In vitro translation of polyadenylic acid-free rabbit globin messenger RNA

H. Soreq*, U. Nudel, R. Salomon, M. Revel, U. Z. Littauer

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

74 Scopus citations

Abstract

A specific method has been developed for the removal of polyadenylic acid-rich sequences from messenger RNA. The method is based on the processive phosphorolysis of mRNA using molar excess of Escherichia coli polynucleotide phosphorylase at 0 ° C in the presence of 1 m-NaCl. It also enables the determination of the location, length and gross base composition of the poly(A)-rich segment. Under these conditions, it was established that the poly(A)-rich sequence of rabbit globin mRNA is located at the 3 ′ -OH terminus and has an average size of 149 nucleotide residues. After removal of the poly(A)-rich sequence from the mRNA, the remainder of the molecule fails to bind to oligo(dT)-cellulose columns. The poly(A)-rich sequence of rabbit globin mRNA does not interact strongly with other regions of the molecule, as it is phosphorolyzed at the same rate as free poly (A). On the other hand, phosphorolysis of the molecule beyond the poly(A)-rich sequence is rather slow, indicating that this region of the mRNA has a stable secondary structure. The sequence beyond the poly(A)-rich segment was found to be rich in guanosine and uridine residues. The poly(A)-rich sequence of the mRNA present as part of a specific polysomal ribonucleoprotein complex, is protected from the action of polynucleotide phosphorylase, indicating that the attachment of the protecting protein(s) extends to the 3 ′ end of the mRNA. The poly(A)-free mRNA can still be translated in a Krebs ascites tumor cellfree extract. The initial rate of translation of poly(A)-free mRNA, is virtually identical to that found with poly(A)-containing mRNA, in the presence or absence of reticulocyte ribosomal wash fluid. The only difference in the template activity of the mRNA preparations, appears at longer periods of incubation and in the presence of reticulocyte ribosomal wash fluid, when the rate of protein synthesis tends to level off more strongly with poly(A)-free mRNA than with native poly(A)-containing mRNA. On electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate or on cellulose acetate strips the product of the reaction has the same mobility as that of free globin.

Original languageEnglish
Pages (from-to)233-240,IN33-IN35,241-245
JournalJournal of Molecular Biology
Volume88
Issue number1
DOIs
StatePublished - 5 Sep 1974
Externally publishedYes

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