World

Journal

of Microbiology

and Biotechnology

9. 421432

Special Topic Review

Post-transcriptional control of gene expression : bacterial mRNA degradation C.M. Arraiano Many biological processes cannot be fully understood without detailed knowledge of RNA metabolism. The continuous breakdown and resynthesis of prokaryotic mRNA permit rapid production of new kinds of proteins. In this way, mRNA levels can regulate protein synthesis and cellular growth. Analysing mRNA degradation in prokaryotes has been particularly difficult because most mRNA undergo rapid exponential decay. Prokaryotic mRNAs differ in their susceptibility to degradation by endonucleases and exonucleases, possibly because of variation in their sequencing and structure. In spite of numerous studies, details of mRNA degradation are still largely unknown. This review highlights those aspects of mRNA metabolism which seem most influential in the regulation of gene expression. Key words: mRNA

degradation,

post-transcriptional

control,

Most prokaryotic mRNA undergo rapid exponential decay, the average mRNA having a half life of 1.3 min at 37’C (Ingraham et al. 1983). This mRNA instability best explains the rapid adaptation of microorganisms to a changing environment. The level of expression of a gene is determined primarily by three elements: the rate of transcription; the stability of the RNA transcript; and the efficiency of translation. Clearly, factors that either modify mRNA or that alter its decay may play a major role in controlling gene expression. An important consequence of the rapid kinetics in microorganisms is that a large fraction of molecules in a message population are decaying. The coupling of transcription, translation and mRNA degradation in prokaryotes also makes the mRNA metabolism less amenable to investigation because the perturbation of many cellular processes can indirectly influence mRNA degradation. Individual mRNA species differ widely with respect to metabolic stability. Earlier studies have established four facts. Firstly, the rate of turnover has no relation to the length of the gene (Blundell et al. 1972). Secondly, the sequences that decay most rapidly may be anywhere on the mRNA (Achord & Kennel1 1974; Von Gabain et al. 1983). Thirdly, the rates of decay of some species can be altered in response to physiological signals such as changes in growth rate C. M. Arraiano is with the lnstituto da Tecnologia Quimica (ITQB). Apt 127, 2780 Oeiras, Portugal; fax: 351 1 4428766. @ 1993 Rapid

Communications

of Oxford

a Biobgica

prokaryotes,

ribonucleases.

(Nilsson et al. 1984). Fourthly, the stability of gene transcripts seems to be dependent on determinants localized to specific mRNA segments, but little is understood about these determinants (Belasco et al. 1986). Apart from these observations, the process of mRNA degradation is still largely unknown.

Enzymes and other mRNA Degradation

Factors

involved

in

The enzymes involved in RNA degradation are poorly characterized. Most of the nucleases involved in the maturation of tRNA and rRNA precursors do not seem to play a major role in mRNA decay (Apirion & Gegenheimer 1984; Deutscher 1988). We have to reject the possibility that each message is recognized by a unique enzyme because it would necessitate 1000 different enzymes for the 1000 or so different messages in the cell. Another possibility would be sequences or structures that are common to all messages and that are attacked by a few ribonucleases. These messages will have different decay rates, reflecting their degree of protection or vulnerability. Exonucleases

While endonucleases can certainly cleave mRNA, bulk degradation of mRNA must depend on exonucleolytic activities. All the exonucleases so far identified in Escher&a

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C. M. Arraiano coli degrade RNA progressively from the 3’ end and no 5-3’ exonuclease activity has yet been identified (Deutscher 1988). Several exonucleases have been partially characterized in E. coli but only two are believed to be the principal enzymes in the degradation of bacterial mRNA to nucleotides (Deutscher 1988). These two exonucleases are polynucleotide phosphorylase (PNPase), encoded for by pnp (Reiner 1969) and ribonuclease II (RNase II), encoded for by mb (Spahr 1964; Singer & Tolbert 1965). Both degrade single-stranded mRNA processively in the 3’ to 5’ direction. PNPase degrades mRNA via a reversible phosphorolytic reaction and generates mononucleotides 5’-diphosphate. RNase II irreversibly hydrolyses mRNA to 5’-monophosphates. Sequences with the potential to form stem-loop structures can stabilize upstream mRNA against 3’-5’ exoribonucleolytic attack in viva by blocking the processive activities of these enzymes (Plamann & Stauffer 1990) but RNase II and PNPase seem to have different specificities in 3’ mRNA decay (Guameros & Portier 1990). Although these structures alone can impede the progress of both enzymes, some data suggest that an additional factor, such as a stem-loop binding protein, might be involved in the stabilization (Mackie 1989; McLaren et al. 1991). Donovan and Kushner isolated a mutant mb allele (mb-500) that encoded a thermolabile RNase II protein (Donovan & Kushner 1986). Double mutant strains (pnp-7 mb-5~0) ceased growth within 30 min after a shift to 44’C. Cessation of growth was accompanied by the in viva accumulation of mRNA fragments of 100 to 1500 nucleotides in length (Kushner et al. 1985; Donovan & Kushner 1986). Little or no change was observed in cellular levels of rRNA. At the non-permissive temperature, the half-life of bulk mRNA was twice as long in a double mutant strain than in single mutants. Single mutant strains grew well at 44°C and did not accumulate partially degraded RNA species. These results suggest that the two enzymes are largely complementary. However, the absence of PNPase was enough to stabilize the Nettrospora crussa catabolic dehydrogenase (qa-2) mRNA in E. coli strains (Hautala et al. 1979). The prrp mRNA is processed endonucleolytically by RNase III (Portier ef al. 1987) and these cleavages facilitate other site-specific endonucleolytic cleavages (Takata et al. 1992) by enzymes like RNAse E (Regnier & Hajnsdorf 1991). Expression of the pnp gene is also autoregulated by PNPase, but this control depends on the cleavage by RNase III (Robert-Le Meur & Portier, 1992). RNase II has been cloned (Donovan & Kushner 1986) but the mb gene has only recently been sequenced and analysed (Zilhao et al. 1993). Further studies on the mb gene will lead to an understanding of its expression and regulation. The possibility remains that other exonucleases exist. If they are regulated and/or have specificity for some

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transcripts determining

then they could gene expression.

be an important

factor

in

Endonucleases The first evidence for endonucleolytic cleavage as an initial event in mRNA degradation came from the gal, lac and frp operons (Achord & Kennel1 1974; Blundell & Kennel1 1974; Schlessinger et al. 1977). In the lac operon it was shown that the full-length molecule is lost much faster than the mRNA mass, and this could only be the result of internal cleavages (Blundell & Kennel1 1974). Inactivation by endonucleolytic attack at the ribosome binding site (RBS) would be a very effective way of stopping protein production and starting mRNA decay. However, very few examples exist of mRNA known to be inactivated this way. One of them is the bacteriophage T4 mofA mRNA that is cut in the RBS (Uzan et al. 1988). There was a need to map the precise endpoints of the degradation intermediates in an attempt to distinguish between inhibition of a processing exonuclease and the result of endonucleolytic cleavages. Some of these endpoints were found in the intercistronic region, between 1acZ and lacy (Cannistraro et al. 1986) and between lacl and 1acZ (Subbarao SK Kennel1 1988); there was evidence that endonucleolytic cuts occurred and that the cleavages usually occurred between Pyr-A residues, in single-stranded regions. The attenuated transcript of the Bacilltrs strbfilis pur operon is endonucleolytically cleaved in putative single-stranded regions and the degradation intermediates have been identified (Ebbole & Zalkin 1988). Melefors & Von Gabain (1988) showed that the 5’-non-coding region of E. coli ompA mRNA is a target for site-specific growth-dependent endonucleolytic attacks. Recently, Arraiano et al. (1993) provided the first map of cleavage sites during the degradation of an entire transcript. They identified complementary upstream and downstream cleavage products of internal endonucleolytic cleavages in the thioredoxin message (frxA) and showed the nucleotide sequence of the cleavage sites. There was no apparent consensus sequence and secondary-structure analysis suggested that the cleavages could occur both in single- and double-stranded regions. It remains to be demonstrated whether the specificity resides in the primary sequences, in the spatial structure or both; it is crucial to analyse other messages in order to elucidate this. If mRNA decay were due to a random endonucleolytic process, some diversity could be provided by the target size. However, there is no relationship between mRNA size and decay rate. For example, the 1acA message decays twice as fast as the 1acZ message which is four times longer (Blundell et al. 1972). In Rhodobacfer capsulafus, the decay of pufL.MX messages appears to be initiated by several endonucleolytic cleavages

Bacterial mRNA degradation within the coding sequences (Chen & Belasco 1990) and the decay of frxA in E. coli also involves a progression of endonucleolytic cleavages in the 3’ to 5’ direction (Arraiano et al. 1993). If endonucleolytic cleavage within coding regions often occurs during mRNA degradation, a significant number of ribosomes translating a message should be blocked on mRNA fragments with no termination codon. This would result in the release of peptidyl tRNA species with partially completed proteins attached, and these could be released by peptidyl tRNA hydrolase and subsequently degraded (Menninger 1976). In fact, estimations suggest that up to 20% of the translational products end prematurely (Yen et al. 1980). Endonucleolytic cleavage may expose free 3’ ends, which are substrates for exonucleases (Guameros & Portier 1990). For example, in phage 1 the regulatory site sib folds into the characteristic stem-loop structure that is recognized and cleaved by RNase III. The cleavage renders the upstream int mRNA more susceptible to degradation by exonucleases (Guameros et al. 1982). However, in early mRNA of phage T7, the RNase III cleavage has the opposite effect; it appears to impart stability to the upstream region which then has the potential to form a RNase III-resistant stem-loop that blocks the degradation by ribonucleases (Panayotatos & Truong 1985). RNase III is an endonuclease that can cleave double-stranded RNA and can therefore recognize stem-loop structures in mRNA (Robertson et al. 1968). Escherichia coli strains deficient in RNase III accumulate precursors to mRNA and rRNA (Robertson 1982). However, there are few examples of post-transcriptional cleavage of E. coli mRNA by RNase III. In the rpl]L-rpoBC operon RNase III cleaves an intergenic site separating cistrons coding for ribosomal proteins from those coding for RNA polymerase subunits and triggers the decay of downstream products (Barry et al. 1980; Portier et al. 1987). RNase III cleaves the primary transcripts of the rpsO-pnp, mc-era-rec0 and mefY-nusA-infB operons of E. coli, upstream of the first translated gene, in hairpin structures formed by the 5’ non-coding leader. In the rpsO-pnp operon the half-life of the pnp mRNA is considerably increased in a RNase III-deficient strain (Portier et al. 1987; Takata et al. 1987). Moreover, PNPase was lo-fold overexpressed in this mutant strain, which shows that unprocessed pnp mRNA is functional. The rpsO-pnp cotranscript is also processed by RNase E but the cleavages by RNase III and RNase E occur independently (RCgnier & Hajnsdorf 1991). Cleavage by RNase III in the transcripts of the metY-nusA-infB operon of E. coli releases the tRNA and initiates the decay of the downstream mRNA (RCgnier & Grunberg-Manago 1989). The mc-era mRNA encoding RNase III and the GTP binding protein Era, is also processed by RNase III (Bardwell et al. 1989); this was the first example of any RNase controlling its own expression. There is some indication that the RNA

processing enzymes could be interdependent (Miczak et al. 1991). RNase III cleavage upstream of the coding sequence destabilizes not only its own mRNA but also the mRNA of PNPase. This raises the hypothesis that these two RNases may be controlled by an important common mechanism. RNase I is an endonuclease involved in rRNA degradation (Gesteland 1966) but the possibility remains that this enzyme can also play a role in mRNA degradation. The expression of the Newospora crassa qa-2 gene in E. coli was increased 2-fold in ma-19 (RNase I-) strains, 20 to 50 fold in pnp-7 (PNPase-) strains, and loo-fold in ma-19, pnp-7 (RNase I-, PNPase-) strains (Hautala et al. 1979). The altered message stability (urns) gene was mapped at minute 23 in the E. coli chromosome, and ams single mutants have an increased mRNA stability (Ono & Kuwano 1979, 1980). Ams seemed to be involved in the coupling of functional inactivation to chemical decay of a very broad class of mRNA. The observed accumulation of specific mRNA cleavage products at the non-permissive temperature has suggested a role for the gene product in the decay of mRNA degradation intermediates (Arraiano ef al. 1988). In a triple mutant deficient in Ams, PNPase and RNase II, discrete mRNA breakdown products were dramatically stabilized during the decay and this mutant has been particularly helpful in the analysis of the degradation process of specific messages (Arraiano et al. 1988, 1993). The urns gene was sequenced (Claverie-Martin et al. 1991) and several studies showed that in fact the gene specifying RNase E (me) and that specifying urns were identical (Mudd ef al. 1990; Babitzke & Kushner 1991; Melefors & Von Gabain 1991; Taraseviciene et al. 1991). All the phenotypes associated with either the me-3071 or the urns-1 thermolabile alleles were complemented by a plasmid carrying ams+. RNase E is the first endonuclease identified as having a general role in the chemical decay of E. coli mRNA. RNase E had been previously defined as a processing endonuclease that catalyses the maturation of the 5s rRNA from its precursors and cleaves RNA I, involved in the copy number control of ColEl plasmids (Tomcsanyi & Apirion 1985). It was purified from E. cali cells (Misra & Apirion 1979) but it was only a couple of years ago that there was evidence of its important role in mRNA turnover. RNase E seems to cleave mRNA in the vicinity of secondary structures (Tomcsanyi & Apirion 1985; Mudd et al. 1988) and 5’ terminal base-pairing can control its activity (Bouvet & Belasco, 1992). Lin-Chao & Cohen (1991) found that an RNase E-processing product of the RNA I transcript carrying a 5’ mono-phosphate decays much faster than an identical transcript carrying a tri-phosphate at its 5’ end. Analysis of all of the known putative RNase E sites suggested a consensus cleavage site sequence of RAUUW (R = A or G; W = A or U) (Ehretsmann et al. 1992).

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CM. Arraiano Other Factors

Other factors or other unknown nucleases can also influence the rate at which a given species of mRNA is degraded. The relative stability of some bacteriophage T4 transcripts can be attributed to a 5’ leader sequence but the stabilization requires interaction of the leader sequence with trans-acting factor(s) produced only in the infected cells (Gorski et al. 1985). Translational repression causeschanges in the half-life of ribosomal protein mRNA and may be considered as a reversible non-nucleolytic inactivation of mRNA (Singer & Nomura 1985). For instance, the half-life of LI1 mRNA increased 5-fold when the translational feedback regulation by LI was abolished. The change in half-life seems to be a consequence of LI blocking translation of LIl mRNA and not due to some nucleolytic activity mediated by Ll (Cole & Nomura 1986). Similar effects can exist in other operons where encoded proteins may regulate their own synthesis and the decay of their mRNA. In Klebsiella pneumoniae, the production of active nitrogenase requires the expression of at least 15 linked genes (the nif genes) arranged in seven operons (Brill 1980). The nif transcripts (except @IA) are very stable under derepressing conditions. The specific post-transcriptional control of nif mRNA stability is an important feature in the regulation of nifgene expression, and this specificity requires a nif-encoded protein, the nifL product, which is involved in nif mRNA destabilization in response to 0, and, to a lesser extent, NH,. However, it should be noted that the means by which the nifL product recognizes and responds to 0, and NH, is not known. Autocatalytic splicing is well established in eukaryotes (Bass & Cech 1984) and it has been shown that an RNA molecule from bacteriophage T4 can undergo a specific self-cleavage reaction (Watson et al. 1984). We can only speculate at present but the autocatalytic properties of certain RNA molecules may also be important in mRNA decay. Anti-sense mRNA could also be involved in protecting some regions of mRNA by conferring to them a double-strandedness. A small RNA [mRNA interfering complementary (MIC) RNA] has been shown to inhibit translation by hybridizing to the E. coli ompF mRNA (Mizuno et al. 1984). This form of regulation may apply to other mRNA but so far we lack evidence. However, translational repression by anti-sense RNA is expected to be essentially irreversible because the hybrid formed with the mRNA is cleaved by nucleases that recognize double-stranded regions. These are only a few of the many examples of other factors which could interfere in prokaryotic mRNA degradation.

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Particular Configurations at the 3’ Termini and Role of Secondary Struchue in mRNA Decay Secondary structural features at the 3’ termini can serve as a barrier to exonucleases and influence the rate at which these exonucleases degrade a given speciesof mRNA. It is known that in vitro secondary structures are resistant to attack by the 3’-5’ exonucleases present in the cell (Gupta et al. 1977) and the role of secondary structure has also been demonstrated in viuo (Belascoet al. 1985; Mott et al. 1985; Klug et al. 1987; Newbury et al. 1987a; Chen et al. 1988; Pate1et al. 1990; Plamann & Stauffer 1990). The deletion of rho-independent terminators or other stem-loop structures can result in an accelerated decay of upstream mRNA. Upstream mRNA segments in E. coli can be stabilized and their expression enhanced by insertion of a foreign transcription terminator derived from the crystal protein (cy) gene of Bacillusfhttringiensis(Wong & Chang 1986) or from a bacteriophage T7 DNA segment encoding an RNase III cleavage site (Panayotatos & Truong 1985). However, it appears that not all potentially stable stem-loop structures serve to stabilize mRNA (Wong & Chang 1986) and the stable mRNA is not always translationally active. In two mRNA speciesof bacteriophage PhiX174 there is also a sequence with a potential six-base-paired hairpin structure upstream of the terminator site (Hayashi & Hayashi 1985). The effect of this element on mRNA half-life is independent of other PhiX174 encoded activities; it was cloned at the 3’ ends of different genes in a pBR322 derivative plasmid and the stabilized mRNA were functionally active. This study shows that mRNA stabilizing sequences are an important tool in maximizing gene expression from cloned genes. Repetitive extragenic palindromic (REP) sequences are large inverted repeats which are highly conserved in sequence and have the potential to form a stable stem-loop structure (Higgins et al. 1982). These sequences are not terminators of transcription and they have been identified in a large number of genes, both in E. coli (present in about 25% of all transcripts) and Salmonella fyphimuriwn (Gilson et al. 1984; Stem et al. 1984). The REP sequences are invariably transcribed and are located either in intergenic regions of multicistronic operons or in the 3’untranslated region upstream of the terminator. They can stabilize upstream RNA, by protection from 3’-5’ exonucleolytic attack, and they are frequently responsible for the differential stability of different segments of mRNA within an operon (Newbury et al. 1987a). In certain casesat least, the mRNA is stabilized in a translationally active form. Deletion of REP sequences from the E. co/i malEFG operon not only destabilizes upstream malE mRNA, but also results in a 9-fold reduction in the synthesis of MalE protein (Newbury et al. 1987b). Other hairpin structures also appear to stabilize selected

regions of polycistronic mRNA (Burton ef al. 1983). In the Bacillus subtilis pur operon, several degradation products of the attenuated mRNA are protected from rapid degradation by secondary structures; some of them accumulate to an estimated steady-state level up to do-fold greater than in the intact attenuated transcript (Ebbole & Zalkin 1988). In the puf operon of ~~odu~acfe~ capsttlafw (formerly referred to as the t-x&A operon of ~~odupseudo~o~ ca~a~afa) there is a potential stem-loop that appears to impart stability to the upper transcripts (Belasco et al. 198.5; Klug ef al. 1987). This differential stability plays a role in the differential expression of genes in this operon and there is a IO:1 ratio of polypeptides synthesized from the 5’ and 3’ genes of the operon. However, Chen et al. (1988) showed that decay of the 3’ segment begins with endonucleolytic cleavage in which the intercistronic stem-loop structure does not participate. They concluded that this mRNA hairpin is necessary but insufficient for the stability of the mRNA upstream of it, and that it functions in message degradation solely as an mRNA decay terminator that protects upstream mRNA segments from degradation by 3’ exoribonucleases. Their findings may explain why E. coli mRNA segments upstream of REP elements are found to decay at different rates (Newbury et al, 1987a, b); these disparate mRNA lifetimes probably reflect differential susceptibility to endonudeolytic cleavage. Additional data also indicate that the efficacy of RNA stem-loop structures as 3’-exonuclease barriers is reduced when they are located in translated regions of the messages (Chen & Belasco 1990). In retroregulation, the destruction of the sib stem-loop structure by RNase III leaves the transcript with an exposed 3’ terminus that leads to its rapid decay (Guameros et al. 1982; Schmeissneref al. 1984). However, RNase III single cleavage at a bacteriophage T7 site has the opposite effect of stabilizing the upstream mRNA (Panayotatos & Truong 1985). Panayotatos & Truong (1985) proposed that single cleavage leaves part of the phage T7 RNase III site in a folded structure at the generated 3’ end and stabilizes the upstream mRNA th at is active, thereby increasing protein levels. Double cleavage at the inf site removes the folded structure and acceleratesdegradation, reducing the synthesis of Int protein. Thus, RNase III cleavages in stem-loop structures may either stabilize mRNA and stimulate gene expression or destabilize a messenger and prevent protein synthesis. This is also another example where the differential expression of genes within an operon is controlled by differential mRNA stability occasioned by different secondary structures.

Role of 5’ Terminal Degradation

Regions

in mRNA

Sequencesat the 5’ end of mRNA may also be involved in the decay process. Most of these regions contain about

twice the length of the leader needed for the expected ribosome recognition site (-5 to - 15 region) and so they are probably more than a short end to contain the Shine-Dalgarno (Shine & Dalgarno 1974) sequence for ribosome binding. Minor variations in the leader region not only result in a differential rate of translation, but also dramatically affect the steady state amount of full-length mRNA (Stanssens et al. 1986; Cho & Yanofsky 1988). Alterations that increase the potential for formation of stable secondary structures in the vicinity of the 1acZ ribosome binding site severely reduce the functional stability of the message without significantly affecting the measurable translation efficiency (Petersen 1991). McCarthy et al. (1986) showed that RNA sequences upstream of the ribosome binding site influence the loading of ribosomes, and depriving a transcript of its ribosomes can destabilize the transcript (Nilsson ef at. 1987). The ribosome loading site would be an excellent candidate for an inactivating target. Also, it could be preferred because this region must have specificity, since it is recognized for initiation from other regions of the mRNA. However, although the sequence is specific it is not unique. The isolation of RNase mutants with slower functional inactivation of messageswould be very helpful in this issue. If decay commences only after the 3’ end is completed then there will be a delay between synthesis and degradation. Cannistraro & Kennel1 (1985) showed that the 5’ end starts to decay very soon after it is made: it is inactivated predominantly by attacks near the ribosomebinding site (Petersen 1991). In the rpsO-pup operon the sequence involved in the stabilization of the pup mRNA is also located at the 5’ end of this message (Portier et al. 1987). RNase III cleavage triggers the decay of the transcripts downstream and in this case the RNase III-deficient mutants have their mRNA half-life considerably increased (Regnier & Gnu&erg-Manago 1990). Belasco et al. (1986) have shown that the relatively long half-life of the E. co/i ompA transcript is determined by a 5’ leader segment that includes the ribosomal binding site and the first few codons. Melefors & Von Gabain (1988) showed that the 5’ non-coding region of ompA mRNA is a target for growth-dependent endonucleolytic attacks, and that the half-life of this mRNA follows the rate of endonucleolytic cleavage. However, the mechanism of mRNA stabilization is not related to the spacing between translating ribosomes (Emory & Belasco 1990). When the 5’ non-coding region of the fi-lactamase (&la) transcript is replaced by the same region of the ompA message the resultant hybrid is four times more stable than the normal &la mRNA (Belascoet al. 1986). The stabilization effect of this monocistronic message appears to involve ribosome interaction, as suggested by the fact that a hybrid transcript with a te~nation codon at the end of the 5’ ompA segment is not stabilized. However, the presence of a stem-loop no more than two

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C. M. Arraiano to four nucleotides from the extreme 5’ terminus of this RNA segment is crucial to its stabilizing influence, whereas the sequence is relatively unimportant (Emory et al. 1992; Bouvet & Belasco 1992). Deletion or change in the location of this stem-loop reduces the half-life of ompA mRNA by a factor of 3. A single-stranded RNA segment, the 3’ terminus of which contains the ribosome-binding site, is another domain that seems to be important for mRNA stabilization. The relative stability of some bacteriophage T4 transcripts can also be attributed to a 5’ leader sequence (Gorski et al. 1985). Transfer of this 5’ leader sequence to an otherwise unmodified lac operon mRNA confers lo-fold stabilization on the hybrid RNA. Duvoisin et al. (1986) took advantage of these properties and constructed hybrid plasmid vectors that contain the promoter region and start codon of T4 gene 32. However, the 5’ stabilization required interaction of the leader sequence with factor(s) produced only in the infected cells (Gorski et al. 1985). The studies of Lin-Chao & Cohen (1991) on the RNA I transcript regulating ColEl plasmid replication indicated that the phosphorylation state of the mRNA 5’ end may be also a determinant of this type of decay. Nevertheless, some processed transcripts are very stable (Mudd et al. 1988; Nilsson & Uhlin 1991) so other factors may also influence S-3’ decay. 5’ Terminal regions can definitely be very important in mRNA degradation but their role is not completely defined. The half-lives of mRNA may vary depending on the susceptibility of sites near the 5’ ends to endonuclease attack, and cleavage at these sites may be influenced by the efficiency of ribosome binding and other factors that can bind to these 5’ regions.

Translation

and mRNA

Stability

The presence or absence of ribosomes and the rate of translation may also affect the half-life of mRNA molecules. If this is a means of control, then differing codon usage might also affect some mRNA (Petersen 1984; Varenne et al. 1984). The reduction in translation rates in streptomycindependent cells (Gupta & Schlessinger 1976), the depletion of ribosomes by heat shock (Har-El et al. 1979) and the blocking of translation with chloramphenicol (Graham et al. 1982) or kasugamycin (Schneider et al. 1978) indicate that translation may play an active role in mRNA degradation. However, antibiotics and heat shock may well perturb the cell and make the results difficult to interpret. For instance, chloramphenicol causes ribosomes to stall but they remain bound to the mRNA, and they could stabilize the mRNA by protecting it from nuclease attack. Studies of translational inhibition are also complicated by the generation of polarity

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distal to the peptide bond site that is affected by the inhibitor. The message distal to the translation block may not be completed (Imarnoto & Kano 1971) and may decay abnormally, thus obscuring the normal decay processes. Von Gabain et al. (1983) showed that the most stable portion of the E. coli ompA transcript lies in the 5’ leader region, which is not covered by ribosomes. The finding that large segments of mRNA can be unprotected by ribosomes without being excessively unstable indicates endonucleolytic target sites (Nilsson et al. 1987; Klug et al. 1987). The ribosome structure surrounding the mRNA could prevent some enzymes from penetrating but smaller enzymes could still have access to cleave the mRNA at specific sites. Actually, enzymatic accessibility experiments show that a few sites along the ribosome-bound mRNA are still accessible to ribonucleases (Kang & Cantor 1985). The position and accessibility of these sites reveal unusual features of ribosome-bound mRNA structure and ribosome-mRNA interactions. The stability of ribosomal protein (r-protein) mRNA in E. coli decreases under conditions in which its translation is feed-back inhibited by repressor r-protein (Singer & Nomura 1985). This shows that translational repression can change the half-life of messenger RNA and altering the site where the operon-specific translational repressor protein interacts can result in a 5-fold increase in half-life (Cole & Nomura 1986). With this elucidation of the translational regulation of r-protein synthesis, it is now apparent that measurement of the mRNA remaining after blocking transcription with rifampicin or other inhibitors may cause complications and inaccuracies in feedback regulated operons. These inhibitors may have other effects on regulatory mechanisms that are important in determining mRNA half-life. In the his operon of Salmonella fyphimurittm the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis-acting element and translation of the cistron immediately downstream of the 5’ end of the processed species (Alifano ef al. 1992). Schneider et al. (1978) said: “The results suggest that neither ribosomes nor translation play an active role in the degradative process. Rather, targets can be protected by the proximity of a ribosome, and without nearby ribosomes the probability of cleavage becomes very high. During normal growth there is a certain probability that any message is in such a vulnerable state, and the fraction of vulnerable molecules determines the inactivation rate of that species”. These affirmations are still valuable 15 years later; many experiments have given controversial results but so far there is no compelling evidence demonstrating that ribosomes play anything more than a passive role in mRNA degradation. Features may vary from transcript to transcript (and indeed there appears to be no general correlation between translational frequency and mRNA stability) and

Bacterial mRNA degradation large fragments remain untranslated without being excessively unstable (Stanssens et al. 1986). Distinguishing between functional inactivation and chemical decay has been useful in studies of mRNA turnover. Functional inactivation is an alteration of an mRNA species that renders it unsuitable for further translation. Chemical decay, on the other hand, refers to the degradation of an mRNA species to oligo- or mononucleotides. It was shown that the two processes are temporally distinct in 1970 (Schwartz et al. 1970). Petersen et al. (1978) calculated the functional half-life of individual mRNA species from E. co/i by measuring the decay in their capacity to synthesize proteins after complete inhibition of transcription. They found that the decay rates measured showed a large spectrum of half-lives ranging from 40 s to approximately 20 min. Chemical decay is thought to result largely from the action of 3’ to 5’ exonucleases, starting from the 3’ end of the message or from internal, endonuclease induced breaks or from both (Lim & Kennel1 1979). Translational inactivation probably involves specific endonucleolytic cleavage at the 5’ end of an mRNA molecule, resulting in the destruction (or blockage) of the ribosome-binding site or proximal coding sequences (Cannistraro et al. 1986). In the ams-1 temperature sensitive mutant the chemical mRNA half-life is increased at the non-permissive temperature but the functional half-life is unaltered (Ono & Kuwano 1979). If functional inactivation precedes mRNA decay it may be the result of a cleavage and this cleavage might be the initial event in mRNA degradation. The inactivated messages could have an increased chemical half-life, explaining why increased stability does not necessarily lead to increased expression. It is important to mention that mechanisms of functional inactivation other than endonucleolytic cleavage are possible. For example, the binding of a protein or RNA to the ribosome-binding site of an mRNA could block further ribosome addition (Singer & Nomura 1985). Processing of mRNA by RNase III regulates expression of gene 7.2 of bacteriophage T7; the cleavage exposes the 3’ end of mRNA which can hybridize to an upstream ribosome-binding site, inhibiting translation (Saito & Richardson 1981). Another type of functional inactivation can be mediated by an anti-sense RNA if it hybridizes to the ribosome-binding site (Mizuno et al. 1984). This form of regulation by translational inhibitors may apply to other groups of mRNA. The isolation of RNase mutants with slower functional inactivation of messages would be extremely useful in understanding mRNA decay. Results so far are more consistent with models in which the rate-determining event in mRNA decay is functional inactivation, with a critical competition and interplay between proteins and possibly mRNA that determine the loading rate of ribosomes at initiation codons.

Directionality

of mRNA

Decay

The chemical decay of many mRNA cistrons occurs in a net Y-3 direction, even though no Y-3’ exonuclease has ever been purified from E. cofi. Endonucleolytic cleavage towards the 5’ end of a molecule, followed by 3’-5’ exonucleolytic attack would result in an apparent S-3’ direction; this could be a reasonable explanation for the results observed (Apirion & Gegenheimer 1984). In the gal operon, mRNA from the middle cistron, galT, has a shorter half-life than that of either of the two flanking genes (Achord & Kennel1 1974). This data also supports the idea that degradation occurs by the concerted action of endonucleases and exonucleases. An exclusive 3’ to 5’ decay would be very inefficient biologically. Many proteins would be incomplete and inactive and the cell would waste much energy in a useless protein synthesis. However, an inactivation at the 3’ end would block new functional protein synthesis immediately, while attack at the 5’ coding region would not result in cessation of synthesis until the runoff of all the ribosomes. In bacteriophage infection, the rapidity with which the synthesis of a certain protein is shut off might be more important than energy efficiency. In fact, the int message is inactivated by RNase III cleavage at the 3’ end and then the message is degraded 3’ to 5’ (Schmeissner et al. 1984). In the B. subtilis pur operon, Ebbole & Zalkin (1988) proposed that the degradation of the attenuated transcript is initiated by an endonucleolytic cleavage in a single-stranded region in the middle of the transcript. The half-life measurements indicated that the degradation of the 3’-half proceeded faster than that of the 5’ end and the proposed scheme of decay involved a series of endonucleolytic cleavages followed by processive 3’ exonuclease trimming up to a base-paired secondary structure. The degradation of E. coli trxA mRNA also involves specific endonucleolytic cleavages (Arraiano et al. 1993). The 5’ ends remain intact in the majority of the degradation products and so the 3’ ends are preferentially cleaved. The decay process proceeds through a progression of endonucleolytic cleavages in a net 3’ to 5’ direction. The structural determinants claimed to be stabilizing influences can be located at either end of the messenger. This implies that degradation can start either at the 5’ or 3’ end depending on the RNA species. The mRNA decay can be a multicomponent process in which many RNases could intervene. In any multihit process the rate will only become faster if the slowest event, or rate-limiting step, becomes faster. The directionality of the decay process would be determined by the localization of the rate-limiting target sites.

Concluding

Remarks

The degradation of mRNA can depend on determinants localized on specific mRNA fragments and the determinants

CM. Arraiano claimed to be stabilizing influences can be located at either end of the transcript. However, other factors, such as the presence of ribosomes or bound proteins, may be involved in the regulation of the decay mechanism. The selectivity of mRNA decay is best explained by the action of specific factors that recognize unique sites on the mRNA chains. A rate-limiting endonucleolytic cut, triggered by such an interaction, would be followed by rapid destruction of the mRNA. It is the combination of endo- and exo-nucleolytic degradation which leads to the wide variety of degradation patterns which is proving so difficult to unravel (see Figure 1).

Secondary structure can modulate gene expression. The combination of the mRNA sequence with its potential for secondary structures will give the particular degradation profile of that message. In some cases,secondary structures protect the RNA; this is apparently the case for tRNA and rRNA which otherwise might be unstable. Particular configurations at the 3’ termini appear to impart stability to upstream mRNA segments. Sequenceslocated proximal to a cistron can also influence its half-life. These possibilities need not be mutually exclusive and sequencesat the 3’ end and at the 5’ end could operate upon a single cistron.

I

Repressor PNPase

Figure 1. The figure illustrates diagrammatically the perspective of mRNA decay presented in this review. Several pathways can be outlined, according to the initial and rate-limiting step in decay. (A) Possible ways of degrading the 5’ end. No 5’ exonucleases have been identified for bacterial mRNA degradation and so the nature of a processive decay in the 5’ to 3’ direction is completely unknown. However, some studies suggested that the phosphorylation state of the mRNA 5’ end may be important and in some cases species carrying a 5’ mono-phosphate decay faster than those that carry a 5’ tri-phosphate (1). Enzymes like RNase Ill can cleave endonucleolytically secondary structures located at the 5’ non-translated region (2). Other endonucleases, such as RNase E, can also cleave in this leader region, and their cleavages may require active translation and/or the proximity of stem-loops (3). Factors such as repressors, secondary structures and ribosome loading may also interfere with the degradation of the 5’ region, accelerating or stabilizing its decay process (4). (B) Degradation of the internal segment. A combination of endonuclease cleavages follow 3’ exonucleolytic degradation in the translated region. The cleavages may either be directly inactivating or they may trigger functional inactivation, removing barriers protecting against processive degradation reactions. Changes in the spacing of ribosomes on the message will influence its rate of degradation according to the extent to which they occlude sites of rate-determining endonucleolytic cleavage. (C) Possible ways of degrading the 3’ end. An mRNA 3’ end is generated by transcription termination, and can be degraded by the 3’ to 5’ end exonucleases RNase II and PNPase (1). The processive progression of the exonuclease RNase II is easily stopped by terminal stem-loops, but in some cases PNPase can still overcome these secondary structures (2). The rate at which these exonucleases penetrate this barrier is slow and RNA binding proteins seem to be involved in delaying this decay (3). Sometimes upstream endonucleolytic cleavages become the initial and rate limiting step in the degradation of the 3’ end (4). This upstream cleavage is probably followed by 3’ exonucleolytic digestion of the oligonucleotides generated.

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Bacterial mRNA degradation Secondary structures can be cleaved by double-strandspecific RNases such as RNase III. RNase III, in addition to being autoregulatory, is involved in the expression of PNPase, a 3’-processive exonuclease. The synthesis of the two RNases may be controlled by a common mechanism and it is possible that other genes or operons also belong to what can be called the RNase III regulon. Every mRNA has a sequence that is complementary to some sequence at the 3’ end of the 165 rRNA, but the exact sequence, as well as the number of nucleotides to which it is complementary, varies from rRNA to mRNA; that is, it is a specific sequence but not a unique one. The availability of this sequence as a target could depend on several factors, such as secondary structure or protection by ribosomes, and these could modulate its vulnerability. So far there are no consensus sequences or structures near the starts of E. coli messages, that would provide a common target for functional inactivation. Efforts to isolate RNase mutants with slower functional inactivation of messages have been unsuccessful. If nothing is unique in terms of sequence or structure, then it would follow that there is no enzyme that is specific for the inactivation of mRNA, i.e. the activity has a broad recognition specificity. Faster degradation cannot result from an increase in the size of the target, in terms of number of nucleotides, but from its vulnerability. mRNA in bacteria may be inactivated by a number of parallel mechanisms acting independently on different target sites. In this case no single step is rate-limiting; the observed rate of inactivation is given by the sum of the contributions from the individual mechanisms and thus will be dominated by the fastest mechanism that can be rate-determining (Petersen 1991). To analvse mRNA turnover it is extremelv useful to construct single and multiple isogenic mutants deficient in factors involved in this mechanism. Factors that control mRNA decay can be important elements in the post-transcriptional regulation of gene expression. By comparing the wild type with each mutant constructed specific functions can be assigned to each gene studied. mRNA stability has been shown to be very important for differential gene expression. As there are many prokaryotic operons in which the genes are expressed at disparate levels, it certainly seems likely that there will be many other examples in which differential mRNA stability determines relative expression. Analysis of the stabilizing sequences can lead to the construction of new vectors that can stabilize mRNA and maximize gene expression from cloned genes. It seems likely that very stable hybrid mRNA may be constructed for most genes by the addition of efficient barriers against 5’ and 3’ processive decay, and by fusion to ribosome binding sites having little or no potential for the formation of inhibitory secondary structures. Future work will involve the identification and study of I

the mechanism of action of more RNases, relating these RNases to mRNA decay through the isolation of mutants, and assessing whether the various reactions are regulated. Novel approaches involving the use of cell-free systems, and the identification of cleavage sites that trigger mRNA decay, will be required to achieve further comprehension of this important aspect of gene expression. Much more work will be required to fully understand the process of mRNA degradation.

Acknowledgement I thank Rita Zilhso for useful help with order to draw the figure.

the computer

in

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(Accepted

5 April

1993)

Post-transcriptional control of gene expression: bacterial mRNA degradation.

Many biological processes cannot be fully understood without detailed knowledge of RNA metabolism. The continuous breakdown and resynthesis of prokary...
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