W.Min Jou

Analysis of 32P-labeled bacteriophage MS2 RNA by a mini-fingerprinting procedure.

Abstract A modified procedure is described for the analysis of enzymatic digests of 32P-labeled RNA on thin layer plates. The first dimension consists of conventional electrophoresis at pH 3.5 on cellulose acetate and the second dimension is according to chain length by chromatography with an appropriate homomixture. Ribonuclease T1-digests are run on polyethyleneimine thin layer plates and pancreatic ribonuclease digests on diethylaminoethyl-plates. The procedure combines high sensitivity (due to the small pin-size spots) with excellent resolution. Moreover, the composition of nearly all spots can be deduced from their position. As an illustration, the analyses of enzymatic digests of MS2 RNA (chain length 3569) are presented.

Recent progress in the sequence determination of bacteriophage MS2 RNA

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.

Studies on the bacteriophage MS2. Nucleotide fragments from the coat protein cistron

Partial hydrolysis with ribonuclease T 1 has already been used for the isolation of fragments derived from different regions of R17 [1-4] or of MS2 RNA [5,6]. In this paper, we describe the isolation and purificat.ion of several RNA fragments coding for different parts of the amino acid sequence of the coat protein:. The structural data obtained upon sequence analysis of the purified fragments (not presented here) are compared with the nucleotide sequences from the corresponding parts of R17 RNA [1-3]. The polynucleotides studied have a hairpin-like structure with considerable base pairing, as was found for all the RNA segments isolated from partial digests until now [1-6]. The results further support the concept that the evolutionary process has taken advantage of the degeneracy of the genetic code in order to maximize secondary structure in this type of messenger RNA, as proposed originally in reference [1].

Studies on the bacteriophage MS2. Some nucleotide sequences from the RNA-polymerase gene☆

The knowledge of the amino acid sequence from the coat protein of the RNA bacteriophages f2, R 17 and MS2 [ l-31 allowed the identification of several RNA fragments as being derived from this gene. They were all obtained by partial ribonuclease hydrolysis of R17 RNA [4-61, f2 RNA [7] or MS2 RNA [8, 91. For MS2, these sequences could be further extended, and the complete sequence of the coat gene was established [lo]. Furthermore, this se- quence was followed by a 36 nucleotides long inter- cistronic region and by the ribosomal binding site of the next gene, the RNA-polymerase. As the amino acid sequences for the two other gene prod- ucts, the A-protein and the viral RNA-polymerase, are not known, no such simple test exists to identify polynucleotides corresponding to the latter two genes. In this communication we present data on three nucleotide fragments derived from the polymerase gene. This conclusion is based on direct overlaps with known sequences [ lo] , on the established gene order for these phages [ 11, 121, and on the mapping of a series of polypurine tracts in two fragments of the RNA molecule [ 131 . 2. Methods All RNA fragments described in this study were *

Studies on the bacteriophage MS2 nucleotide sequence of a 3′-terminal fragment (n = 104)

Previous studies from this laboratory have established the 3 -terminal nucleotide sequence of Bacteriophage MS2 RNA up to position w-16 [ 1,2] . For the closely related bacteriophage R17, the terminal sequence up to position o-51 has been reported [3]. Using another approach, viz. in vitro synthesis of a complementary strand, Weissmann and collaborators [4] were able to deduce the 3’terminal sequence of the distantly related Bacteriophage @ up to position w-52. By hydrolysis of 32P-labelled MS2 RNA with ribonuclease T1 under milder conditions, we were able to obtain a set of larger fragments, all derived from the 3’-end. A partial sequence was recently presented [5], while we are now able to report the complete sequence of a 104 nucleotide long fragment.

The 3'-terminal nucleotide sequence (n = 16) of bacteriophage MS2 RNA.

1. Intoduction The 3’-terminal nucleotide sequence of viral RNA is of particular interest. Indeed, as replication proceeds in an antiparallel direction, it is likely that the former region contains the recognition signal required for specific interaction with the viral RNA polymerase. Alternatively, or in addition, one may hope to find a termination signal for protein synthesis for the cistron nearest the 3’end of the molecule, in casu RNA poly- merase [ 1,2]. De Wachter and Fiers [3] have previously reported that MS2 RNA ends in (G) U-U-A-C-C-A-C-C- C-A, and the same sequence was confirmed for the bacteriophage f2 RNA [4,5] and R17 RNA [5], which are serologically closely related to MS2 RNA. Bac- teriophage QP RNA has a very different 3’-terminal sequence (5,6]. All four sequences, however, share

Sequence determination of Gp-rich oligonucleotides by means of the kethoxal modification

The first steps in RNA sequence analysis are usually degradation of the RNA with the base specific rihonuclease T1 which hydrolyzes only Gp-N diester bonds, and with pancreatic ribonuclease which cleaves after pyrimidine nucleotides. The sets of T1 and P-oligonucleotides are separated and the sequence of each product is determined. For this purpose, the oligonucleotide is degraded with the complementary enzyme (pancreatic ribonuclease for T1 -oligonucleotides and vice versa). Further characterization was initially almost totally dependent on partial degradation with venom and spleen exonuclease [l-3]. This method poses several problems of its own: partial digestion conditions are critical, the radioactivity is distributed over several intermediates, products are often also produced by nonspecific endonucleolytic cleavage. Now, RNA sequencing methodology has become much more versatile, especially for the structure determination of T1-oligonucleotides. UZ ribonuclease, a purine-specific enzyme, will create (Pyp), Pup sequences [4]. A ribonuclease from Physwum polycephalum gives rise to (Cp), Np pieces [5]. Although they lack a precise base specificity, spleen acid ribonuclease ([6], our unpublished results) and silkworm endonuclease [7,8] also give useful products. Uracil and guanine residues can be modified by a