Recent progress in the sequence determination of bacteriophage MS2 RNA

Abstract

Summary By hydrolysis with specific ribonucleases we have previously established the terminal sequences of MS-2 RNA as pppG—G—G—U........G—U—U—A—C—C—A—C—C—C—A 3′oh . All sequences of the general structure (Purine nucleotide) n Pyrimidine nucleotide, released by pancreatic ribonuclease hydrolysis and with n ≥ 6 (23 in total) were also sequenced. The longer ones could be assigned either to the right one third of the left two thirds of the entire chain. More recent studies were carried out by partial enzymatic hydrolysis, which allowed the isolation of fragments with chain length 30 to 250. Among these, a 5′-terminal fragment containing 125 nucleotides was identified and completely sequenced. It follows from these results that initiation for protein synthesis at the first cistron, which codes for the A-protein, starts at position 130. The preceding stretch remains untranslated and functions perhaps as a recognition mechanism in replication. The 3′-terminal was isolated as another fragment, which was sequenced up to position ω-70. It does not seem to contain the termination signal for the RNA-polymerase cistron, and suggests that also here an extended, untranslated sequence exists. Five hairpin-like structures, derived from the coat protein cistron, have been isolated and characterized. They can be unambiguously identified, as the sequence of the 129 amino acids of the coat polypeptide is known. These hairpins were subsequently found in still larger fragments. At present 75 p. cent of the nucleotide sequence of this cistron has been established, and only two gaps remain unsolved. All these sequences have a high degree of secondary structure. In the case of the coat cistron, it is clear that in many cases third letters of degenerate codons are chosen on the basis of their ability to participate in base pairing. As expected, the genetic code dictionary, which can now be directly derived from a natural messenger, is in full agreement with the generally accepted code, largely derived from in vitro studies. 35 of the 61 no-nonsense code words have so far been assigned. It is of interest that some degenerate code words seem to be avoided. Another fragment, presumably derived from the polymerase cistron, has also been sequenced. It consists of two polynucleotides, 84 and 34 nucleotides in length respectively. It is of interest, because the outline of the secondary structure seems to be more complex than a simple hairpin. Six more codons, so far not found in the coat cistron, can now be assigned. Some of these, like ACA for threonine, CAA for glutamine and AGU for serine may perhaps play a role in a modulation type control mechanism. Other codons, like AUA for isoleucine and UAU for tyrosine are perhaps not used in Escherichia coli at all.