V S
VS
Bondar
E S
ES
Vysotski
J E
JE
Maximov
H
H
von Doehren
Y B
YB
Alakhov
D002474:Cell-Free System; D007093:Imidazoles; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis; D017510:Protein Folding; D011719:Pyrazines / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013997:Time Factors
D007093:Imidazoles; D008164:Luminescent Proteins; D011719:Pyrazines; D012333:RNA, Messenger; C010497:obelin; C017144:coelenterazine