Stabilization of discrete mRNA breakdown products in ams pnp rnb multiple mutants of Escherichia coli K-12.
The degradation of mRNA in Escherichia coli is thought to occur through a series of endonucleolytic and exonucleolytic steps. By constructing a series of multiple mutants containing the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and ams-1 (altered message stability) alleles, it was possible to study general mRNA turnover as well as the degradation of specific mRNAs. Of most interest was the ams-1 pnp-7 rnb-500 triple mutant in which the half-life of total pulse-labeled RNA increased three- to fourfold at the nonpermissive temperature. RNA-DNA hybridization analysis of several specific mRNAs such as trxA (thioredoxin), ssb (single-stranded-DNA-binding protein), uvrD (DNA helicase II), cat (chloramphenicol acetyltransferase), nusA (N utilization substance), and pnp (polynucleotide phosphorylase) demonstrated two- to fourfold increases in their chemical half-lives. A new method for high-resolution Northern (RNA) analysis showed that the trxA and cat mRNAs are degraded into discrete fragments which are significantly stabilized only in the triple mutant. A model for mRNA turnover is discussed.
Arraiano
C M
CM
Department of Genetics, University of Georgia, Athens 30602.
Yancey
S D
SD
Kushner
S R
SR
D015152:Blotting, Northern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D005095:Exoribonucleases / Q000502:physiology; D005798:Genes, Bacterial; D009154:Mutation; D011117:Polyribonucleotide Nucleotidyltransferase / Q000502:physiology; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*
D012329:RNA, Bacterial; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase; D011117:Polyribonucleotide Nucleotidyltransferase; D005095:Exoribonucleases; C022535:exoribonuclease II