title: The 5' ends of RNA oligonucleotides in Escherichia coli and mRNA degradation.
abstract: The 5' ends of RNA oligonucleotides in Escherichia coli were identified to assess the contributions of specific endoribonucleases to the cleaving of bulk mRNA. About 60% of the total 5' ends have a 5' OH, and 40% a phosphate. Of those oligonucleotides with a 5'-OH end, 55% of the larger-sized molecules started with 5'-OH-A. With decreasing size there was a progressive decrease in its relative abundance, reaching 33% for the mononucleotide pool, close to its content in E. coli mRNA. In a mutant lacking RNase I* (a form of RNase I), the fraction starting with 5'-OH-A was even higher; 65-70% for oligonucleotides of any size, as well as the mononucleotides, whereas only 3-5% started with 5'-OH-U. Oligonucleotides with a 5'-P end were analyzed after pulse-labeling growing cells with 32Pi. Virtually all of them had a 5'-ppp-purine end which would result from transcription initiations, and there were four-times more G than A starts. The fraction of 5' ends with a monophosphate (5'-pN) was too low to measure. The known degradative enzymes of E. coli (RNases I, I*, M and R) release a 5'-OH oligonucleotide upon cleavage, whereas known processing endoribonucleases, e.g. RNases E, H, P and III, generate 5'-P oligonucleotides. Among these enzymes, RNase M is the only one known to enrich for 5'-OH-A ends, since its preference is for pyrimidine-A bonds [Cannistraro, V. J. & Kennell, D. (1989) Eur. J. Biochem. 181, 363-370]. It also gives a very low level of 5'-OH-U ends. These results are consistent with generalizations derived from our previous studies [Cannistraro, V. J., Subbaro, M. N. & Kennell, D. (1986) J. Mol. Biol. 192, 257-274] and suggest that RNase M is a primary endoribonuclease for mRNA degradation in E. coli. The results also indicate that RNase I* contributes a smaller fraction of cleavages to larger RNA oligonucleotides and accounts for most of the degradation of the very small oligonucleotides and almost all degradation of dinucleotide to mononucleotide.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110.
authors.forename: V J
authors.initials: VJ
authors.lastname: Cannistraro
authors.forename: D
authors.initials: D
authors.lastname: Kennell
mesh_terms: D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005095:Exoribonucleases; D006868:Hydrolysis; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D009841:Oligonucleotides; D012329:RNA, Bacterial; D012333:RNA, Messenger; D004722:Endoribonucleases; D005095:Exoribonucleases; C022538:ribonuclease M; C080868:ribonuclease R; D012259:Ribonuclease, Pancreatic