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Purification and characterization of Escherichia coli RNase I. Comparisons with RNase M
J Meador1, B Cannon, V J Cannistraro
1Department of Microbiology and Immunology, Washington University School of Medicine, St. Louis, MO 63110.
Abstract:
The endoribonuclease, RNase I, was purified from the periplasm of Escherichia coli. Based on PAGE, it has molecular mass of approximately 27 kDa with a migration rate indistinguishable from that of the recently reported RNase M from E. coli. The amino acid sequence of the two enzymes must be very similar based on two-dimensional mapping of their tryptic peptides and suggests either a post-transcriptional modification to yield different proteins from the same gene or evolution of two genes by gene duplication. However, while RNase I could degrade each of the four ribonucleotide homopolymers, only poly(U) or poly(C) were good substrates for RNase M with possibly some hydrolysis of poly(A). The reaction rate for poly(C) hydrolysis with RNase M was about ten times faster than for poly(U), while for RNase I the rates were about equal. Besides differences in specificity, RNase M was only located in the spheroplasts while RNase I found in the periplasm of growing cells. In terms of function, RNase I is known to cause degradation of rRNA during periods of stress or non-growth, whereas it has been proposed that RNase M is the endonuclease for mRNA degradation in growing cells.
Insights
Two endoribonucleases, RNase I and RNase M from Escherichia coli, show similar molecular masses but distinct substrate specificities and cellular locations. Their close relationship suggests either post-transcriptional modification or gene duplication, impacting RNA degradation in E. coli.
Area of Science:
- Molecular Biology
- Enzymology
- Bacteriology
Background:
- RNase I is a periplasmic endoribonuclease in Escherichia coli.
- RNase M is a recently identified E. coli endoribonuclease.
Purpose of the Study:
- To compare the biochemical properties and cellular localization of RNase I and RNase M.
- To investigate the relationship between RNase I and RNase M.
Main Methods:
- Purification of RNase I from E. coli periplasm.
- Polyacrylamide gel electrophoresis (PAGE) for molecular mass determination.
- Two-dimensional mapping of tryptic peptides to assess amino acid sequence similarity.
- Enzymatic assays using ribonucleotide homopolymers to determine substrate specificity and reaction rates.
- Cellular fractionation to determine enzyme localization.
Main Results:
- RNase I and RNase M exhibit indistinguishable molecular masses (~27 kDa) but possess different substrate specificities.
- RNase I degrades all four ribonucleotide homopolymers, while RNase M preferentially degrades poly(U) and poly(C).
- RNase I is localized in the periplasm, whereas RNase M is found in spheroplasts.
- Tryptic peptide mapping indicates high similarity between RNase I and RNase M, suggesting a common origin.
Conclusions:
- RNase I and RNase M are closely related enzymes, likely originating from gene duplication or post-transcriptional modification.
- Distinct substrate specificities and localizations suggest specialized roles in RNA metabolism: RNase I in stress-induced rRNA degradation and RNase M in mRNA degradation during growth.