028020033.Pdf by Guest on 30 September 2021 Nuclein Contained Large Amounts of Phosphorus and No Sulphur, Characteristics That Differentiated It from Proteins1

special feature Biochemical Journal classic papers

The messenger: the structure of RNA

In the early part of the 20th Century, the nature of nucleic acid and what its role was within the by Richard Reece cell were a bit of a mystery. DNA itself was first isolated as far back as 1869 by the Swiss chemist (University of Manchester, UK) Johann Friedrich Miescher. He separated nuclei from the cytoplasm of cells and then isolated an acidic substance from these nuclei that he called nuclein1. Chemical tests by Miescher showed that Downloaded from http://portlandpress.com/biochemist/article-pdf/28/2/33/6410/bio028020033.pdf by guest on 30 September 2021 nuclein contained large amounts of phosphorus and no sulphur, characteristics that differentiated it from proteins1. The first step in determining the structure of nucleic acid (either DNA or RNA) would be to identify its precise composition. RNA was considered a more approachable target for composition analysis because the simple treatment of RNA with hydroxide rapidly and completely hydrolyses the molecule to its individual component nucleotides. DNA, on the other hand, is resistant to such treatment.

In 1909, Phoebus Levene observed that yeast the influence of a high voltage, the ribonucleotides ribonucleic acid was composed of approximately and oligoribonucleotides migrate across the moist equal amounts of the two purines (adenine and paper with mobilities that decrease with increasing guanine) and the two pyrimidines (cytosine and size. The methods they described represented the uracil)2. Based on this, and by determining the type first analysis of individual components of nucleic of linkages that joined the nucleotides together, acid. These papers not only paved the way for Levene and Henry Simms proposed a tetranu- the more precise analysis of RNA and DNA mol- cleotide structure to explain the chemical arrange- ecules, but also provide intriguing insights into ment of nucleotides within nucleic acids3. In this theory, they proposed a very simple four- 5′ RNA 5′ RNA Base Base nucleotide unit that was repeated many times to O O form long nucleic acid molecules. The simplistic H H H H nature of the tetranucleotide structure led to the H H widespread acceptance that nucleic acids would be O O :B O O H B+ incapable of providing the chemical variation H P - O P O - expected of the genetic material. We would have to O O + wait until the seminal experiments of Oswald O + Avery, Colin MacLeod and Maclyn McCarty were A H RNAA 3' A: H O published in 1944 to discover otherwise4. RNAA 3' In the late 1940s and early 1950s, Roy Markham and John D. Smith, working at the Agricultural 5′ RNA 5′ RNA Base Base Research Council’s Virus Research Unit at the O O Molteno Institute, in Cambridge, published a raft H H H H of papers — predominately in the Biochemical H H

Journal — in which they described techniques for O O H B+ OH O :B P H the analysis of nucleotides formed by the hydro- H - - O P O lysis of RNA. In these articles, they described O O - how to separate, by paper electrophoresis, single O A: HO A+ H nucleotides and small oligonucleotides obtained H from the hydrolysis of RNA from the genomes of plant and animal viruses. They showed that under Figure 1. The transphosphorylation (top) and hydrolysis (bottom) reactions catalysed by ribonucleases

Figure 2. (Fig1 from Markham and Lord Alexander Todd6, was correct. The & Smith5) showing the hydrolysis reaction proceeds in two steps: ‘museum jar’ set up. transphosphorylation of RNA to form a 2′,3′- cyclic phosphodiester (N>p) intermediate and hydrolysis of this cyclic intermediate to form a 3′- phosphomonoester (Np) (Figure 1). The second of these papers investigated the smaller products of ribonuclease digestion7. Several dinucleotides were identified and it was discovered that the cyclic forms of the dinucleotides were formed first and that the subsequent hydrolysis was slow.

Subsequent work from Markham’s laboratory Downloaded from http://portlandpress.com/biochemist/article-pdf/28/2/33/6410/bio028020033.pdf by guest on 30 September 2021 showed that the natural configuration of the purine nucleotides in RNA was 3′–5′ rather than the alternative suggestion of 2′–5′8. Further evi- dence for this type of link was obtained from subsequent studies of the action of nucleases on the nature of the chemical bonds contained mononucleotide esters carried out by others9. within nucleic acids themselves. Reading through the Markham and Smith The first of this pair of papers that appeared in papers is an absolute joy. I came away with an the Biochemical Journal (‘The structure of ribonu- overwhelming feeling of pioneering work taking cleic acid. I. Cyclic nucleotides produced by ribonu- place. The deductions of the linkages that must clease and by alkaline hydrolysis’) describes the occur between nucleotides within the RNA chain methods employed for the analysis of the products based on their breakdown products was certainly of the digestion of RNA both with ribonuclease an important advance in understanding a long, and through hydrolysis with alkali5. The cyclic repetitive molecule. Others who subsequently nucleotides (2′,3′-monohydrogen phosphate esters solved the three-dimensional structure of nucleic of nucleosides) produced by this treatment and acids may have attracted bigger accolades, but, analysed by ‘paper strip electrophoresis’ proved without the pioneering work to determine the that the previously published chemical scheme for precise chemical linkages, those structures may the alkali hydrolysis of RNA, by Daniel Brown not have been calculated until much later.

References

1. Miescher, F. (1871) Über die chemische Zusammensetzung der Eiterzellen. Hoppe- 9. Brown, D.M., Heppel, L.A. and Hilmoe, R.D. (1954) Nucleotides. Part XXIV. The action of Seyler’s medicinisch-chemische Untersuchungen 4, 441–460 some nucleases on simple esters of monoribonucleotides.J.Chem. Soc., 40–46 2. Levene, P.A. (1909) Über die Hefenucleinsäure.Biochem. Z. 17,120–131 10. Zamenhof, S., Brawerman, G. and Chargaff, E. (1952) On the desoxypentose nucleic 3. Levene, P.A. and Simms, H.S. (1926) Nucleic acid structure as determined by electro- acids from several microorganisms. Biochim. Biophys. Acta 9, 402–405 metric titration data.J.Biol. Chem. 70,327–341 11. Chargaff, E., Lipshitz, R. and Green, C. (1952) Composition of the desoxypentose nucleic 4. Avery, O.T., MacLeod, C.M. and McCarty, M. (1944) Studies on the chemical nature of the acids of four genera of sea-urchin.J.Biol. Chem. 195,155–160 substance inducing transformation of pneumococcal types.J.Exp. Med. 79, 137–158 12. Wilkins, M.H.F., Stokes, A.R. and Wilson, H.R. (1953) Molecular structure of deoxypentose 5. Markham, R. and Smith, J.D. (1952) The structure of ribonucleic acids. 1. Cyclic nucleotides nucleic acids. Nature (London) 171, 738–740 produced by ribonuclease and by alkaline hydrolysis.Biochem. J. 52, 552–557 13. Franklin, R.E. and Gosling, R.G. (1953) Molecular configuration in sodium thymo- 6. Brown, D.M. and Todd, A.R. (1952) Nucleotides. Part X. Some observations on the nucleate. Nature (London) 171, 740–741 structure and chemical behaviour of the nucleic acids.J.Chem. Soc., 52–58 14. Watson, J.D. and Crick, F.C.H. (1953) Molecular structure of nucleic acids: a structure 7. Markham, R. and Smith, J.D. (1952) The structure of ribonucleic acids. 2. The smaller for deoxyribose nucleic acid. Nature (London) 171, 737–738 products of ribonuclease digestion.Biochem. J. 52, 558–565 15. Sanger, F. and Coulson, A.R. (1975) A rapid method for determining sequences in DNA 8. Heppel, L.A., Markham, R. and Hilmoe, R.J. (1953) Natural configuration of the purine by primed synthesis with DNA polymerase.J.Mol. Biol. 94, 441–448 nucleotides in ribonucleic acids. Nature (London) 171, 1151–1152

The methods initiated by Markham and Smith On a personal note, reading through these were widely and rapidly disseminated. Most famously, classic papers makes me realize just how lucky perhaps, the paper chromatography method was we are to be able to practice science today. The employed by Erwin Chargaff to perform experiments description of a how to build a 1000 V power to address the chemical composition of DNA at supply for the electrophoresis experiments5 seems Columbia University, New York. He hydrolysed alien to those of us who have gone through our entire DNA into its constituent nucleotides by treatment scientific careers by being able to dip into catalogues with strong acid, and then separated the nucleotides to buy the ‘standard’ molecular biology equipment using similar methods to those described by Markham off-the-shelf. In addition, the electrophoresis appara- and Smith. As we all know, Chargaff’s experiments tus itself — being constructed of three museum jars showed that the relative ratios of the four bases in with paper wicks sticking out of them — has a decid-

DNA were not equal, but were also not random. The edly Heath Robinson feel to it and the chromatograms Downloaded from http://portlandpress.com/biochemist/article-pdf/28/2/33/6410/bio028020033.pdf by guest on 30 September 2021 number of adenine (A) residues in all DNA samples produced have a distinctly smeary nature to them7. was equal to the number of thymine (T) residues, However, as with many pioneering experiments, the while the number of guanine (G) residues equalled interpretation of what may not necessarily be the the number of cytosine (C) residues10,11. This finding, clearest data can lead to important breakthroughs. combined with the X-ray diffraction data from Richard Reece is at the University of 12 13 Maurice Wilkins and Rosalind Frankin , formed the Manchester.After undergraduate studies at the University of Leeds, he obtained basis of Watson and Crick’s 1953 iconic double-helical his PhD from the University of Leicester. He was a postdoctoral fellow at Harvard 14 DNA structure model . Much later, the procedures University. His research interests focus on the regulation of transcription in developed within the pages of Markham and Smith’s yeast by small molecule metabolites using biochemical and structural 5,7 papers were modified into the separation of nucleic approaches. He has been the General Editor of The Biochemist since 2002. acid fragments by gel electrophoresis for the analysis [email protected] and sequencing of both RNA and DNA15.