The effects of 5'-capping, 3'-polyadenylation and leader composition upon the translation and stability of mRNA in a cell-free extract derived from the yeast Saccharomyces cerevisiae.
A new modular expression system was developed to direct the in vitro synthesis of defined transcripts that were used as templates for translation in yeast cell-free extracts. The system was used to examine the influence of 5'-capping, 3'-polyadenylation and leader sequence upon the translation and stability of the synthetic Tn9 cat (chloramphenicol acetyl transferase), yeast PGK (phosphoglycerate kinase) and yeast HSP26 (heat-shock protein 26) mRNAs. The addition of a methylated cap (m7Gppp) or of a poly(A) tail enhanced translation and stabilized the mRNA. The dependence of translation upon capping was reduced in the presence of the HSP26 leader sequence. This may indicate the existence of a translational mechanism that enhances cap-independent translation. The enhancement of the translation and stability of mRNA was relatively insensitive to changes in the position of the poly(A) tail relative to the reading frame.
Gerstel
B
B
Department of Gene Expression, Gesellschaft für Biotechnologische Forschung (GBF), Braunschweig, Germany.
Tuite
M F
MF
McCarthy
J E
JE
D001483:Base Sequence; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism*; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000378:metabolism; D012315:RNA Caps; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D021382:Protein Sorting Signals; D012315:RNA Caps; D012331:RNA, Fungal; D012333:RNA, Messenger; D011061:Poly A