Studies on the bacteriophage MS2: IV. The 3′-OH terminal undecanucleotide sequence of the viral RNA chain

Abstract

The 3′-OH terminus of bacteriophage MS2 RNA was selectively labelled with 3H. This was achieved by oxidation of the free 2′, 3′-diol group with sodium periodate to a dialdehyde, and reduction of the latter with tritiated sodium borohydride. Treatment of this RNA with alkali and separation of the hydrolysis products confirmed that only the terminal adenosine residue had been labelled. For the determination of the 3′-OH terminal sequence, RNA randomly labelled with 32P was admixed with terminally tritiated RNA, or, alternatively, the 32P-labelled RNA itself was terminally tritiated. Hydrolysis with ribonuclease T 1 resulted in a mixture of oligonucleotides terminating in guanylic acid and a decanucleoside nonaphosphate containing the 3H label. This terminal oligonucleotide was isolated by ion-exchange chromatography on DEAE-Sephadex and DEAE-cellulose columns at different pH values and in the presence of 7 m-urea. The 3H label served to localize the terminal sequence and to estimate the purity. Alkaline hydrolysis of the isolated product indicated the composition (2 Ap, 2 Up, 5 Cp)A while pancreatic ribonuclease hydrolysis showed that each adenylic acid residue was followed by cytidylic acid. The complete sequence was established by partial hydrolysis with snake venom phosphodiesterase. The stepwise degradation products so obtained were labelled with 3H in the terminal nucleoside by the same procedure used for the entire RNA molecule, and then separated according to chain length. In this way, the nucleotide composition of each product could be determined on the basis of 32P activity, while its terminal nucleoside was determined on the basis of 3H activity. Both sets of data confirmed each other. The results, together with the known specificity of the ribonuclease T 1, which had released the sequence, establish that MS2 RNA ends in …GpUpUpApCpCpApCpCpCpA. It is suggested that the termination signal for the translation into polypeptides is located at the left of this sequence and that the latter may be important for the initiation of replication.