Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile.

Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria.

Both temperature and medium composition regulate RNase E processing efficiency of the rpsO mRNA coding for ribosomal protein S15 of Escherichia coli.

Cleavage by RNase E is believed to be the rate-limiting step in the degradation of many RNAs. These cleavages are modulated by 5' end-phosphorylation, folding and translation of the mRNA in question. Here, we present data suggesting that these cleavages are also regulated by environmental conditions. We report that rpsO mRNA, 15 minutes after a shift to 44 degrees C, is stabilized in cells grown in minimal medium. This stabilization is correlated with a reduction in the efficiency of the RNase E cleavage which initiates its decay. We also observe the appearance of RNA fragments previously detected following RNase E inactivation and a defect in the adaptation of RNase E concentration. These observations, coupled to the fact that RNase E overproduction slightly reduces the accumulation of the rpsO mRNA, suggest that this stabilization is caused in part by a limitation in RNase E concentration. An increase in the steady-state level of rpsT mRNA is also observed following a shift to 44 degrees C in minimal medium; however, processing of the 9 S rRNA precursor is not affected under these conditions. We thus propose that RNase E concentration changes in the cell in response to environmental conditions and that these changes can selectively affect the processing and the stability of individual mRNAs. Our data also indicate that the efficiency of cleavage of the rpsO mRNA by RNase E is modified by other factor(s) which remain to be identified.

RNase II removes the oligo(A) tails that destabilize the rpsO mRNA of Escherichia coli.

Polyadenylation controls mRNA stability in procaryotes, eucaryotes, and organelles. In bacteria, oligo(A) tails synthesized by poly(A) polymerase I are the targets of the 3'-to-5' exoribonucleases: polynucleotide phosphorylase and RNase II. Here we show that RNase II very efficiently removes the oligo(A) tails that can be used as binding sites by PNPase to start degradation of the rpsO mRNA. Both enzymes are impeded by the secondary structure of the transcription terminator at the 3' end of the mRNA. RNase II mostly generates tailless transcripts harboring 2 unpaired nt downstream of the transcription terminator hairpin, whereas PNPase releases molecules that exhibit a single-stranded stretch of 5-7 nt terminated by a tail of 3-5 As. The rpsO mRNAs whose oligo(A) tails have been removed by RNase II are more stable than oligoadenylated molecules that occur in strains deficient for RNase II. Moreover, the rpsO mRNA is stabilized when RNase II is overproduced. This modulation of mRNA stability by RNase II is only observed when poly(A) polymerase I is active. These in vivo data demonstrate that RNase II protects mRNAs ending by stable terminal hairpins, such as primary transcripts, from degradation by poly(A)-dependent ribonucleases.

Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I.

Current evidence suggests that the length of poly(A) tails of bacterial mRNAs result from a competition between poly(A) polymerase and exoribonucleases that attack the 3' ends of RNAs. Here, we show that host factor Hfq is also involved in poly(A) tail metabolism. Inactivation of the hfq gene reduces the length of poly(A) tails synthesized at the 3' end of the rpsO mRNA by poly(A) polymerase I in vivo. In vitro, Hfq stimulates synthesis of long tails by poly(A) polymerase I. The strong binding of Hfq to oligoadenylated RNA probably explains why it stimulates elongation of primers that already harbor tails of 20-35 A. Polyadenylation becomes processive in the presence of Hfq. The similar properties of Hfq and the PABPII poly(A) binding protein, which stimulates poly(A) tail elongation in mammals, indicates that similar mechanisms control poly(A) tail synthesis in prokaryotes and eukaryotes.

E. coli RpsO mRNA decay: RNase E processing at the beginning of the coding sequence stimulates poly(A)-dependent degradation of the mRNA.

The rpsO mRNA of E. coli encoding ribosomal protein S15 is destabilized by poly(A) tails posttranscriptionally added by poly(A)polymerase I. We demonstrate here that polyadenylation also contributes to the rapid degradation of mRNA fragments generated by RNase E. It was already known that an RNase E cleavage occurring at the M2 site, ten nucleotides downstream of the coding sequence of rpsO, removes the 3' hairpin which protects the primary transcript from the attack of polynucleotide phosphorylase and RNase II. A second RNase E processing site, referred to as M3, is now identified at the beginning of the coding sequence of rpsO which contributes together with exonucleases to the degradation of messengers processed at M2. Cleavages at M2 and M3 give rise to mRNA fragments which are very rapidly degraded in wild-type cells. Poly(A)polymerase I contributes differently to the instability of these fragments. The M3-M2 internal fragment, generated by cleavages at M3 and M2, is much more sensitive to poly(A)-dependent degradation than the P1-M2 mRNA, which exhibits the same 3' end as M3-M2 but harbours the 5' end of the primary transcript. We conclude that 5' extremities modulate the poly(A)-dependent degradation of mRNA fragments and that the 5' cleavage by RNase E at M3 activates the chemical degradation of the rpsO mRNA.

The rpsO mRNA of Escherichia coli is polyadenylated at multiple sites resulting from endonucleolytic processing and exonucleolytic degradation.

The rpsO monocistronic messenger, encoding ribosomal protein S15, is destabilized upon polyadenylation occurring at the hairpin structure of the transcription terminator t1. We report that mRNA fragments differing from the monocistronic transcript by their 3' termini are also polyadenylated in the absence of polynucleotide phosphorylase and RNase II. Some of these 3' extremities result from endonucleolytic cleavages by RNase E and RNase III and from exonucleolytic degradation. Most of these mRNA fragments are destabilized upon polyadenylation with the exception of the RNA species generated by RNase III. RNase E appears to reduce the amount of poly(A) added at the transcription terminator t1.

Polynucleotide phosphorylase is required for the rapid degradation of the RNase E-processed rpsO mRNA of Escherichia coli devoid of its 3' hairpin.

The monocistronic transcript of rpsO undergoes an endonucleolytic cleavage downstream of the coding sequence, which removes the hairpin of the transcription terminator and initiates the rapid degradation of the message. We demonstrate here that the two rne-dependent cleavages, on both sides of the transcription terminator, are catalysed by RNase E in vitro and that the RNase E-processed rpsO message is rapidly degraded by polynucleotide phosphorylase, while RNase II produces stable decay intermediates. Moreover, we show that RNase E cuts in vitro the coding sequence of the rpsO mRNA at several sites which are not detected in vivo.

Multiple degradation pathways of the rpsO mRNA of Escherichia coli. RNase E interacts with the 5' and 3' extremities of the primary transcript.

The degradation process of the rpsO mRNA is one of the best characterised in E coli. Two independent degradation pathways have been identified. The first one is initiated by an RNase E endonucleolytic cleavage which allows access to the transcript by polynucleotide phosphorylase and RNase II. Cleavage by RNase E gives rise to an rpsO message lacking the stabilising hairpin of the primary transcript; this truncated mRNA is then degraded exonucleolytically from its 3' terminus. This pathway might be coupled to the translation of the message. The second pathway allows degradation of polyadenylated rpsO mRNA independently of RNase II, PNPase and RNase E. The ribonucleases responsible for degradation of poly(A) mRNAs under these conditions are not known. Poly(A) tails have been proposed to facilitate the degradation of structured RNA by polynucleotide phosphorylase. In contrast, we believe that removal of poly(A) by RNase II stabilises the rpsO mRNA harbouring a 3' hairpin. In addition to these two pathways, we have identified endonucleolytic cleavages which occur only in strains deficient for both RNase E and RNase III suggesting that these two endonucleases protect the 5' leader of the mRNA from the attack of unidentified ribonuclease(s). Looping of the rpsO mRNA might explain how RNase E bound at the 5' end can cleave at a site located just upstream the hairpin of the transcription terminator.

Improved recombinant tandem expression of translation initiation factor IF2 in RNASE E deficient E. coli cells.

The prokaryotic translation initiation factor IF2 exists in a varying number of nested forms in different species. In E. coli three natural forms exist, IF2 alpha, IF2 beta and IF2 gamma differing only in the N-terminal: IF2 beta and IF2 gamma lack 158 and 165 amino acid residues, respectively, as compared to IF2 alpha. We have earlier shown that the smaller forms of IF2 are not the result of a specific proteolysis of IF2 alpha, but produced from individual translation initiation sites in the mRNA. However it has not been known whether the expression in E. coli of IF2 beta and IF2 gamma is dependent on or related to a posttranscriptional processing of the polycistronic nusA operon, containing infB, the gene for IF2. Here we have used S1 mapping to study the existence of such mRNA processing in the region between the initiation sites for IF2 alpha and IF2 beta/IF2 gamma. The results show a Ribonuclease E cleavage site at position +200 in the infB mRNA between the translation initiation sites. However, studies of the overexpression of the different forms of IF2 show that the relative expression of IF2 alpha and IF2 beta/IF2 gamma is independent of RNase E activity. Thus E. coli exhibits a true tandem translation of intact infB mRNA with multiple in-frame translation initiation sites resulting in gene products of different sizes. An additional observation is a significant increase in the level of overexpression of IF2 in cells devoid of RNase E activity. We conclude that due to lack of RNase E activity, the amount of plasmid-transcribed infB mRNA available for translation is accumulated, resulting in an elevated amount of recombinant IF2. This observation may have a more general application within the field of recombinant protein production and expression efficiency.

Polyadenylylation destabilizes the rpsO mRNA of Escherichia coli.

The rpsO mRNA, encoding ribosomal protein S15, is only partly stabilized when the three ribonucleases implicated in its degradation--RNase E, polynucleotide phosphorylase, and RNase II--are inactivated. In the strain deficient for RNase E and 3'-to-5' exoribonucleases, degradation of this mRNA is correlated with the appearance of posttranscriptionally elongated molecules. We report that these elongated mRNAs harbor poly(A) tails, most of which are fused downstream of the 3'-terminal hairpin at the site where transcription terminates. Poly(A) tails are shorter in strains containing 3'-to-5' exoribonucleases. Inactivation of poly(A) polymerase I (pcnB) prevents polyadenylylation and stabilizes the rpsO mRNA if RNase E is inactive. In contrast polyadenylylation does not significantly modify the stability of rpsO mRNA undergoing RNase E-mediated degradation.

Roles of RNase E, RNase II and PNPase in the degradation of the rpsO transcripts of Escherichia coli: stabilizing function of RNase II and evidence for efficient degradation in an ams pnp rnb mutant.

The Escherichia coli rpsO gene gives rise to different mRNA species resulting either from termination of transcription or from processing of primary transcripts by RNase E and RNase III. The main degradation pathway of these transcripts involves a rate-limiting RNase E cleavage downstream of the structural gene which removes the 3' terminal stem-loop structure of the transcription terminator. This structure protects the message from the attack of 3'-5' exonucleases and its removal results in very rapid degradation of the transcript by polynucleotide phosphorylase and RNase II. Polynucleotide phosphorylase is also able to degrade slowly the mRNA harboring the 3' terminal hairpin of the terminator. In contrast, RNase II appears to protect the rpsO mRNA species which retains the 3' hairpin structure. Rapid degradation of the rpsO mRNA is observed after inactivation of RNase II even in a strain deficient for RNase E and polynucleotide phosphorylase. The enzyme(s) involved in this degradation pathway is not known. We detected an unstable elongated rpsO mRNA presumably resulting from the addition of nucleotides at the 3' end of the transcript.

Nucleolytic inactivation and degradation of the RNase III processed pnp message encoding polynucleotide phosphorylase of Escherichia coli.

The two cleavages made by RNase III in the transcripts of the pnp gene of Escherichia coli, 80 nucleotides upstream of the coding sequence of polynucleotide phosphorylase, were previously demonstrated to trigger the rapid degradation of the pnp messenger. In this paper, we demonstrate that the 5' end of the RNase III processed pnp mRNA is attacked by ribonucleases more efficiently than the rest of the molecule. Several 5' extremities resulting from cleavages occurring in the first 500 nucleotides of the pnp transcript have been identified. Three of them referred to as X, Y and W occur in the wild-type strain at the beginning of the coding sequence of the pnp mRNA. The mRNA appears to be cleaved more efficiently at the X site, proximal to the initiation codon, than at sites Y and W located downstream. In vitro, the maturation at X is catalysed by RNase E but not by RNase III. Accumulation of RNA processed at X in RNase E deficient strains leads us to postulate that X is a high affinity primary site which is slowly cleaved by the residual activity of thermosensitive RNase E at non-permissive temperature and that secondary sites located downstream are processed less efficiently than X. Taken together, our results suggest that in wild-type E. coli the degradation of the RNase III processed mRNA is mediated by RNase E.

Decay of mRNA encoding ribosomal protein S15 of Escherichia coli is initiated by an RNase E-dependent endonucleolytic cleavage that removes the 3' stabilizing stem and loop structure.

The transcripts of the rpsO-pnp operon of Escherichia coli, coding for ribosomal protein S15 and polynucleotide phosphorylase, are processed at four sites in the 249 nucleotides of the intercistronic region. The initial processing step in the decay of the pnp mRNA is made by RNase III, which cuts at two sites upstream from the pnp gene. The other two cleavages are dependent on the wild-type allele of the rne gene, which encodes the endonucleolytic enzyme RNase E. The cuts are made 37 nucleotides apart at the base of the stem-loop structure of the rho-independent attenuator located downstream from rpsO. The cleavage downstream from the attenuator generates an rpsO mRNA.nearly identical with the monocistronic attenuated transcript, while the cleavage upstream from the transcription attenuator gives rise to an rpsO mesage lacking the terminal 3' hairpin structure. The rapid degradation of the processed mRNA in an rne+ strain, compared to the slow degradation of the transcript that accumulates in an rne- strain, suggests that RNase E initiates the decay of the rpsO message by removing the stabilizing stem-loop at the 3' end of the RNA.

An electrophoretic method for the purification of RNA regions involved in protein crosslinking.

Direct information about structural interactions in ribonucleoprotein complexes can be obtained from crosslinking data. The purification of specific complexes, i.e., their separation from noncrosslinked proteins, from free RNA, and from other complexes, is essential for the identification of the bound proteins and the precise localization of their attachment sites in RNA. We describe a two-dimensional denaturing gel system which achieves this purification; in the first dimension basic proteins do not enter the gel and RNA--protein complexes are slowed down compared to protein free RNA, and in the second dimension sodium dodecyl sulfate improves the separation between the different complexes on the basis of their protein content.

New RNA-protein crosslinks in domains 1 and 2 of E. coli 30S ribosomal subunits obtained by means of an intrinsic photoaffinity probe.

Functionally active 70S ribosomes containing 4-thiouridine (s4U) in place of uridine were prepared by a formerly described in vivo substitution method. Proteins were crosslinked to RNA by 366 nm photoactivation of s4U. We observe the systematic and characteristic formation of 30S dimers; they were eliminated for analysis of RNA-protein crosslinks. M13 probes containing rDNA inserts complementary to domains 1 and 2 of 16S RNA from the 5'end up to nucleotide 868 were used to select contiguous or overlapping RNA sections. The proteins covalently crosslinked to each RNA section were identified as S3, S4, S5, S7, S9, S18, S20 and S21. Several crosslinks are compatible with previously published sites for proteins S5, S18, S20 and S21; others for proteins S3, S4, S7, S9, S18 correspond necessarily to new sites.

Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA. Its use for E. coli ribosome structural analysis.

In vivo incorporation of the uridine-photoactivable analogue, 4-thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated. It is highly dependent on the exogenous uridine and 4-thiouridine concentrations as well as on temperature. We have defined conditions allowing the substitution of 13 +/- 2% of the uridine residues in bulk RNA by 4-thiouridine. On a high-Mg2+ sucrose gradient, 33 +/- 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non-associated 50S and 30S particles containing immature rRNA. The thiolated 70S ribosomes tolerate a 4-5% substitution level (40 thiouridine molecules/particle). Surprisingly, 3-4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4-thiouridine-substituted rRNA. Specific 366-nm photoactivation increased this proportion to 10-12%, i.e. up to six or seven ribosomal protein molecules/particle. The photochemical cross-linking proceeds with apparent first-order kinetics with a quantum yield close to 5 X 10(-3). Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross-linking were independent of oxygenation conditions. The thiolated (4.5%) 70S ribosomes allowed the poly(U)-directed poly(Phe)synthesis at 48% the control rate. Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively.

Identification of form III conformers in tRNAPhe from Escherichia coli by intramolecular photo-cross-linking.

In the absence of divalent cations, at neutral pH, low ionic strength, and low to moderate temperature, tRNAs are known to be in a denatured form, designated form III in the tRNA phase diagram by Cole et al. [Cole, P. E., Yang, S. R., & Crothers, D. M. (1972) Biochemistry 11, 4358-4368]. Form III tRNAPhe from Escherichia coli has been studied at pH 7, 5 mM Na+, and 10 degrees C. As judged from ethidium bromide intercalation, it exhibits extensive secondary structure. 4-Thiouridine in position 8 of the tRNAPhe sequence was used as a built-in photoaffinity probe. Spectroscopic and spectrofluorometric analysis in the near-UV range of form III tRNAPhe irradiated with broad-band near-UV light to completion of the reaction before or after reduction with NaBH4 revealed that the Pdo(4-5)Cyt (8-C) and Pdo(4-5)Urd (8-U) adducts form in equimolar yield. In different experiments, the overall yield of s4U conversion to these adducts varies between 20 and 40%. The remaining s4U is photolyzed to weakly absorbing product(s) in the near-UV range. The disappearance of s4U follows biexponential kinetics while the 8-C adduct formation follows monoexponential kinetics, indicating the presence of at least two tRNA classes of conformers, not in equilibrium on the time scale of the reaction. Migration on a denaturing polyacrylamide gel of irradiated form III tRNAPhe revealed three main bands, D1, D2, and D3, and no slowly migrating tRNA dimers. D1 migrates at the control position and presumably contains the photolysis product(s) P. The fast-migrating D2 and D3 bands contain 8-Pyr cross-links which were identified by sequence analysis as 8-(66-68) in D2 and 8-(40-43) and 8-(59-62) in D3. On the basis of these data, it is proposed that the minor poorly photoreactive class II conformers are the cloverleaf and close variants, whereas the major class I cross-linkable conformers are essentially long-extended secondary structures. Clearly, our data demonstrate the polymorphism of form III tRNAPhe.

Multiple crosslinks of proteins S7, S9, S13 to domains 3 and 4 of 16S RNA in the 30S particle.

Functionally active 70S ribosomes containing 4-thiouracil in place of uracil (substitution level 2%) were prepared by an in vivo substitution method. RNA-protein crosslinks were introduced by 366 nm photoactivation of 4-thiouracil in the purified 30S subunits. Seven single stranded M13 probes containing rDNA inserts complementary to domains 3 and 4 of 16S RNA were constructed. These inserts approximately 100 nucleotides long starting at nucleotide 868 and ending at the 3' OH terminus were used to select contiguous RNA sections. The proteins covalently linked to each selected RNA section were identified by 2D gel electrophoresis. Proteins S7, S9, S13 were shown to be efficiently crosslinked to multiple sites belonging to both domains.