Ribonuclease v of Escherichia Coli, I. Dependence on Ribosomes and Translocation

Abstract

Abstract.-A new RNase activity, tentatively named RNase V, was found in cell-free extracts of E. coli. This activity requires ribosomes, G and T factors, tRNA, K+ or NH4+, Mg2+, GTP, and a sulfhydryl compound to degrade poly U, poly A, T4 phage mRNA, or E. coli mRNA. RNase V is specific for mRNA; it does not attack ribosomal RNA. It is inhibited by antibiotics that decrease breakdown of mRNA in vivo, such as chloramphenicol and streptomycin, and by such agents as 5'-#, 'y-methylene-guanosine triphosphate, and fusidic acid, which inhibit ribosome-dependent GTPase and translocation of ribosomes along mRNA. The evidence suggests that RNase V is either an integral part of the ribosome or is tightly associated with it, and that it selectively degrades mRNA in intact cells.The instability of messenger RNA is central to many questions in biology.-What determines how long a molecule of mRNA persists, and how does it break down? The mechanisms have remained unknown, though recent work has suggested that at least in some cases, both functional inactivation and degradation of mRNA proceed from the 5' to the 3' end of the molecule.1' 2 A clue to the mechanism and regulation of mRNA breakdown has been obtained from studies of rnRNA metabolism in antibiotic-treated cultures of E. coli.3-5 When protein synthesis is blocked by chloramphenicol or streptomycin, mIRNA synthesis continues, but mRNA breakdown is inhibited. This implies that mRNA breakdown is linked to some feature of protein synthesis. Therefore, we have looked for an RNase activity linked to the protein synthetic machinery. Here we report the finding of such an activity, tentatively designated RNase V, and describe some of its properties. It is specific for mRNA and requires ribosomes, tRNA, T factor, and G factor.6"9 RNase V is inhibited by chloramphenicol and streptomycin, which block mRNA breakdown in vivo, [3][4][5] and by fusidic acid4-5 10-13 and 5'-3,By-methylene-guanosine triphosphate"4 which block translocation. These properties suggest that RNase V is an mRNase in the cell.Materials and Methods.-Strains: The strains used were all derived from E. coli K12.N11 (met-his-ura-RNase I10-) was isolated from strain D10 (met-RNase Ilo-

Electronmicroscopy of Genetic Activity

“…The direction of exonucleolytic degradation in such a scheme would have to be from the 5' to the 3' end of the RNA, in agreement with several biochemical studies (19)(20)(21) . Since regular short-to-long polyribosome gradients usually are seen, mRNA degradation is likely not totally random, contrary to the suggestion by Baker & Yanofsky (18).…”

Regulation of Ribonucleic Acid Synthesis in Escherichia coli During Diauxie Lag: Accumulation of Heterogeneous Ribonucleic Acid

“…3). In terms of the frequently expressed notion of an obligate coupling between the breakdown of an mRNA and its translation (5,9), it is possible that the decreased rate of mRNA turnover during diauxie lag is the result of a generally decreased rate of protein synthesis.…”

Messenger Ribonucleic Acid Synthesis and Degradation in Escherichia coli During Inhibition of Translation

“…These are based on the 'Present address: Department of Bacteriology, University of Bristol, Bristol, England. findings that (i) degradation of mRNA occurs in the same direction (5' to 3') as its synthesis and translation (23, 24); (ii) inhibition of protein synthesis by chloramphenicol or streptomycin inhibits mRNA breakdown (18,20); and (iii) a ribosome-bound 5' exonucleolytic activity (RNase V) was reported (15) which is at least partially dependent upon active translation. Though the existence of RNase V has been questioned (1,9), the notion of exonucleases degrading mRNA behind translating ribosomes is widely accepted.…”