The nature of the 5'-linked 5' nucleotide sequence at the 5' end of rabbit globin messenger ribonucleic acid

Abstract

Poly(A)-containing mRNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has 2 oxidizable end groups per molecule of MW 220,000. When the mRNA is subjected to stepwise degradation by .beta.-elimination, only 1 oxidizable end-group is found. This indicates that 1 of the 2'',3'' hydroxyl end-groups is linked through the normal 3''-5'' phosphodiester bond, but that the other is linked in such a way that after stepwise degradation no new 2'',3'' hydroxyl group is revealed. This structure could be a 5''-linked 5''-phospho di- or tri-ester. On digestion with RNase the isoniazid-labeled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3'' end plus fragements that are not found after stepwise degradation. These fragments have a charge of -6 and -8 from pancreatic RNase or -7 from RNase T1 digestion. These charges are changed to -3.4 and -4.1 after pancreatic RNase, RNase T2 and alkaline phosphatase digestion. Methyl-3H-labeled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labeled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3H-labeled nucleotides with the electrophoretic mobility of pm7G and pAm. Globin mRNA probably has the 5'' sequences m7G(5'')ppp''AmpYpGp... and m7G(5'')pppAmpApGpYp..