Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli.
Crude extracts from Escherichia coli were screened for any broad-specificity endoribonuclease after the cell proteins were fractionated by size. In a mutant lacking the gene for RNase I (molecular mass, 27,156 Da), the only such activities were also in the size range of 23 to 28 kDa. Fractionation by chromatography on a strong cation-exchange resin revealed only two activities. One of them eluted at a salt concentration expected for RNase M and had the specificity of RNase M. It preferred pyrimidine-adenosine bonds, could not degrade purine homopolymers, and had a molecular mass of approximately 27 kDa (V. J. Cannistraro and D. Kennell, Eur. J. Biochem. 181:363-370, 1989). A second fraction, eluting at a higher salt concentration, was active against any phosphodiester bond but was about 100 times less active than are RNase I and RNase I* (a form of RNase I) in the wild-type cell. On the basis of sizing-gel chromatography, this enzyme had a molecular mass of approximately 24 kDa. We call it RNase R (for residual). RNase R is not an abnormal product of the mutant rna gene; a cell carrying many copies of that gene on a plasmid did not synthesize more RNase R. Our search for broad-specificity endoribonucleases was prompted by the expectation that the primary activities for mRNA degradation are expressed by a relatively small number of broad-specificity RNases. If correct, the results suggest that the endoribonucleases for this major metabolic activity reside in the 24- to 28-kDa size range. Endoribonucleases with much greater specificity must have as primary functions the processing of specific RNA molecules at a very limited number of sites as steps in their biosynthesis. In exceptional cases, these endoribonucleases inactivate a specific message that has such a site, and they can also effect total mRNA metabolism indirectly by a global disturbance of the cell physiology. It is suggested that a distinction be made between these processing and degradative activities.
Srivastava
S K
SK
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Cannistraro
V J
VJ
Kennell
D
D
D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D011131:Polyribonucleotides / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D011131:Polyribonucleotides; D012333:RNA, Messenger; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; C022538:ribonuclease M; C022543:ribonuclease T(2)