Effects of alkylation of phosphodiesters and of bases of infectivity and stability of tobacco mosaic virus RNA.

Abstract

Upon ethyl nitrosourea treatment of RNA of tobacco mosaic virus, up to four phosphodiester groups may be alkylated per molecule without chain breakage, as shown by sucrose gradient centrifugation. This indicates that ribophosphotriesters are quite stable. However, when this alkylation reaction is of longer duration and 6 to 10 triesters are formed, then an average of 1 to 2 breaks occurs and little or no intact RNA can be isolated. Methyl nitrosourea is less effective in forming triesters (about 25% of total alkyl groups compared to about 65% for ethyl nitrosourea), and a greater number of alkyl groups can, therefore, be introduced before breaks occur. Diethyl sulfate and dimethyl sulfate, which alkylate almost only the bases of nucleic acids, do not cause significant degradation of RNA of tobacco mosaic virus, even when as many as 70 alkyl groups are bound.All types of alkylation cause similar losses in viral infectivity at low levels of alkylation. Thus, an average of two chemical events leads to one lethal event, regardless of the nature of the alkylating reaction, which, for example, is with dimethyl sulfate about 65% on the N-7 guanine while with ethyl nitrosourea it is about 65% on phosphodiesters. It is thus concluded that all alkyl groups, whether on the base or on the phosphate, have the same potential to cause inactivation and that inactivation of RNA can result from phosphotriester formation per se.Alkylation of nucleic acids by ethyl nitrosourea (EtNU) in neutral aqueous solution has been shown to occur primarily on the 06 of guanine and on the oxygen of phosphodiesters (1, 2). About 65% of the total alkylation of RNA in vitro and of HeLa cell DNA in vitro and in vivo is in the form of phosphotriesters, while 8-12% is in the form of 06-ethylguanine (1, 2). EtNU is a very potent carcinogen (3, 4), and the mechanism of its biological effect has been studied by Goth and Rajewsky (5-7), Lijinsky et al. (8), and Swann and Magee (9). All agree that there is no correlation between tumor formation in rats and the amount of 7-ethylguanine found in the RNA or DNA of various tissues. Goth and Rajewsky (6,7) propose that ethylation of the 06 of guanine is the significant biological event, but they do not consider phosphate alkylation, although their data can now be interpreted as indicating about 70% phosphotriester formation (1,2).In the course of a general study of the alkylation of nucleic acids, this laboratory has now focused on the chemical and biological effects of phosphate alkylation. In this paper we present data on the stability of ribophosphotriesters in tobacco mosaic virus (TMV) RNA and the effects of triester formation on the infectivity of intact (30S) TMV RNA.It has long been believed that, in contrast to deoxyribophosphotriesters, ribophosphotriesters were extremely unstable, and as soon as a triester was formed, chain scission occurred (10-13). This was based primarily on indirect evidence, since no ribophosphotriester had been isolated or synthesized while deoxyribophosphotriesters ha...

Can Phosphate‐Branched RNA Persist under Physiological Conditions?

“…An RNA phosphotriester linkage embedded within an oligonucleotide structure can, however, be substantially more stable,5 even if the oligonucleotide does not form a welldefined structure such as a double helix (Scheme , compound 2 ) 6. In fact, such a structure, the so‐called RNA X (Scheme ), has been reported as a product of a model reaction of eukaryotic splicing.…”

Reconstitution of rods from tobacco mosaic virus protein and RNA modified with bulky carcinogens.

“…TMV, TMV RNA, and TMV A-protein were prepared by the usual procedures (4)(5)(6) aliquots containing about 30-40 jig of RNA were removed after 0.33-24 hr. These were diluted 1:20 in H20 at 0°C and were subjected to three cycles of differential centrifugation (10 min at 8,000 rpm, followed by 2 hr at 40,000 rpm).…”

Characterization of the catalytic activity of U2 and U6 snRNAs

“…For example, it has been observed that treatment of RNA with alkylating agents results in formation of molecules containing multiple phosphotriesters throughout their length. These RNAs can be subjected to shearing forces and other stresses, for example through multiple rounds of centrifugation and extended incubations at room temperature, without backbone breakage (Singer et al 1975). Therefore, the relative stability of RNA X does not preclude the presence of a phosphotriester linkage.…”

“…Phosphotriesters have long been known to occur in both DNA and RNA as a result of exposure to certain toxins and chemicals (e.g., see Bannon and Verly 1972;Singer et al 1975). Formation of a phosphotriester similar to the type proposed to exist in RNA X was observed to occur as a side reaction during chemical synthesis of DNA (Weimann and Khorana 1962).…”