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. Copyright © 1993, Wiley Blackwell. All rights reserved