Requirements for efficient in vitro transcription and translation: a study using yeast invertase as a probe.
Factors for efficient synthesis of mRNA in vitro and its subsequent translation in cell free lysates from reticulocyte and wheat germ were studied using yeast invertase as a probe. Among various transcription systems tested, containing either SP6, T5, T7 or a bacterial synthetic consensus promoter, the T7 system was superior both from a quantitative and qualitative point of view. Transcription with SP6 polymerase, but not with the other enzymes, resulted in premature transcript termination, which is ascribed to a sensitivity of the SP6 polymerase towards a hairpin loop structure in the invertase coding region. In-frame fusion of the critical DNA sequence to a different gene promoted premature transcription termination of the resulting chimeric template, which in its original form is transcribed correctly. Transcripts with additional sequences 5' upstream of the natural translation start revealed a diminished protein synthesis presumably due to the presence of out of frame ATG codons. In contrast, no influence on translation was found when additional sequences at the 3' end were present or when the stop codon was missing. Capping of transcripts was essential for translation in wheat germ lysates, whereas protein synthesis in reticulocytes was only reduced in the absence of a cap. The influence of polyadenylation on translation was studied using transcripts with engineered poly(A) tracts of different size. Increasing poly(A) chain length abolished translation in vitro in both translation systems. Inhibition was poly(A)-specific and is discussed as interference of the poly(A) sequences with a crucial component(s) of the protein synthesis machinery.
Roitsch
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T
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, F.R.G.
Lehle
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D001435:Bacteriophages / Q000201:enzymology; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000235:genetics*; D010957:Plasmids; D011061:Poly A / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012314:RNA Cap Analogs / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013604:T-Phages / Q000235:genetics; D014158:Transcription, Genetic; D043324:beta-Fructofuranosidase
D012314:RNA Cap Analogs; D012333:RNA, Messenger; D011061:Poly A; D012321:DNA-Directed RNA Polymerases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase