Identification of endonucleolytic cleavage sites involved in decay of Escherichia coli trxA mRNA

Arraiano, Cecília M.; Yancey, S D; Kushner, Sidney R.

doi:10.1128/jb.175.4.1043-1052.1993

Cited by 35 publications

(37 citation statements)

References 46 publications

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“…The in vivo cleavage specificity in E. coli is for Pyd-N bonds, with a preference for Pyd-Ade, not for a UUAU sequence, which has been an incorrect interpretation of the data (7,8).…”

Section: Rnase Mmentioning

confidence: 99%

“…A sampling of RNA with reported RNase E sites shows only one or a small number of such sites on a given RNA. For example, besides the large 9S RNA with two sites and RNA I with one, the mRNAs for S20 (86), for rpsO (52,53), for trxA (8), for the rpsU-dnaG-rpoD operon (140), for the pap operon (105), and for phage sites (for T4 [42] and polycistronic f1 [71]) generally have only one RNase E site within 200 or more nucleotides. They cannot account for the many cleavages along the entire mRNA described above unless a great many RNase E sites on these messages have not been detected.…”

Section: What Is the Rnase E Recognition Site?mentioning

confidence: 99%

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Processing Endoribonucleases and mRNA Degradation in Bacteria

Kennell

2002

J Bacteriol

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Forty years have passed since the dramatic identification of mRNA, the unstable carrier of genetic information from DNA to protein (15,48,56). During the last decade, there have been scores of papers and reviews that assume that RNase E is the central enzyme for degradation of mRNA. (There have been hundreds of papers and many reviews on RNase E and mRNA degradation, with at least 60 just in the last 2 years. I regret the unintentional omission of worthy ones but only refer to examples in this short presentation.) It is appropriate to consider evidence for and against this conclusion since it bears on our understanding of overall pathways of metabolism. RNase E was identified in 1978 by Apirion et al. (4,44,96) as an endoribonuclease (endo-RNase) that catalyzed the maturation of 5S rRNA by two sequential cleavages at specific sites of the 9S RNA of Escherichia coli. About the same time, Kuwano (recently from training with Apirion) et al. isolated an unusual temperature-sensitive mutant called the ams (for altered mRNA stability) mutant (73,107). About a decade later, it was shown that the ams mutation maps in the gene for RNase E, rne (9,95,99,130). This identification contributed to the now widely held view that RNase E is the principal RNase for initiation of mRNA decay (e.g., see references 33, 34, 35, 49, 57, 87, 122, and 124). FUNCTIONAL DECAY AND MASS DECAY SHOULD BE CONSIDERED SEPARATELYIt is important to recognize the useful compartmentalization of the degradation of an mRNA population for a specific protein (message) into two distinct components: inactivation or loss of function (the ability to synthesize protein) as opposed to loss of mass. It is also important to recognize that decay rates are measured on populations of millions of molecules and are used to infer how a single molecule is degraded. Loss of mass of a heterogeneous population of mRNAs is defined empirically by loss of trichloroacetic acid-precipitable material; the lost fraction generally includes oligonucleotides of less than 12 to 15 nucleotides (nt). For mRNA for a specific protein (message), it has been defined by loss of oligonucleotides that cannot form stable hybrids with their cDNA. The minimal size depends on the stringency of the hybrid reaction and the base composition but is often in the 15-to 30-nt range. A 500-nt mRNA population would only show loss of mass if cleavages occurred near the ends or if it were degraded by an exonuclease. Other terms have been used, such as "bulk decay" and, most commonly, "chemical decay." Mass decay is more specific, since "chemical" could refer to any number of parameters from chemical modifications to structural alterations, as well as loss of mass.Functional decay (inactivation) is defined here by loss of the "capacity" of an mRNA molecule to participate in the initiation of synthesis that leads to the normal functional protein product. It is the rate-limiting event for determining the amount of functional protein from the message, as well as for the onset of rapid degradation of the inact...

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“…The in vivo cleavage specificity in E. coli is for Pyd-N bonds, with a preference for Pyd-Ade, not for a UUAU sequence, which has been an incorrect interpretation of the data (7,8).…”

Section: Rnase Mmentioning

confidence: 99%

Section: What Is the Rnase E Recognition Site?mentioning

confidence: 99%

Processing Endoribonucleases and mRNA Degradation in Bacteria

Kennell

2002

J Bacteriol

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“…RNase E is the only endoribonuclease implicated thus far in general mRNA turnover (Arraiano et al 1988;Mudd et al 1990b;Babitzke and Kushner 1991). Many mRNAs were shown to contain sites whose cleavage was abolished in strains harboring temperature-sensitive rne mutations at the nonpermissive temperature (Kokoska et al 1990;Mudd et al 1990a;Gross 1991;Lin-Chao and Cohen 1991;Nilsson and Uhlin 1991; Rdgnier and Hajnsdorf qnstituto de Tecnologia Quimica e Biol6gica, Universit~ Nova Lisboa, 2780 Oeiras, Portugal. 1991; Patel and Dunn 1992;Arraiano et al 1993;Klug 1993;Mudd and Higgins 1993;Carpousis et al 1994). rne-dependent cleavages might produce relatively stable processed transcripts, such as the T4 gene 32 (Mudd et al 1988) and the dicF (Faubladier et al 1990) and the ghX mRNAs (Brunet al 1990).…”

Section: Mrnamentioning

confidence: 99%

Ribonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA.

Alifano¹,

Rivellini²,

Piscitelli³

et al. 1994

Genes Dev.

144

102

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The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coil strains carrying mutations in several genes for exo-and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized me-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.[Key Words: Escherichia coli; Salmonella typhimurium; his operon; mRNA processing; translation; RNase E; RNase P] Received June 2, 1994; revised version accepted October 18, 1994. mRNA decay in bacteria is mediated by the coordinated action of exonucleases and endonucleases (for review, see Petersen 1992). Polynucleotide phosphorylase (PNPase) and ribonuclease II (RNase II) are the two major 3'--* 5' exonucleases involved in mRNA turnover (Donovan and Kushner 1986). Endoribonuclease III (RNase III) does not affect bulk mRNA stability (Babitzke et al. 1993) and is mostly involved in processing of the 30S rRNA precursor even though RNase III can initiate the functional decay of a few specific mRNAs (Court 1993). Endoribonuclease E (RNase E)was initially characterized as the enzyme that processes the precursor of 5S rRNA (Ghora and Apirion 1978). RNase E is the only endoribonuclease implicated thus far in general mRNA turnover (Arraiano et al. 1988;Mudd et al. 1990b;Babitzke and Kushner 1991). Many mRNAs were shown to contain sites whose cleavage was abolished in strains harboring temperature-sensitive rne mutations at the nonpermissive temperature (Kokoska et al. 1990;Mudd et al. 1990a;Gross 1991 1991; Patel and Dunn 1992;Arraiano et al. 1993;Klug 1993;Mudd and Higgins 1993;Carpousis et al. 1994). rne-dependent cleavages might produce relatively stable processed transcripts, such as the T4 gene 32 (Mudd et al. 1988) and the dicF (Faubladier et al. 1990) and the ghX mRNAs (Brunet al. 1990). In different studies ribonuclease P (RNase P) and other nucleases responsible for stable RNA processing did not seem to be involved in mRNA degradation (Deutscher 1988).We have previously identified in Salmonella typhimurium a rather stable 3900-nucleotide-long mRNA molecule...

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“…To study mRNA decay in Escherichia coli and its role in gene regulation, our laboratory used a genetic approach to examine RNases that might participate in mRNA decay (1)(2)(3)(4) Consequently, we set out to examine additional genes that might be involved. The identification of pcnB, the structural gene for poly(A) polymerase I (PAP I) (5), has allowed us to determine whether polyadenylylation affects mRNA decay in E. coli the same way it does in eukaryotes (6).…”

mentioning

confidence: 99%

Polyadenylylation helps regulate mRNA decay in Escherichia coli.

O'Hara

Chekanova²,

Ingle³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

237

273

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As part of our genetic analysis of mRNA decay in Escherichia coli K-12, we Consequently, we set out to examine additional genes that might be involved. The identification of pcnB, the structural gene for poly(A) polymerase I (PAP I) (5), has allowed us to determine whether polyadenylylation affects mRNA decay in E. coli the same way it does in eukaryotes (6). For many eukaryotic mRNAs, the degradation of poly(A) tails initiates mRNA decay (7-9). If it does so in E. coli, then, presumably, mRNA stability would increase in the absence of PAP I.To test whether the degradation of poly(A) tails initiates mRNA decay in E. coli, a series of isogenic strains containing mutations in PAP I (10), RNase E(mne) (1), PNPase (pnp) (1), and RNase II (mb) (1) were constructed. Northern analysis of the trxA, ompA, and lpp mRNAs showed that when polyadenylylation was almost completely eliminated, their half-lives always increased significantly and their decay patterns changed. We also determined the number and size of poly(A) tails in the total bacterial RNA population. In wild-type E. coli, poly(A) tails ranged from 10 to >50 nt. Where there was no PNPase and there was reduced RNase II activity, both the number and the size of the tails increased significantly. More than 90% of the poly(A) tails were absent in ApcnB mutants. We discuss the implications of these results below.MATERIALS AND METHODS Bacterial Strains. All strains were derived from E. coli MG1693 (thyA715), provided by B. Bachmann (E. coli Genetic Stock Center, Yale University). SK5704 (pnp-7 mb-500 me-i thyA 715) (1) was constructed by phage P1-mediated transduction (11). SK7988 (ApcnB thyA715) and SK8901 (lApcnBpnp-7 mb-500 me-I thyA 715) were derived from MG1693 and SK5704, respectively. A kanamycin-resistance (KmR) determinant in the ApcnB gene (10) was used as a selectable transduction marker. SK7988 and SK8901 each grew more slowly than their pcnB+ parents. The absence of poly(A) polymerase in SK8901 did not affect the conditional lethality associated with inactivating RNase II, RNase E, and PNPase.RNA Isolation. Seven-milliliter samples were mixed with an equal volume of crushed frozen TM buffer (10 mM Tris, pH 7.2/5 mM MgCl2) containing 20 mM NaN3 and chloramphenicol at 0.4 mg/ml. The cells were then centrifuged and the pellet was suspended in 0.34 ml of TM buffer with lysozyme at 0.3 mg/ml and DNase I at 32 units/ml. After three freezethaw cycles, one-sixth volume of 20 mM acetic acid was added, followed by an equal volume of Catrimox-14 (Iowa Biotechnology, Oakdale, IA).The resulting precipitate of protein, DNA, and RNA was spun in a microcentrifuge at medium speed to form a soft pellet. That pellet was completely suspended in 1 ml of 2 M LiCl in 35% ethanol. In this solution protein and DNA dissolve but RNA does not. The resulting RNA precipitate was centrifuged at high speed for 5 min to form a pellet. It was then suspended in 2 M LiCl in distilled water and centrifuged again. The pellet was washed with 70% ethanol and resuspended in distilled w...

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Identification of endonucleolytic cleavage sites involved in decay of Escherichia coli trxA mRNA

Cited by 35 publications

References 46 publications

Processing Endoribonucleases and mRNA Degradation in Bacteria

Processing Endoribonucleases and mRNA Degradation in Bacteria

Ribonuclease E provides substrates for ribonuclease P-dependent processing of a polycistronic mRNA.

Polyadenylylation helps regulate mRNA decay in Escherichia coli.

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