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

Abstract

Poly(A)-containing messenger RNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has two oxidizable end groups per molecule of mol. wt. 220000. When the mRNA is subjected to stepwise degradation by beta-elimination, only one oxidizable end-group is found. This indicates that one 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 ribonuclease the isoniazid-labelled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3′ end plus fragments that are not found after stepwise degradation. These fragments have a charge of -6 and -8 from pancreatic ribonuclease or -7 from ribonuclease T1 digestion. These charges are changed to -3.4 and -4.1 after pancreatic ribonuclease, ribonuclease T2 and alkaline phosphatase digestion. methyl-3H-labelled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labelled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3 H-labelled nucleotides with the electrophoretic mobility of pm7G and pAm. It is concluded that globin mRNA has the 5′ sequences m7G(5′)ppp′AmpYpGp ... and m7G(5′)pppAmpApGpYp.