Photochemical cleavage of phosphodiester bonds in oligoribonucleotides.

The release of inorganic phosphate from a variety of mononucleotides and the generation of new phosphomonoester end groups as a result of chain cleavage in a number of oligoribonucleotides have been studied quantitatively as a function of irradiation with 254-nm light. The reaction cross sections for adenosine 5'-phosphate, guanosine 2'(3')-phosphate, cytidine 5'-phosphate, cytidine 3'-phosphate, cytidine 2'(3')-phosphate, uridine 5'-phosphate, uridine 2'(3')-phosphate, dihydrouridine 5'-phosphate, and ribose 5-phosphate are close to 2 X 10(-7) m2/J. The value for UpU is similar. The reaction cross sections, sigma, for (Ap)n where n = 3-10 as well as for the oligonucleotides ApUpGp, m1ApCpUpCpGp, CpCpCpCpCpGp, and DpDpDpApApGp increased linearly as a function of the number of phosphodiester bonds and gave values close to 6.4 X 10(-7) m2/J per bond. The cross sections for (Up)n were also about 6.4 X 10(-7) m2/J per bond for n = 2-5 and then, unexpectedly, increased rapidly for n = 6-10. By analogy to the carefully studied release of phosphate from ethyl phosphate and several sugar phosphates by 254-nm light [Halmann, M., & Platzner, I. (1965) J. Chem. Soc., 5380-5385], we conclude that the photolysis reactions we have observed are induced by absorption of photons by the sugar phosphate groups rather than the purine or pyrimidine rings. It follows that the quantum yields for chain cleavage of both RNA and DNA have been seriously underestimated since these calculations were based on the assumption that the observed photochemistry is due to absorption of photons by the purine and pyrimidine rings, and the absorption cross section of these rings is roughly 10 000 times greater than that of the sugar phosphate group itself.

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.

Functional inactivation and appearance of breaks in RNA chains caused by gamma irradiation of Escherichia coli ribosomes.

70S ribosomes and 30S ribosomal subunits from Escherichia coli MRE 600 were exposed to gamma irradiation at -80szC. Exponential decline of activity with dose was observed when the ability of ribosomes to support the synthesis of polyphenylalanine was assayed. Irradiated ribosomes showed also an increased thermal lability. D(37) values of 2.2 MR and 4.8 MR, corresponding to radiation-sensitive molecular weights of 3.1 x 10(5) and 1.4 x 10(5), were determined for inactivation of 70S ribosomes and 30S subunits, respectively. Zone sedimentation analysis of RNA isolated from irradiated bacteria or 30S ribosomal subunits showed that at average, one chain scission occurs per four hits into ribosomal RNA. From these results it was concluded that the integrity of only a part of ribosomal proteins (the sum of their molecular weights not exceeding 1.4 x 10(5)) could be essential for the function of the 30S subunit in the polymerization of phenylalanine. This amount is smaller if the breaks in the RNA chain inactivate the ribosome.