Effective non-viral leader for cap-independent translation in a eukaryotic cell-free system.
The 61 nt 5'-untranslated region (5'-UTR) of mRNA encoding for a light-emitting protein of hydroid polyp Obelia longissima, obelin, is shown to provide a high level of cap-independent translation of heterologous mRNAs in cell-free translation systems based on wheat germ extracts. The inhibition of translation typically observed when excess mRNA is present or produced in a eukaryotic system (the so-called self-inhibition phenomenon) is found abated with mRNA constructs carrying the obelin mRNA leader. The role of the sequestration of a limiting initiation factor, probably eIF4F, in the self-inhibition phenomenon and the possible independence of the obelin mRNA leader from eIF4F are discussed. We propose the obelin mRNA leader be used for effective cap-independent translation in eukaryotic cell-free systems, including combined transcription-translation systems with uncontrolled phage polymerase-catalyzed accumulation of mRNA.
Shaloiko
L A
LA
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia 142290.
Granovsky
I E
IE
Ivashina
T V
TV
Ksenzenko
V N
VN
Shirokov
V A
VA
Spirin
A S
AS
D020121:5' Untranslated Regions / Q000235:genetics; D002474:Cell-Free System / Q000502:physiology*; D005057:Eukaryotic Cells / Q000502:physiology; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D012314:RNA Cap Analogs / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014908:Triticum / Q000196:embryology / Q000235:genetics* / Q000378:metabolism*; D014780:Viruses / Q000235:genetics
D020121:5' Untranslated Regions; D008164:Luminescent Proteins; D010940:Plant Proteins; D012314:RNA Cap Analogs; D011994:Recombinant Proteins; C010497:obelin