期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1974
卷号：71
期号：11
页码：4635-4639
DOI：10.1073/pnas.71.11.4635
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Highly purified RNase H (RNA{middle dot}DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34 ) from calf thymus was used to specifically remove the poly(A) sequences of purified rabbit globin mRNA after its hybridization with poly(dT). The deadenylylated globin mRNA was repurified by a one-step procedure including a nitrocellulose column. The poly(A) size and the content of unmodified mRNA were determined by hybridization with [3H]-poly(U), and it could be shown that the RNase H digestion method effectively removes this terminal poly(A) sequence. No difference in activity was found between mRNAs with and without poly(A) to initiate, elongate, terminate, and release newly synthesized globin chains in exogenous-mRNA-dependent, cell-free, protein-synthesizing systems from wheat embryo, ascites Krebs II cells, and rat liver. Furthermore, poly(A)-free globin mRNA competed with the same efficiency as authentic globin mRNA against chick ovalbumin mRNA when translated under total mRNA saturation conditions. It is apparent that the 3'-terminal poly(A) sequence is not necessary to maintain the translationally active secondary and tertiary configuration of the globin mRNA molecule. Preincubation of intact and deadenylylated globin mRNA in the Krebs II ascites translational system indicates that the presence of the poly(A) sequence may stabilize the translationally active mRNA molecule.
关键词：poly(dT) ; nitrocellulose column ; cell-free protein synthesis systems ; mRNA competition ; mRNA stability
