Mechanism and physiological significance of autoregulation of the Escherichia coli hfq gene.

The RNA chaperone Hfq plays a critical role in sRNA-mediated gene regulation in enteric bacteria. The major role of Hfq is to stimulate base-pairing between sRNAs and target mRNAs by binding both RNAs through three RNA-binding surfaces. To understand the post-transcriptional network exerted by Hfq and its associated sRNAs, it is important to know how the cellular concentration of Hfq is regulated. While an early study showed that hfq translation is repressed by Hfq, the detailed mechanism and biological significance of the hfq autoregulation remain to be studied. Here, we show that the synthesis of Hfq is strictly autoregulated to maintain the cellular concentration of Hfq within a limited range even when the hfq mRNA is overexpressed from a plasmid-borne hfq gene. Mutational and biochemical studies demonstrate that Hfq represses its own translation primarily by binding to the hfq mRNA through the distal face. The growth of cells harboring the hfq plasmid is markedly inhibited due to an increased Hfq level when the distal face of Hfq is mutated or the 5'-UTR of hfq is mutated. A mutation in the rim suppresses the growth inhibition caused by the distal face mutation, suggesting that the interaction of Hfq with undefined RNAs through the rim is responsible for the growth inhibition by the increased Hfq level. In addition, the data suggest that the hfq autoregulation operates not only in cells harboring a multicopy hfq gene but also in the wild-type cells.

Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs.

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3' end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs.

Inhibition of Phagocytic Killing of Escherichia coli in Drosophila Hemocytes by RNA Chaperone Hfq.

An RNA chaperone of Escherichia coli, called host factor required for phage Qß RNA replication (Hfq), forms a complex with small noncoding RNAs to facilitate their binding to target mRNA for the alteration of translation efficiency and stability. Although the role of Hfq in the virulence and drug resistance of bacteria has been suggested, how this RNA chaperone controls the infectious state remains unknown. In the present study, we addressed this issue using Drosophila melanogaster as a host for bacterial infection. In an assay for abdominal infection using adult flies, an E. coli strain with mutation in hfq was eliminated earlier, whereas flies survived longer compared with infection with a parental strain. The same was true with flies deficient in humoral responses, but the mutant phenotypes were not observed when a fly line with impaired hemocyte phagocytosis was infected. The results from an assay for phagocytosis in vitro revealed that Hfq inhibits the killing of E. coli by Drosophila phagocytes after engulfment. Furthermore, Hfq seemed to exert this action partly through enhancing the expression of σ(38), a stress-responsive σ factor that was previously shown to be involved in the inhibition of phagocytic killing of E. coli, by a posttranscriptional mechanism. Our study indicates that the RNA chaperone Hfq contributes to the persistent infection of E. coli by maintaining the expression of bacterial genes, including one coding for σ(38), that help bacteria evade host immunity.

Insights into transcription termination of Hfq-binding sRNAs of Escherichia coli and characterization of readthrough products.

The genes encoding Hfq-dependent sRNAs possess a typical Rho-independent transcription terminator. Here, we have studied the molecular events occurring at Rho-independent terminators of sRNA genes, focusing on two well-characterized Hfq-binding sRNAs, SgrS and RyhB. We constructed several hybrid genes in which the DNA sequence corresponding to a strong Rho-independent terminator was placed just downstream from the Rho-independent terminators of sRNA genes. By using this system, we demonstrate that transcripts frequently read through the Rho-independent terminators of sgrS and ryhB in normally growing cells. We show that Hfq does not affect the transcriptional readthrough event itself. We also find that the readthrough products no longer bind to Hfq in vivo. We have developed a competition assay based on a biotin-streptavidin system to analyze the interaction of Hfq and a particular RNA molecule in vitro. By using this method, we verify that the 3'-extended form of SgrS does not bind to Hfq in vitro. Finally, we demonstrate that transcription termination is significantly enhanced under stress conditions where transcription initiation of sRNA genes on the chromosome is induced. We conclude that the production of sRNAs is regulated not only at the step of transcription initiation but also at the step of transcription termination. The mechanism by which transcription termination is enhanced under stress conditions remains to be understood.

Photo-induced regulation of the chromatic adaptive gene expression by Anabaena sensory rhodopsin.

Rhodopsin molecules are photochemically reactive membrane-embedded proteins, with seven transmembrane α-helices, which bind the chromophore retinal (vitamin A aldehyde). They are roughly divided into two groups according to their basic functions: (i) ion transporters such as proton pumps, chloride pumps, and cation channels; and (ii) photo-sensors such as sensory rhodopsin from microbes and visual pigments from animals. Anabaena sensory rhodopsin (ASR), found in 2003 in the cyanobacterium Anabaena PCC7120, is categorized as a microbial sensory rhodopsin. To investigate the function of ASR in vivo, ASR and the promoter sequence of the pigment protein phycocyanin were co-introduced into Escherichia coli cells with the reporter gene crp. The result clearly showed that ASR functions as a repressor of the CRP protein expression and that this is fully inhibited by the light activation of ASR, suggesting that ASR would directly regulate the transcription of crp. The repression is also clearly inhibited by the truncation of the C-terminal region of ASR, or mutations on the C-terminal Arg residues, indicating the functional importance of the C-terminal region. Thus, our results demonstrate a novel function of rhodopsin molecules and raise the possibility that the membrane-spanning protein ASR could work as a transcriptional factor. In the future, the ASR activity could be utilized as a tool for arbitrary protein expression in vivo regulated by visible light.

Detection of sRNA-mRNA interactions by electrophoretic mobility shift assay.

Electrophoretic mobility shift assay is a simple, rapid, and sensitive technique to analyze the RNA-RNA interaction. A (32)P-labeled RNA is incubated with another unlabeled RNA and subjected to electrophoresis on a native polyacrylamide gel. If two RNA molecules base pair stably, the movement of the probe RNA through the gel is retarded resulting in a characteristic band corresponding to the RNA duplex. Here, we describe the methods to study the interaction of an Hfq-binding small RNA (sRNA) and its target mRNA. Although we focus on the interaction of SgrS and its target ptsG mRNA, the methods can be applied to the analysis of base pairing between any sRNAs and their targets.

The functional Hfq-binding module of bacterial sRNAs consists of a double or single hairpin preceded by a U-rich sequence and followed by a 3' poly(U) tail.

Hfq-dependent sRNAs contain, at least, an mRNA base-pairing region, an Hfq-binding site, and a Rho-independent terminator. Recently, we found that the terminator poly(U) of Escherichia coli sRNAs is essential for Hfq binding and therefore for riboregulation. In this study, we tried to identify additional components within Hfq-binding sRNAs required for efficient Hfq binding by using SgrS as a model. We demonstrate by mutational and biochemical studies that an internal hairpin and an immediately upstream U-rich sequence also are required for efficient Hfq binding. We propose that the functional Hfq-binding module of SgrS consists of an internal hairpin preceded by a U-rich sequence and a Rho-independent terminator with a long poly(U) tail. We also show that the Rho-independent terminator alone can act as a functional Hfq-binding module when it is preceded by an internal U-rich sequence. The 3' region of most known sRNAs share the features corresponding to either a double- or single-hairpin-type Hfq-binding module. We also demonstrate that increasing the spacing between the base-pairing region and the Hfq-binding module reduces or impairs the silencing ability. These findings allowed us to design synthetic Hfq-binding sRNAs to target desired mRNAs.

PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action.

Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs.

RNase E action at a distance: degradation of target mRNAs mediated by an Hfq-binding small RNA in bacteria.

A major class of bacterial small RNAs (sRNAs), along with RNA-binding protein Hfq and endoribonuclease RNase E, acts on target mRNAs through base-pairing, leading to translational repression and rapid degradation of the mRNAs. In this issue of Genes & Development, Prévost and colleagues (pp. 385-396) demonstrate by using the well-characterized sRNA RyhB that RNase E cleavage at sites distal from the pairing region triggers degradation of target mRNAs. The study has provided an important insight into the initial events of sRNA-induced degradation of target mRNAs.

Hfq binding at RhlB-recognition region of RNase E is crucial for the rapid degradation of target mRNAs mediated by sRNAs in Escherichia coli.

An RNA chaperon Hfq along with Hfq-binding sRNAs stably binds to RNase E in Escherichia coli. The role of the Hfq-RNase E interaction is to recruit RNase E to target mRNAs of sRNAs resulting in the rapid degradation of the mRNA-sRNA hybrid. The C-terminal scaffold region of RNase E is responsible for the interaction with Hfq. Here, we demonstrate that the scaffold region can be deleted up to residue 750 without losing the ability to cause the rapid degradation of target mRNAs mediated by Hfq/sRNAs. The truncated RNase E750 can still bind to Hfq although the truncation significantly reduces the Hfq-binding ability. We conclude that the subregion between 711 and 750 is sufficient for the functional interaction with Hfq to support the rapid degradation of ptsG mRNA although additional subregions within the scaffold are also involved in Hfq binding. Deletion of the 702-750 region greatly impairs the ability of RNase E to cause the degradation of ptsG mRNA. In addition, a polypeptide corresponding to the scaffold region binds to Hfq without the help of RNA. Finally, we demonstrate that overexpression of RhlB partially inhibits the Hfq binding to RNase E and the rapid degradation of ptsG mRNA.

A minimal base-pairing region of a bacterial small RNA SgrS required for translational repression of ptsG mRNA.

Escherichia coli SgrS is an Hfq-binding small RNA that is induced under glucose-phosphate stress to cause translational repression and RNase E-dependent rapid degradation of ptsG mRNA encoding the major glucose transporter. A 31-nt-long stretch in the 3' region of SgrS is partially complementary to the translation initiation region of ptsG mRNA. We showed previously that SgrS alone causes translational repression when pre-annealed with ptsG mRNA by a high-temperature treatment in vitro. Here, we studied translational repression of ptsG mRNA in vitro by synthetic RNA oligonucleotides (oligos) to define the SgrS region required for translational repression. We first demonstrate that a 31 nt RNA oligo corresponding to the base-pairing region is sufficient for translational inhibition of ptsG mRNA. Then, we show that RNA oligo can be shortened to 14 nt without losing its effect. Evidence shows that the 14 nt base-pairing region is sufficient to inhibit ptsG translation in the context of full-length SgrS in vivo. We conclude that SgrS 168-181 is a minimal base-pairing region for translational inhibition of ptsG mRNA. Interestingly, the 14 nt oligo efficiently inhibited ptsG translation without the high-temperature pre-treatment, suggesting that remodelling of structured SgrS is an important mechanism by which Hfq promotes the base pairing.

Analysis of nonstop mRNA translation in the absence of tmRNA in Escherichia coli.

tmRNA, a product of ssrA gene, plays a crucial role in the quality control system that eliminates aberrant products of nonstop mRNAs in prokaryotes. Although tmRNA recycles ribosomes stalled at the 3' end of nonstop mRNAs, the fate of ribosomes that stall at the 3' end in the absence of tmRNA has not been extensively examined. Here we report our analysis of the translation status of nonstop mRNAs. Polysome analysis showed that nonstop mRNAs were translated efficiently, and peptidyl-tRNA was not found in any fraction in a DeltassrA strain. In vitro translation experiments using PURESYSTEM revealed that ribosomes translating nonstop mRNAs were dissociated from the 3' end of mRNA, and the peptidyl-tRNA was only weakly hydrolyzed in the monosome. These results suggest that the peptidyl-tRNA of a nonstop mRNA is hydrolyzed by an unknown factor(s) in vivo, thereby allowing a nonstop mRNA to be translated as efficiently as a normal mRNA. Possible factors involved in the hydrolysis of the peptidyl-tRNAs of nonstop mRNAs are discussed.

RNA, but not protein partners, is directly responsible for translational silencing by a bacterial Hfq-binding small RNA.

SgrS is an Hfq-binding small RNA that is induced under glucose phosphate stress in Escherichia coli. It forms a specific ribo nucleo protein complex with Hfq and RNase E resulting in translational repression and rapid degradation of ptsG mRNA, encoding the glucose transporter. Here, we report translational silencing of ptsG mRNA in a defined in vitro system. We demonstrate that SgrS and Hfq are the minimum components for translational silencing to faithfully reproduce the reaction in cells. We show that ptsG-SgrS base pairing is sufficient to cause translational repression when the ptsG mRNA is forced to base pair with SgrS without the help of Hfq. The extent of translational repression correlates with the extent of duplex formation. We conclude that base pairing itself but not Hfq is directly responsible for translational silencing and the major role of Hfq in gene silencing is to stimulate the base pairing between SgrS and ptsG mRNA. This simple mechanism is in striking contrast to miRNA action in eukaryote in which the RNA is believed to act only as a guide of protein partners.

Cleavage of mRNAs and role of tmRNA system under amino acid starvation in Escherichia coli.

We have shown previously that ribosome stalling during translation caused by various reasons leads to mRNA cleavage, resulting in non-stop mRNAs that are eliminated in a tmRNA-dependent manner. Amino acid starvation is a physiological condition in which ribosome stalling is expected to occur more frequently. Here we demonstrate that mRNA cleavage is induced by amino acid starvation, resulting in accumulation of truncated mRNAs in cells lacking tmRNA. The truncated mRNAs are eliminated in wild-type cells, indicating that the tmRNA system rapidly degrade the truncated mRNAs. The cleavage pattern of model mRNAs in which serine codons were replaced with threonine codons indicated that mRNA cleavage occurs near serine codons in response to serine starvation. Cells lacking all of the five known toxin loci were proficient in mRNA cleavage, showing that toxin-antitoxin systems are not responsible for the cleavage. A mild serine starvation caused a significant growth inhibition in cells lacking tmRNA but not in wild-type cells. The ribosome-mediated mRNA cleavage along with the tmRNA system is an important mechanism that enables cells to adapt to amino acid starvation conditions.

Analyses of mRNA destabilization and translational inhibition mediated by Hfq-binding small RNAs.

A major class of bacterial small RNAs binds to an RNA chaperone Hfq and acts via imperfect base pairing to regulate the translation and stability of target mRNAs under specific physiological conditions. SgrS, an example for this class of small RNAs, is induced in response to the accumulation of glucose phosphates and downregulates the ptsG mRNA, which encodes the glucose transporter IICB(Glc) in Escherichia coli. SgrS forms a specific ribonucleoprotein complex with RNase E through Hfq. The regulatory outcomes of SgrS are the inhibition of translation and RNase E-dependent degradation of ptsG mRNA. Translational inhibition is the primary event for gene silencing. The crucial base pairs for the action of SgrS are confined to the 6-nt region overlapping the Shine-Dalgarno sequence of the target mRNA. Hfq accelerates the rate of duplex formation between SgrS and the target mRNA. Membrane localization of the target mRNA contributes to efficient SgrS action by competing with ribosome loading. Here, we describe major experimental methods and results used to study functions of Hfq-binding small RNAs in our laboratory. These are illustrated using the regulation of ptsG mRNA by SgrS is used as an example.

An oncoprotein from the plant pathogen agrobacterium has histone chaperone-like activity.

Protein 6b, encoded by T-DNA from the pathogen Agrobacterium tumefaciens, stimulates the plant hormone-independent division of cells in culture in vitro and induces aberrant cell growth and the ectopic expression of various genes, including genes related to cell division and meristem-related class 1 KNOX homeobox genes, in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b is found in nuclei and binds to several plant nuclear proteins. Here, we report that 6b binds specifically to histone H3 in vitro but not to other core histones. Analysis by bimolecular fluorescence complementation revealed an interaction in vivo between 6b and histone H3. We recovered 6b from a chromatin fraction from 6b-expressing plant cells. A supercoiling assay and digestion with micrococcal nuclease indicated that 6b acts as a histone chaperone with the ability to mediate formation of nucleosomes in vitro. Mutant 6b, lacking the C-terminal region that is required for cell division-stimulating activity and interaction with histone H3, was deficient in histone chaperone activity. Our results suggest a relationship between alterations in nucleosome structure and the expression of growth-regulating genes on the one hand and the induction of aberrant cell proliferation on the other.

Mechanism of RNA silencing by Hfq-binding small RNAs.

The stress-induced small RNAs SgrS and RyhB in Escherichia coli form a specific ribonucleoprotein complex with RNAse E and Hfq resulting in translation inhibition, RNAse E-dependent degradation of target mRNAs. Translation inhibition is the primary event for gene silencing and degradation of these small RNAs is coupled with the degradation of target mRNAs. The crucial base-pairs for action of SgrS are confined to the 6 nt region overlapping the Shine-Dalgarno sequence of the target mRNA. Hfq accelerates the rate of duplex formation between SgrS and the target mRNA. Membrane localization of target mRNA contributes to efficient SgrS action by competing with ribosome loading.

Reduced action of polypeptide release factors induces mRNA cleavage and tmRNA tagging at stop codons in Escherichia coli.

Certain C-terminal sequences of nascent peptide cause an efficient protein tagging by tmRNA system at stop codons in Escherichia coli. Here, we demonstrate that both mRNA cleavage and tmRNA tagging occur at UAG stop codon recognized specifically by polypeptide release factor 1 (RF-1) when the activity of RF-1 is reduced by a mutation in the prfA gene without requirement of particular C-terminal sequences of nascent peptide. The tmRNA tagging and mRNA cleavage in the prfA mutant were eliminated when the wild-type RF-1 but not RF-2 was supplied from plasmid. In addition, depletion of either RF-1 or RF-2 induces endonucleolytic cleavage and tmRNA tagging at UAG or UGA stop codons respectively. We conclude that ribosome stalling at the cognate stop codon caused by reduced activity or expression of RF-1 or RF-2 is responsible for mRNA cleavage. The present data along with our previous studies strongly suggest that ribosome stalling leads to endonucleolytic cleavage of mRNA in general resulting in non-stop mRNA and that the 3' end of non-stop mRNA is probably only target for the tmRNA system.

Base-pairing requirement for RNA silencing by a bacterial small RNA and acceleration of duplex formation by Hfq.

SgrS is an Hfq-binding small antisense RNA that is induced upon phosphosugar stress. It forms a ribonucleoprotein complex with RNase E through Hfq to mediate silencing of the target ptsG mRNA encoding the membrane component of the glucose-specific phosphoenolpyruvate phosphotransferase system. Although SgrS is believed to act on ptsG mRNA through base pairing between complementary regions, this was not previously tested experimentally. We addressed the question of whether SgrS indeed forms an RNA-RNA duplex with ptsG mRNA to exert its regulatory function. Specific single nucleotide substitutions around the Shine-Dalgarno (SD) sequence of ptsG completely eliminated SgrS action while compensatory mutations in SgrS restored it. A systematic mutational analysis of both ptsG and SgrS RNAs revealed that six base pairs around SD sequence of ptsG are particularly important for SgrS action. We also showed in vitro that SgrS forms a stable duplex with the ptsG mRNA, and that Hfq markedly facilitates the rate of duplex formation.