Exonucleolytic Attack Research Articles

Super-resolution optical microscopy study of telomere structure.

Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5?-TTAGGG-3? in humans) repeated more than a thousand times, a short 3? single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3? overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded “t-loop.” Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

A Circular RNA (circRNA) ciRS‐7 in Alzheimer's disease (AD) targets miRNA‐7 trafficking and promotes deficits in the expression of the ubiquitin conjugase (UBE2A) and the epidermal growth factor receptor (EGFR)

Circular RNAs (circRNAs) represent a recently described category of novel, single stranded ribonucleic acid (ssRNA) abundantly represented in the eukaryotic transcriptome. Non‐polyadenylated and intrinsically resistant to exonucleolytic attack, circRNAs are a highly stable ssRNA species significantly enriched in human brain and retinal tissues. The biological evolution and function of most central nervous system (CNS) circRNAs, however, remains poorly understood. Established methods of ssRNA detection and quantitation that require a free 3′ or 5′ ribonucleotide end may have significantly underestimated circRNA abundance and importance in CNS function in both health and disease. One highly represented circRNA in the human brain and retina is a ~1400 nucleotide (nt) ciRS‐7 that contains about ~70 tandem anti‐miRNA‐7 sequences; ciRS‐7 thereby acts as a kind of endogenous, competing, anti‐complementary miRNA inhibitor or “sponge” to adsorb, and hence quench, the normal function(s) of miRNA‐7 (hsa‐miRNA‐7‐1; 23 nt; chr 9q21.32). Using DNA and miRNA arrays, RNA‐sequencing, enhanced LED‐Northern, ELISA and Western blot hybridization, the circularity‐sensitive probe RNaseR, energy of association (EA) indexing and miRNA‐mRNA bioinformatics algorithms, we here provide initial evidence of a mis‐regulated miRNA‐7‐anti‐miRNA‐7‐circRNA system in sporadic Alzheimer's disease (AD) brain. Deficits in ciRS‐7, and ciRS‐7 “sponging activities” might be expected to increase ambient miRNA‐7 levels in AD‐affected tissues, as is observed, and these ultimately contribute to the down‐regulation of selective miRNA‐7‐sensitive messenger RNA (mRNA) targets. We report here that the up‐regulation of ambient miRNA‐7 in AD brain, due to a deficiency in ciRS‐7 and ciRS‐7‐mediated “sponging” effects, in turn down‐regulates several AD‐relevant mRNA targets and their expression, including the ubiquitin conjugase protein (UBE2A; miRNA‐7‐UBE2A mRNA 3′UTR EA = −22.9 kcal/mol) and the epidermal growth factor receptor (EGFR; miRNA‐7‐EGFR mRNA 3′UTR EA = −23.2 kcal/mol). UBE2A (chr Xq24; ~17kDa), a central effector in the ubiquitination cycle that helps to orchestrate the clearance of amyloid peptides via phagocytosis, is depleted in sporadic AD brain and retina and contributes to amyloidogenesis. Similarly, decreased levels of the transmembrane glycoprotein EGFR (chr 7p11.1; ~134kDa) found in AD appears to be linked to deficits in brain cell proliferation, cytoskeletal rearrangement and angiogenesis. Our conceptual understanding of the highly specialized functions for non‐coding ssRNA such as circRNAs in the human CNS continues to evolve and their presence and activities underscore the increasing complexity of miRNA‐mRNA‐mediated genetic regulatory networks.Support or Funding InformationResearch on miRNA signaling in the Lukiw laboratory involving the innate‐immune response in AD, AMD and in other forms of neurological or retinal disease, amyloidogenesis, ubiquitination, synaptogenesis and brain inflammation was supported through an unrestricted grant to the LSU Eye Center from Research to Prevent Blindness (RPB); the Louisiana Biotechnology Research Network (LBRN) and NIH grants NEI EY006311 and NIA AG038834 (WJL).

SOT1, a pentatricopeptide repeat protein with a small MutS-related domain, is required for correct processing of plastid 23S-4.5S rRNA precursors in Arabidopsis thaliana.

Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid-localized pentatricopeptide repeat (PPR) protein with a small MutS-related domain, is required for maturation of the 23S-4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5' end of the 23S-4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA 'footprint' associated with this site in sot1 mutants. We found that more than half of the 23S-4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5' and 3' ends, and that the endonucleolytic cleavage product normally released from the 5' end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5' extremity of the 23S-4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5' and 3' ends.

Way to go, RNA.

Way to go, RNA.

Inactivation of the Sas2 histone acetyltransferase delays senescence driven by telomere dysfunction

Changes in telomere chromatin have been linked to cellular senescence, but the underlying mechanisms and impact on lifespan are unclear. We found that inactivation of the Sas2 histone acetyltransferase delays senescence in Saccharomyces cerevisiae telomerase (tlc1) mutants through a homologous recombination-dependent mechanism. Sas2 acetylates histone H4 lysine 16 (H4K16), and telomere shortening in tlc1 mutants was accompanied by a selective and Sas2-dependent increase in subtelomeric H4K16 acetylation. Further, mutation of H4 lysine 16 to arginine, which mimics constitutively deacetylated H4K16, delayed senescence and was epistatic to sas2 deletion, indicating that deacetylated H4K16 mediates the delay caused by sas2 deletion. Sas2 normally prevents the Sir2/3/4 heterochromatin complex from leaving the telomere and spreading to internal euchromatic loci. Senescence was delayed by sir3 deletion, but not sir2 deletion, indicating that senescence delay is mediated by release of Sir3 specifically from the telomere repeats. In contrast, sir4 deletion sped senescence and blocked the delay conferred by sas2 or sir3 deletion. We thus show that manipulation of telomere chromatin modulates senescence caused by telomere shortening.

Exonucleolysis is required for nuclear mRNA quality control in yeast THO mutants

Production of aberrant messenger ribonucleoprotein particles (mRNPs) is subject to quality control (QC). In yeast strains carrying mutations of the THO complex, transcription induction triggers a number of interconnected QC phenotypes: (1) rapid degradation of several mRNAs; (2) retention of a fraction of THO-dependent mRNAs in transcription site-associated foci; and (3) formation of a high molecular weight DNA/protein complex in the 3'-ends of THO target genes. Here, we demonstrate that the 3'-5' exonucleolytic domain of the nuclear exosome factor Rrp6p is necessary for establishing all QC phenotypes associated with THO mutations. The N terminus of Rrp6p is also important presumably through its binding to the Rrp6p co-factor Rrp47p. Interestingly, the 3'-5' exonucleolytic activity of Dis3p, the only other active exonuclease of the nuclear exosome, can also contribute to RNA QC in THO mutants, while other nuclear 3'-5' exonucleases cannot. Our data show that exonucleolytic attack by the nuclear exosome is needed both for provoking mRNP QC and for its ensuing elimination of faulty RNA.

Mutational analysis of a viral RNA element that counteracts rapid RNA decay by interaction with the polyadenylate tail

We previously demonstrated that the Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA contains a 79-nt cis-acting element, the ENE, which allows intronless polyadenylated transcripts to accumulate to high nuclear levels by protecting them from rapid degradation. We proposed a model based on the predicted structure of the ENE in which a U-rich internal loop hybridizes with the 3'-polyadenylate (polyA) tail to sequester it from exonucleolytic attack. We have tested this model by mutational analysis of the ENE. Point mutations in the predicted U-rich internal loop and in the flanking stems abolish the ENE's ability to (i) interact with the polyA tail, (ii) inhibit deadenylation in vitro, and (iii) stabilize transcripts in vivo. In all but one case, compensatory mutations in the flanking stems restore ENE activities, demonstrating the importance of these stems and uncovering a unique role for the loop-proximal G-C base pair in the lower stem. Increasing the U content of the U-rich internal loop surprisingly decreases stability in vivo but does not affect deadenylation in vitro, comparable to the effects of deleting certain "unstructured" regions of the ENE. Taken together, our data support the formation of the proposed ENE secondary structure in vivo and argue that the specific ENE structure inhibits rapid RNA decay in cis by engaging in a limited set of base-pairing interactions with the polyA tail.

Enzymatic processing of replication and recombination intermediates by the vaccinia virus DNA polymerase

Poxvirus DNA polymerases play a critical role in promoting virus recombination. To test if vaccinia polymerase (E9L) could mediate this effect by catalyzing the post-synaptic processing of recombinant joint molecules, we prepared substrates bearing a nick, a 3′-unpaired overhang, a 5′ overhang, or both 3′ and 5′ overhangs. The sequence of the 5′ overhang was also modified to permit or preclude branch migration across the joint site. These substrates were incubated with E9L, and the fate of the primer strand characterized under steady-state reaction conditions. E9L rapidly excises a mispaired 3′ strand from a DNA duplex, producing a meta-stable nicked molecule that is a substrate for ligase. The reaction was not greatly affected by adding an unpaired 5′ strand, but since such molecules cannot be processed into nicked intermediates, the 3′-ended strand continued to be subjected to exonucleolytic attack. Incorporating homology into the 5′ overhang prevented this and permitted some strand assimilation, but such substrates also promoted strand-displacement DNA synthesis of a type predicted by the 1981 Moyer and Graves model for poxvirus replication. Single-strand annealing reactions are used by poxviruses to produce recombinant viruses and these data show that virus DNA polymerases can process DNA in such a manner as to both generate single-stranded substrates for such reactions and to facilitate the final processing of the reaction products.

SsrA-mediated trans-translation plays a role in mRNA quality control by facilitating degradation of truncated mRNAs

An important unsolved question regarding the bacterial SsrA system is the fate of target mRNAs replaced by SsrA RNA during trans-translation. The aim of the present study is to address the potential role of SsrA system in mRNA quality control, focusing on truncated mRNAs that are expected to arise from 3'-to-5' exonucleolytic attack. We found that significant amounts of truncated mRNAs and polypeptides were produced from genes lacking a rho-independent terminator in SsrA-deficient cells. These truncated mRNAs, hence truncated polypeptides, were no longer observed in the presence of SsrA RNA. The data indicate that the SsrA system facilitates degradation of "nonstop" mRNAs by presumably removing the stalled ribosomes. Furthermore, analysis of affinity-purified proteins indicated that truncated polypeptides could be produced even from a gene with an intact rho-independent terminator, although less efficiently, implying that C-terminally truncated proteins and 3'-truncated mRNA may be produced from virtually all protein-coding genes. We conclude that the SsrA system not only promotes the degradation of incomplete polypeptides but also minimizes the synthesis of incomplete polypeptides by facilitating the degradation of truncated mRNAs that are produced in cells.

Polyadenylation can regulate ColE1 type plasmid copy number independently of any effect on RNAI decay by decreasing the interaction of antisense RNAI with its RNAII target

Replication of ColE1-type plasmids is regulated by RNAI, an antisense RNA that interacts with the replication pre-primer, RNAII. Exonucleolytic attack at the 3 ′ end of RNAI is impeded in pcnB mutant bacteria, which lack poly(A) polymerase I—the principal RNA polyadenylase of E. coli; this leads to accumulation of an RNAI decay intermediate (RNAI −5) and dramatic reduction of the plasmid copy number. Here, we report that polyadenylation can also affect RNAI-mediated control of plasmid DNA replication by inhibiting interaction of RNAI −5 with RNAII. We show that mutation of the host pcnB gene profoundly affects the plasmid copy number, even under experimental conditions that limit the effects of polyadenylation on RNAI −5 decay. Moreover, poly(A) tails interfere with RNAI/RNAII interaction in vitro without producing any detectable alteration of RNAI secondary structure. Our results establish the existence of a previously undetected mechanism by which RNA polyadenylation can control plasmid copy number.

The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum have short A tails.

The mitochondrial genome of Plasmodium falciparum encodes highly fragmented rRNAs. Twenty small RNAs which are putative rRNA fragments have been found and 15 of them have been identified as corresponding to specific regions of rRNA sequence. To investigate the possible interactions between the fragmented rRNAs in the ribosome, we have mapped the ends of many of the small transcripts using primer extension and RNase protection analysis. Results obtained from these studies revealed that some of the rRNA transcripts were longer than the sequences which encode them. To investigate these size discrepancies, we performed 3' RACE PCR analysis and RNase H mapping. These analyses revealed non-encoded oligo(A) tails on some but not all of these small rRNAs. The approximate length of the oligo(A) tail appears to be transcript-specific, with some rRNAs consistently showing longer oligo(A) tails than others. The oligoadenylation of the rRNAs may provide a buffer zone against 3' exonucleolytic attack, thereby preserving the encoded sequences necessary for secondary structure interactions in the ribosome.

Reconstitution of the degradation of the mRNA for ribosomal protein S20 with purified enzymes

Previous work has implicated poly(A) polymerase I (PAP I), encoded by the pcnB gene, in the decay of a number of RNAs from Escherichia coli. We show here that PAP I does not promote the initiation of decay of the rpsT mRNA encoding ribosomal protein S20 in vivo; however, it does facilitate the degradation of highly folded degradative intermediates by polynucleotide phosphorylase. As expected, purified degradosomes, a multi-protein complex containing, among others, RNase E, PNPase, and RhlB, generate an authentic 147-residue RNase E cleavage product from the rpsT mRNA in vitro. However, degradosomes are unable to degrade the 147-residue fragment in the presence of ATP even when it is oligoadenylated. Rather, both continuous cycles of polyadenylation and PNPase activity are necessary and sufficient for the complete decay of the 147-residue fragment in a process which can be antagonized by the action of RNase II. Moreover, both ATP and a non-hydrolyzable analog, ATPγS, support the PAP I and PNPase-dependent degradation of the 147-residue intermediate implying that ATPase activity, such as that which may reside in RhlB, a putative RNA helicase, is not necessarily required. Alternatively, the rpsT mRNA can be degraded in vitro by a second 3′-decay pathway which is dependent on PAP I, PNPase and ATP alone. Our results demonstrate that a hierarchy of RNA secondary structures controls access to exonucleolytic attack on 3′ termini. Moreover, decay of a model mRNA can be reconstituted in vitro by a small number of purified components in a process which is more dynamic and ATP-dependent than previously imagined.

The Xenopus intron-encoded U17 snoRNA is produced by exonucleolytic processing of its precursor in oocytes.

U17 is a small nucleolar RNA encoded in the introns of the Xenopus laevis gene for ribosomal protein S7 (formerly S8, see Note). To study the mechanisms involved in its in vivo processing from S7 transcripts, various in vitro synthesized RNAs embedding a U17 sequence have been microinjected into the germinal vesicle of Xenopus oocytes and their processing analysed. In particular, the Xenopus U17 gene copies a and f and a U17 gene copy from the pufferfish Fugu rubripes have been used. Information about the nature of the processing activities involved in U17 RNA maturation have been sought by injecting transcripts protected from exonucleolytic attack at their 5'-end by capping and/or lengthened at their 3'-end by polyadenylation. The results obtained indicate that U17 RNA processing is a splicing-independent event and that it is mostly or entirely due to exonucleolytic degradation at both the 5'- and 3'-ends of the precursor molecules. Moreover, it is concluded that the enzymes involved are of the processive type. It is suggested that the apparatus for U17 RNA processing is that responsible for the degradation of all excised and debranched introns. Protection from exonucleolytic attack, due to the tight structure and/or to the binding of specific proteins, would be the mechanism by which U17 RNA is produced.

Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.

Two thermostable DNA polymerases with proofreading activity--Vent DNA polymerase and Pfu DNA polymerase--have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the oligodeoxynucleotides serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction.

Multiple determinants of functional mRNA stability: sequence alterations at either end of the lacZ gene affect the rate of mRNA inactivation

The Escherichia coli lacZ gene was used as a model system to identify specific sequence elements affecting mRNA stability. Various insertions and substitutions at the ribosome-binding site increased or decreased the rate of mRNA inactivation by up to fourfold. Deletion of a dyad symmetry, which may give rise to a very stable secondary structure in the mRNA immediately downstream of the gene, decreased the functional stability of the lacZ message. The magnitude of the latter effect was strongly dependent on the sequences at the ribosome-binding site, ranging from practically no effect for the most labile transcripts to a threefold decrease in stability for the most stable one. The results suggest that the wild-type lacZ message is inactivated predominantly by attacks near the ribosome-binding site, presumably in part because the putative secondary structure downstream of the gene protects against 3'-exonucleolytic attack. Taken together, the data for all of the modified variants of lacZ were shown to be quantitatively compatible with a general model of mRNA inactivation involving multiple independent target sites.

MINIREVIEW: Insights into Hormonal Control of Messenger RNA Stability

Until recently most studies of the regulation of eukaryotic mRNA levels have focused on the control of gene transcription. However, the level of a cellular mRNA actually represents a balance between its rates of synthesis and degradation. In the last few years the regulation of cytoplasmic mRNA stability has emerged as an important control point in a variety of biological systems (reviewed in Refs. 1–8). In this review we try to relate what is known about the regulation of mRNA stability in several intensively studied systems to the emerging area of hormonal control of mRNA stability. A useful working hypothesis is that most mRNAs are relatively stable in cells until subjected to an initial endonucleolytic cleavage. The 5′ and 3′ ends of most eukaryotic mRNAs are protected from exonucleolytic attack by the presence of a 5′ cap structure (9) and a 3′ poly(A) tract (10) complexed to a poly(A)-binding protein (11). Intramolecular cleavage by an endonuclease or removal of the protective poly(A) tract from the...

Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation.

We show that upon induction of Agrobacterium tumefaciens, free linear double-stranded T-DNA molecules as well as the previously described T-strands are generated from the Ti plasmid. A majority of these molecules are bound to a protein. We show that this protein is the product of the virulence gene virD2. This protein was found to be attached to the 5' terminus of processed T-DNA at the right border and to the rest of the Ti plasmid at the left border. The protein remnant after Pronase digestion rendered the right end of the double-stranded T-DNA resistant to 5'----3' exonucleolytic attack in vitro. The protein-DNA association was resistant to SDS, mercaptoethanol, mild alkali, piperidine, and hydroxylamine, indicating that it involves a covalent linkage. The possible involvement of this T-DNA-protein complex in replication, transduction to the plant, nuclear targeting, and integration into the plant nuclear DNA is discussed.

Plasmid-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sbcBC mutant. Involvement of RecF recombination pathway genes

Apparent plasmid instability, i.e. progressive plasmid loss in a bacterial culture growing in the absence of selection for the plasmid, in an Escherichia coli recBC sbcBC mutant was investigated with two different ColE1 derivatives (pMB9 and pBR322) and a mini-F plasmid. The instability was most striking for pMB9 and much less, but still significant, for pBR322 and the mini-F. It was also dependent upon a subset of the genes involved in the RecF recombination pathway: in addition to the previously reported recA, recF and recJ mutations, a recO and a recQ mutation showed a total and a partial suppression, respectively, of the instability. Other recF-family mutations, recN and ruv, were without such an effect. Population analyses of the recBC sbcBC strain carrying pMB9 or the mini-F, as carried out by plating and Coulter counting, revealed marked loss of viability in plasmid-carrying cells, strongly implicating plasmid-mediated cell death in the apparent defect in plasmid maintenance. Analysis of intracellular plasmid DNA by pulsed-field gel electrophoresis combined with the in-agarose cell lysis technique showed that the instability was associated with the formation of plasmid multimers, with a good correlation between the degree of the instability and the amount of the multimers. The multimer formation was also dependent on the same subset of the RecF pathway genes as in the instability phenomenon. These results strongly suggest that the lethality is somehow caused by the multimer formation. Various DNase treatments of cell lysates showed that such multimers of pMB9 DNA comprised molecules of exonuclease-sensitive and exonuclease-resistant types. It was inferred that the former class, which showed electrophoretic mobilities corresponding to plain linear duplexes of approximately 200 x 10(3) to 2200 x 10(3) base-pairs, represented linear multimers possibly carrying circular structures at one end. The latter class, which remained in the origin, was thought to consist of circular multimers and/or linear multimers protected by circular structures at both ends against exonucleolytic attack.

Binding of adenosine diphosphoribosyltransferase to the termini and internal regions of linear DNAs

Binding mechanisms of ADPR-transferase to restricted double-stranded DNA fragments of SV40 and pBR322 DNA were determined by nuclease protection techniques. Top and bottom strands of double-stranded DNA were identified by specific labeling with 32P. Protection against specific exonucleases identified binding of ADPR-transferase to DNA termini, whereas binding to internal regions of linear DNAs was probed by protection against endonucleases. ADPR-transferase protein protected against exonucleolytic attack from lambda exo and exoIII in all DNA fragments tested, demonstrating that the enzyme protein binds indiscriminately to all DNA termini. Extending earlier results [Sastry, S.S., & Kun, E. (1988) J. Biol. Chem. 263, 1505-1512], the modifying effect of the binding of ADPR-transferase to DNA induced changes in DNA conformation, as evident from altered pause sites that appeared following digestion of DNA fragments by lambda exonuclease in the presence of ADPR-transferase. In contrast to the nonselective binding of ADPR-transferase to DNA termini, ADPR-transferase conferred protection endonuclease attack (DNase I and micrococcal nuclease) only to the 209-bp EcoRI-PstI SV40 DNA fragment. These results indicate that binding of ADPR-transferase to relatively rare internal regions of restricted DNA fragments exhibits some degree of specificity. Specificity of binding appears to be related to the coincidental relative A+T-rich regions in DNA, and to DNA bending, both identified in the 209-bp SV40 DNA fragment. Synthetic polydeoxyribonucleotides containing dA-dT bind ADPR-transferase stronger than polydeoxyribonucleotides containing dG-dC. It was deduced from endonuclease protection patterns that binding of the enzyme protein leaves no defined footprints on the 209-bp SV40 DNA fragment, but there is significant modification of DNA structure following binding of the enzyme protein. Methylation protection assays and the prevention of the binding of ADPR-transferase to T4 DNA by its glucosylation indicate that the enzyme binds in the major groove of DNA. The 36-kDa A peptide fragment of ADPR-transferase [Buki, K. G., & Kun, E. (1988) Biochemistry 27, 5990-5995] exhibits the same protection against endonucleolytic enzymes as the intact ADPR-transferase molecule.

Site-specific endonucleolytic cleavages and the regulation of stability of E. coli ompA mRNA

The stability of ompA mRNA is growth-rate dependent. We show that the 5′ noncoding region of this mRNA provides a target for site-specific endonucleases. The rate of degradation of ompA mRNA parallels the rate of these endonucleolytic cleavages, implying that endonucleolytic rather than exonucleolytic attack is the initial step in ompA mRNA degradation. Thus the 5′ noncoding region appears to be a determinant of mRNA stability, and endonucleolytic cleavages in the 5′ noncoding region may well regulate expression of the ompA gene.