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-S00 (RNase II), and ams-l (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 twoto 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.

A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature. Using lambda clones containing DNA fragments from the 23-to 24-min region on the E. coli chromosome, we have isolated a 5.8-kilobase DNA fragment which, when present in a low-copy-number plasmid, complements the conditional lethality and increased mRNA stability associated with the ams-1 mutation. The approximate initiation site and the direction of transcription of the ams gene were determined from the size of truncated polypeptides produced by TnlOOO insertions and Bal 31 deletions. Overexpression of the ams locus by using a T7 RNA polymerase-promoter system permitted the identification of an ams-encoded polypeptide of 110 kilodaltons.In recent years, it has become apparent that Escherichia coli contains a large set of distinct RNases that are involved in the various degradative and processing reactions of RNA metabolism (5,14,15). Whereas many RNases may participate in mRNA decay in vivo, most have yet to be positively identified (see reference 14 for a review). Donovan and Kushner (6) have shown that strains lacking both RNase II and polynucleotide phosphorylase, two enzymes that degrade single-stranded RNA in the 3'-to-5' direction, are inviable.The product of the gene designated ams (altered message stability) is also involved in decay of mRNA (2,17,23). Strains carrying the temperature-sensitive ams-J mutation have a longer chemical half-life of total pulse-labeled RNA at the nonpermissive temperature (17). More recently, Arraiano et al. (2) have shown that the absence of the Ams protein leads to increased chemical half-lives of specific mRNAs and significant stabilization of mRNA breakdown products in ams-J pnp-7 rnb-S00 triple mutants. They suggested that the Ams protein was either an RNase or a positive regulator of other RNases (2).Genetic analysis has shown that the ams gene maps at 23 min (close to the pyrC gene) on the E. coli chromosome (24), distinct from the structural genes for RNase II (rnb), RNase III (rnc), and polynucleotide phosphorylase (pnp). The rne gene maps approximately at 24 min (1), but RNase E is a very specific endoribonuclease involved in the initial 3' and 5' processing of 5S rRNA precursors (21) as well as RNA1 of ColEl (34) and the T4. gene 32 mRNA (22).Chanda et al. (4) cloned an 8.1-kilobase (kb) EcoRI fragment from a wild-type E. coli strain that apparently complemented the ams-l mutation. However, their published nucleotide sequence (4) is in fact identical to that of a segment of the groEL gene (12). The groEL gene maps at 94 min on the E. coli chromosome (9) and has been cloned on an 8.1-kb EcoRI fragment (7). * Corresponding author. In this study, we used lambda clones from the E. coli genomic library of Kohara et al. (16), containing DNA fragments that map close to 23 to 24 min, to isolate the ams gene. ams-J strains carrying the wild-type ams gene on a low-copy-number plasmid grew at 44°C and had norm...

The degradation of individual mRNAs in Escherichia coli has been studied through the use of a multiple mutant carrying the pnp-7 (polynucleotide phosphorylase), rnb-500 (RNase II), and rne-1 (RNase E) alleles. In this triple mutant, discrete mRNA breakdown products are stabilized in vivo at the nonpermissive temperature (Arraiano, C. M., S. D. Yancey, and S. R. Kushner, J. Bacteriol. 170:4625-4633, 1988). In the case of thioredoxin (trxA) mRNA decay, degradation fragments accumulated at early times after a shift to the nonpermissive temperature. Using Northern (RNA) blots, S1 nuclease analysis, and primer extensions, we identified a series of specific endonucleolytic cleavage sites that occur throughout the transcript in both the triple mutant and a wild-type control. The implications of the complex decay patterns observed are discussed.

No abstract

We have identified a gene in Escherichia coli that is required for both the normal decay of mRNA and RNA synthesis. Originally designated mrsC (mRNA stability), themrsC505 mutation described here is, in fact, an allele of the hflB/ftsH locus (R.-F. Wang et al., J. Bacteriol. 180:1929–1938, 1998). Strains carrying the thermosensitivemrsC505 allele stopped growing soon after the temperature was shifted to 44°C but remained viable for several hours. Net RNA synthesis stopped within 20 min after the shift, while DNA and protein synthesis continued for over 60 min. At 44°C, the half-life of total pulse-labeled RNA rose from 2.9 min in a wild-type strain to 5.9 min in the mrsC505 single mutant. In an rne-1 mrsC505double mutant, the average half-life was 19.8 min. InactivatingmrsC significantly increased the half-lives of thetrxA, cat, secG, andkan mRNAs, particularly in an mrsC505 pnp-7 rnb-500 rne-1 multiple mutant. In addition, Northern analysis showed dramatic stabilizations of full-length mRNAs in a variety ofmrsC505 multiple mutants at 44°C. These results suggest that MrsC, directly or indirectly, controls endonucleolytic processing of mRNAs that may be independent of the RNase E-PNPase-RhlB multiprotein complex.