The 5′ ends of RNA oligonucleotides in Escherichia coli and mRNA degradation

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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. Copyright © 1993, Wiley Blackwell. All rights reserved

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CANNISTRARO, V. J., & KENNELL, D. (1993). The 5′ ends of RNA oligonucleotides in Escherichia coli and mRNA degradation. European Journal of Biochemistry, 213(1), 285–293. https://doi.org/10.1111/j.1432-1033.1993.tb17761.x

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