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The degradation of messenger RNA is of universal importance for controlling gene expression. It directly affects protein synthesis by modulating the amount of mRNA available for translation. Regulation of mRNA decay provides an efficient means to produce just the proteins needed and to rapidly alter patterns of protein synthesis. In bacteria, the half-lives of individual mRNAs can differ by as much as two orders of magnitude, ranging from seconds to an hour. Most of what we know today about the diverse mechanisms of mRNA decay and maturation in prokaryotes comes from studies of the two model organisms Escherichia coli and Bacillus subtilis. Their evolutionary distance provided a large picture of potential pathways and enzymes involved in mRNA turnover. Among them are three ribonucleases, two of which have been discovered only recently, which have a truly general role in the initiating events of mRNA degradation: RNase E, RNase J and RNase Y. Their enzymatic characteristics probably determine the strategies of mRNA metabolism in the organism in which they are present. These ribonucleases are coded, alone or in various combinations, in all prokaryotic genomes, thus reflecting how mRNA turnover has been adapted to different ecological niches throughout evolution.

RNase Y is a key endoribonuclease that regulates global mRNA turnover and processing in Bacillus subtilis and likely many other bacteria. This enzyme is anchored to the cell membrane, creating a pseudo-compartmentalization that aligns with its role in initiating the decay of mRNAs primarily translated at the cell periphery. However, the reasons behind and the consequences of RNase Y's membrane attachment remain largely unknown. In our study, we examined a strain expressing wild-type levels of a cytoplasmic form of RNase Y from its chromosomal locus. This strain exhibits a slow-growth phenotype, similar to that of an RNase Y null mutant. Genome-wide data reveal a significant impact on the expression of hundreds of genes. While certain RNA substrates clearly depend on RNase Y's membrane attachment, others do not. We observed no correlation between mRNA stabilization in the mutant strains and the cellular location or function of the encoded proteins. Interestingly, the Y-complex, a specificity factor for RNase Y, also appears also recognize the cytoplasmic form of the enzyme, restoring wild-type levels of the corresponding transcripts. We propose that membrane attachment of RNase Y is crucial for its functional interaction with many coding and non-coding RNAs, limiting the cleavage of specific substrates, and potentially avoiding unfavorable competition with other ribonucleases like RNase J, which shares a similar evolutionarily conserved cleavage specificity.

One presenilin gene (PSEN) is expressed in the sea urchin embryo, in the vegetal pole of the gastrula and then mainly in cilia cells located around the digestive system of the pluteus, as we recently have reported. PSEN expression must be accurately regulated for correct execution of these two steps of development. While investigating PSEN expression changes in embryos after expansion of endoderm with LiCl or of ectoderm with Zn2+ by whole-mount in situ hybridization (WISH) and quantitative PCR (qPCR), we detected natural antisense transcription of PSEN. We then found that Endo16 and Wnt5, markers of endo-mesoderm, and of Hnf6 and Gsc, markers of ectoderm, are also sense and antisense transcribed. We discuss that general gene expression could depend on both sense and antisense transcription. This mechanism, together with the PSEN gene, should be included in gene regulatory networks (GRNs) that theorize diverse processes in this species. We suggest that it would also be relevant to investigate natural antisense transcription of PSEN in the field of Alzheimer's disease (AD) where the role of human PSEN1 and PSEN2 is well known., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The instability of messenger RNA is crucial to the control of gene expression. In Bacillus subtilis , RNase Y is the major decay-initiating endoribonuclease. Here, we show how this key enzyme regulates its own synthesis by modulating the longevity of its mRNA. Autoregulation is achieved through cleavages in two regions of the rny (RNase Y) transcript: (i) within the first ~100 nucleotides of the open reading frame, immediately inactivating the mRNA for further rounds of translation; (ii) cleavages in the rny 5' UTR, primarily within the 5'-terminal 50 nucleotides, creating entry sites for the 5' exonuclease J1 whose progression is blocked around position -15 of the rny mRNA, potentially by initiating ribosomes. This links the functional inactivation of the transcript by RNase J1 to translation efficiency, depending on the ribosome occupancy at the translation initiation site. By these mechanisms, RNase Y can initiate degradation of its own mRNA when the enzyme is not occupied with degradation of other RNAs and thus prevent its overexpression beyond the needs of RNA metabolism.

RNase Y and RNase E are disparate endoribonucleases that govern global mRNA turnover/processing in the two evolutionary distant bacteria Bacillus subtilis and Escherichia coli, respectively. The two enzymes share a similar in vitro cleavage specificity and subcellular localization. To evaluate the potential equivalence in biological function between the two enzymes in vivo we analyzed whether and to what extent RNase E is able to replace RNase Y in B. subtilis. Full-length RNase E almost completely restores wild type growth of the rny mutant. This is matched by a surprising reversal of transcript profiles both of individual genes and on a genome-wide scale. The single most important parameter to efficient complementation is the requirement for RNase E to localize to the inner membrane while truncation of the C-terminal sequences corresponding to the degradosome scaffold has only a minor effect. We also compared the in vitro cleavage activity for the major decay initiating ribonucleases Y, E and J and show that no conclusions can be drawn with respect to their activity in vivo. Our data confirm the notion that RNase Y and RNase E have evolved through convergent evolution towards a low specificity endonuclease activity universally important in bacteria., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

mRNA levels result from an equilibrium between transcription and degradation. Ribonucleases (RNases) facilitate the turnover of mRNA, which is an important way of controlling gene expression, allowing the cells to adjust transcript levels to a changing environment. In contrast to the heterotrophic model bacteria Escherichia coli and Bacillus subtilis , RNA decay has not been studied in detail in cyanobacteria. Synechocystis sp. PCC6803 encodes orthologs of both E. coli and B. subtilis RNases, including RNase E and RNase J, respectively. We show that in vitro Sy RNases E and J have an endonucleolytic cleavage specificity that is very similar between them and also compared to orthologous enzymes from E. coli, B. subtilis , and Chlamydomonas. Moreover, Sy RNase J displays a robust 5'-exoribonuclease activity similar to B. subtilis RNase J1, but unlike the evolutionarily related RNase J in chloroplasts. Both nucleases are essential and gene deletions could not be fully segregated in Synechocystis . We generated partially disrupted strains of Sy RNase E and J that were stable enough to allow for their growth and characterization. A transcriptome analysis of these strains partially depleted for RNases E and J, respectively, allowed to observe effects on specific transcripts. RNase E altered the expression of a larger number of chromosomal genes and antisense RNAs compared to RNase J, which rather affects endogenous plasmid encoded transcripts. Our results provide the first description of the main transcriptomic changes induced by the partial depletion of two essential ribonucleases in cyanobacteria., (Copyright © 2020 Cavaiuolo, Chagneau, Laalami and Putzer.)

Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli , the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli , where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries., (Copyright © 2020 Hamouche et al.)

Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b λx isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of α-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Key Message: Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5' exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation. RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5' end maturation is thought to be achieved by the combined action of a 5' exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5' exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5' exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5' exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.

The metabolic instability of mRNA is fundamental to the adaptation of gene expression. In bacteria, mRNA decay follows first-order kinetics and is primarily controlled at the steps initiating degradation. In the model Gram-positive organism Bacillus subtilis, the major mRNA decay pathway initiates with an endonucleolytic cleavage by the membrane-associated RNase Y. High-throughput sequencing has identified a large number of potential mRNA substrates but our understanding of what parameters affect cleavage in vivo is still quite limited. In vitro reconstitution of the cleavage event is thus instrumental in defining the mechanistic details, substrate recognition, the role of auxiliary factors, and of membrane localization in cleavage. In this chapter, we describe not only the purification and assay of RNase Y but also RNase J1/J2 which shares a similar low-specificity endoribonucleolytic activity with RNase Y. We highlight potential problems in the set-up of these assays and include methods that allow purification of full-length RNase Y and its incorporation in multilamellar vesicles created from native B. subtilis lipids that might best mimic in vivo conditions., (© 2018 Elsevier Inc. All rights reserved.)

Bacteria adopt social behavior to expand into new territory, led by specialized swarmers, before forming a biofilm. Such mass migration of Bacillus subtilis on a synthetic medium produces hyperbranching dendrites that transiently (equivalent to 4 to 5 generations of growth) maintain a cellular monolayer over long distances, greatly facilitating single-cell gene expression analysis. Paradoxically, while cells in the dendrites (nonswarmers) might be expected to grow exponentially, the rate of swarm expansion is constant, suggesting that some cells are not multiplying. Little attention has been paid to which cells in a swarm are actually multiplying and contributing to the overall biomass. Here, we show in situ that DNA replication, protein translation and peptidoglycan synthesis are primarily restricted to the swarmer cells at dendrite tips. Thus, these specialized cells not only lead the population forward but are apparently the source of all cells in the stems of early dendrites. We developed a simple mathematical model that supports this conclusion., Importance: Swarming motility enables rapid coordinated surface translocation of a microbial community, preceding the formation of a biofilm. This movement occurs in thin films and involves specialized swarmer cells localized to a narrow zone at the extreme swarm edge. In the B. subtilis system, using a synthetic medium, the swarm front remains as a cellular monolayer for up to 1.5 cm. Swarmers display high-velocity whirls and vortexing and are often assumed to drive community expansion at the expense of cell growth. Surprisingly, little attention has been paid to which cells in a swarm are actually growing and contributing to the overall biomass. Here, we show that swarmers not only lead the population forward but continue to multiply as a source of all cells in the community. We present a model that explains how exponential growth of only a few cells is compatible with the linear expansion rate of the swarm., (Copyright © 2017 Hamouche et al.)

The instability of messenger RNA is fundamental to the control of gene expression. In bacteria, mRNA degradation generally follows an "all-or-none" pattern. This implies that if control is to be efficient, it must occur at the initiating (and presumably rate-limiting) step of the degradation process. Studies of E. coli and B. subtilis, species separated by 3 billion years of evolution, have revealed the principal and very disparate enzymes involved in this process in the two organisms. The early view that mRNA decay in these two model organisms is radically different has given way to new models that can be resumed by "different enzymes-similar strategies". The recent characterization of key ribonucleases sheds light on an impressive case of convergent evolution that illustrates that the surprisingly similar functions of these totally unrelated enzymes are of general importance to RNA metabolism in bacteria. We now know that the major mRNA decay pathways initiate with an endonucleolytic cleavage in E. coli and B. subtilis and probably in many of the currently known bacteria for which these organisms are considered representative. We will discuss here the different pathways of eubacterial mRNA decay, describe the major players and summarize the events that can precede and/or favor nucleolytic inactivation of a mRNA, notably the role of the 5' end and translation initiation. Finally, we will discuss the role of subcellular compartmentalization of transcription, translation, and the RNA degradation machinery.

RNase Y is a key endoribonuclease affecting global mRNA stability in Bacillus subtilis. Its characterization provided the first evidence that endonucleolytic cleavage plays a major role in the mRNA metabolism of this organism. RNase Y shares important functional features with the RNA decay initiating RNase E from Escherichia coli, notably a similar cleavage specificity and a preference for 5' monophosphorylated substrates. We used high-resolution tiling arrays to analyze the effect of RNase Y depletion on RNA abundance covering the entire genome. The data confirm that this endoribonuclease plays a key role in initiating the decay of a large number of mRNAs as well as non coding RNAs. The downstream cleavage products are likely to be degraded by the 5' exonucleolytic activity of RNases J1/J2 as we show for a specific case. Comparison of the data with that of two other recent studies revealed very significant differences. About two thirds of the mRNAs upregulated following RNase Y depletion were different when compared to either one of these studies and only about 10% were in common in all three studies. This highlights that experimental conditions and data analysis play an important role in identifying RNase Y substrates by global transcriptional profiling. Our data confirmed already known RNase Y substrates and due to the precision and reproducibility of the profiles allow an exceptionally detailed view of the turnover of hundreds of new RNA substrates.

RNase Y is a novel endoribonuclease affecting global mRNA metabolism. We show that this nuclease affects the expression of the Bacillus subtilis infC-rpmI-rplT operon, encoding translation initiation factor IF3 and the ribosomal proteins L35 and L20. This operon is autoregulated by a complex L20-dependent transcription attenuation mechanism. L20 binds to a phylogenetically conserved domain on the 5' untranslated region of the infC mRNA which mimics the L20 binding sites on 23S rRNA. We have identified a second promoter (P1) upstream of the previously identified promoter (P2). The P1, but not the P2, readthrough transcript is stabilized in a strain depleted for RNase Y. However, under these conditions infC biosynthesis is repressed threefold. We show that the unprocessed P1 transcript is non-functional for IF3 translation but fully competent to express the co-transcribed ribosomal protein genes. RNase Y cleavage of the P1 transcript creates an entry site for the 5'-3' exonucleolytic activity of RNase J1 which degrades the infC mRNA when translation initiation efficiency is low. A second RNase Y cleavage is crucial for initiating degradation of the prematurely terminated infC leader RNAs, including the L20 operator complex, which permits efficient recycling of the L20 protein., (© 2011 Blackwell Publishing Ltd.)

The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyper-branched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp+ (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30-40 %), it is dispensable at high humidity. Dendritic migration (flagella- and surfactin-dependent) of strains 168 sfp+ swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (single-cell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication.

The ribosomal protein S1, in Escherichia coli, is necessary for the recognition by the ribosome of the translation initiation codon of most messenger RNAs. It also participates in other functions. In particular, it stimulates the T4 endoribonuclease RegB, which inactivates some of the phage mRNAs, when their translation is no longer required, by cleaving them in the middle of their Shine-Dalgarno sequence. In each function, S1 seems to target very different RNAs, which led to the hypothesis that it possesses different RNA-binding sites. We previously demonstrated that the ability of S1 to activate RegB is carried by a fragment of the protein formed of three consecutive domains (domains D3, D4, and D5). The same fragment plays a central role in all other functions. We analyzed its structural organization and its interactions with three RNAs: two RegB substrates and a translation initiation region. We show that these three RNAs bind the same area of the protein through a set of systematic (common to the three RNAs) and specific (RNA-dependent) interactions. We also show that, in the absence of RNA, the D4 and D5 domains are associated, whereas the D3 and D4 domains are in equilibrium between open (noninteracting) and closed (weakly interacting) forms and that RNA binding induces a structural reorganization of the fragment. All of these results suggest that the ability of S1 to recognize different RNAs results from a high adaptability of both its structure and its binding surface.

An Arabidopsis thaliana leaf-variegated mutant yellow variegated2 (var2) results from loss of FtsH2, a major component of the chloroplast FtsH complex. FtsH is an ATP-dependent metalloprotease in thylakoid membranes and degrades several chloroplastic proteins. To understand the role of proteolysis by FtsH and mechanisms leading to leaf variegation, we characterized the second-site recessive mutation fu-gaeri1 (fug1) that suppressed leaf variegation of var2. Map-based cloning and subsequent characterization of the FUG1 locus demonstrated that it encodes a protein homologous to prokaryotic translation initiation factor 2 (cpIF2) located in chloroplasts. We show evidence that cpIF2 indeed functions in chloroplast protein synthesis in vivo. Suppression of leaf variegation by fug1 is observed not only in var2 but also in var1 (lacking FtsH5) and var1 var2. Thus, suppression of leaf variegation caused by loss of FtsHs is most likely attributed to reduced protein synthesis in chloroplasts. This hypothesis was further supported by the observation that another viable mutation in chloroplast translation elongation factor G also suppresses leaf variegation in var2. We propose that the balance between protein synthesis and degradation is one of the determining factors leading to the variegated phenotype in Arabidopsis leaves.

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.

The T4 RegB endoribonuclease cleaves specifically in the middle of the -GGAG- sequence, leading to inactivation and degradation of early phage mRNAs. In vitro, RegB activity is very weak but can be enhanced 10- to 100-fold by the Escherichia coli ribosomal protein S1. Not all RNAs carrying the GGAG motif are cleaved by RegB, suggesting that additional information is required to obtain a complete RegB target site. In this work, we find that in the presence of S1, the RegB target site is an 11 nt long single-stranded RNA carrying the 100% conserved GGA triplet at the 5' end and a degenerate, A-rich, consensus sequence immediately downstream. Our data support the notion that RegB alone recognizes only the trinucleotide GGA, which it cleaves very inefficiently, and that stimulation of RegB activity by S1 depends on the nucleotide immediately 3' to -GGA-.

The RegB protein, encoded by the T4 bacteriophage genome, is a ribonuclease involved in the inactivation of the phage early messenger RNAs. Its in vitro activity is very low but can be enhanced up to 100-fold in the presence of the ribosomal protein S1. The latter is made of six repeats of a conserved module found in many other proteins of RNA metabolism. Considering the difference between its size (556 amino acids) and that of several RegB substrates (10 nucleotides), we wondered whether all six modules are necessary for RegB activation. We studied the influence of twelve S1 fragments on the cleavage efficiency of three short substrates. RegB activation requires the cooperation of different sets of modules depending on the substrates. Two RNAs are quite well cleaved in the presence of the fragment formed by the fourth and fifth modules, whereas the third requires the presence of the four C-terminal domains. However, NMR interaction experiments showed that, despite these differences, the interactions of the substrates with either the bi- or tetra-modules are similar, suggesting a common interaction surface. In the case of the tetra-module the interactions involve all four domains, raising the question of the spatial organization of this region.

We have isolated the structural gene for translation initiation factor IF2 (infB) from the myxobacterium Myxococcus xanthus. The gene (3.22 kb) encodes a 1,070-residue protein showing extensive homology within its G domain and C terminus to the equivalent regions of IF2 from Escherichia coli. The protein cross-reacts with antibodies raised against E. coli IF2 and was able to complement an E. coli infB mutant. The M. xanthus protein is the largest IF2 known to date. This is essentially due to a longer N-terminal region made up of two characteristic domains. The first comprises a 188-amino-acid sequence consisting essentially of alanine, proline, valine, and glutamic acid residues, similar to the APE domain observed in Stigmatella aurantiaca IF2. The second is unique to M. xanthus IF2, is located between the APE sequence and the GTP binding domain, and consists exclusively of glycine, proline, and arginine residues.

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.

RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets. We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.

According to their role in translation, tRNAs specifically interact either with elongation factor Tu (EFTu) or with initiation factor 2 (IF2). We here describe the effects of overproducing EFTu and IF2 on the elongator versus initiator activities of various mutant tRNAMet species in vivo. The data obtained indicate that the selection of a tRNA through one or the other pathway of translation depends on the relative amounts of the translational factors. A moderate overexpression of EFTu is enough to lead to a misappropriation of initiator tRNA in the elongation process, whereas overproduced IF2 allows the initiation of translation to occur with unformylated tRNA species. In addition, we report that a strain devoid of formylase activity can be cured by the overproduction of tRNAMetf. The present study brings additional evidence for the importance of formylation in defining tRNAMetf initiator identity, as well as a possible explanation for the residual growth of bacterial strains lacking a functional formylase gene such as observed in Guillon, J. M., Mechulam, Y., Schmitter, J.-M., Blanquet, S., and Fayat, G. (1992) J. Bacteriol. 174, 4294-4301.

trans-Diamminedichloroplatinum(II) was used to induce reversible cross-links between Escherichia coli initiation factor 2 (IF-2) and fMet-tRNA(f)(Met). Two distinct cross-links between IF-2 and the initiator tRNA were produced. Analysis of the cross-linking regions on both RNA and protein moieties reveals that the T arm of the tRNA is in the proximity of a region of the C-terminal domain of IF-2 (residues Asn611-Arg645). This cross-link is well-correlated with the fact that the C-domain of IF-2 contains the fMet-tRNA binding site and that the cross-linked RNA fragment precisely maps in a region which is protected by IF-2 from chemical modification and enzymatic digestion. Rather unexpectedly, a second cross-link was characterized which involves the anticodon arm of fMet-tRNA(f)(Met) and the N-terminal part of IF-2 (residues Trp215-Arg237).

The thrS gene in Bacillus subtilis is specifically induced by starvation for threonine and is, in addition, autorepressed by the overproduction of its own gene product, the threonyl-tRNA synthetase. Both methods of regulation employ an antitermination mechanism at a factor-independent transcription terminator that occurs just upstream of the start codon. The effector of the induction mechanism is thought to be the uncharged tRNA(Thr), which has been proposed to base pair in two places with the leader mRNA to induce antitermination. Here we show that the autoregulation by synthetase overproduction is likely to utilize a mechanism similar to that characterized for induction by amino acid starvation, that is by altering the levels of tRNA charging in the cell. We also demonstrate that the base pairing interaction at the two proposed contact points between the tRNA and the leader are necessary but not always sufficient for either form of regulation. Finally, we present evidence that the thrS gene is expressed in direct proportion to the growth rate. This method of regulation is also at the level of antitermination but is independent of the interaction of the tRNA with the leader region.

During the IF2-catalysed formation of the 30S initiation complex, the GTP requirement and its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli. In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E. coli IF2, we have introduced seven single amino acid substitutions into its GTP-binding site (Gly for Val-400; Thr for Pro-446; Gly, Glu, Gln for His-448; and Asn, Glu for Asp-501). These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E. coli strain from which the functional chromosomal copy of the infB gene has been deleted. Only one of the mutated proteins (Asp-501 to Glu) was able to sustain cell viability and several displayed a dominant negative effect. These results emphasize that the amino acid residues we substituted are essential for the IF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation. These findings are discussed in relation to a previously proposed theoretical model for the IF2 G-domain.

The gene infB codes for two forms of translational initiation factor IF2; IF2 alpha (97,300 Da) and IF2 beta (79,700 Da). IF2 beta arises from an independent translational event on a GUG codon located 471 bases downstream from IF2 alpha start codon. By site-directed mutagenesis we constructed six different mutations of this GUG codon. In all cases, IF2 beta synthesis was variably affected by the mutations but not abolished. We show that the residual expression of IF2 beta results from translational initiation on an AUG codon located 21 bases downstream from the mutated GUG. Furthermore, two forms of IF2 beta have been separated by fast protein liquid chromatography and the determination of their N-terminal sequences indicated that they resulted from two internal initiation events, one occurring on the previously identified GUG start codon, the other on the AUG codon immediately downstream. We conclude that two forms of IF2 beta exist in the cell, which differ by seven aminoacid residues at their N terminus. Only by mutating both IF2 beta start codons could we construct plasmids that express only IF2 alpha. A plasmid expressing only IF2 beta was obtained by deletion of the proximal region of the infB gene. Using a strain that carries a null mutation in the chromosomal copy of infB and a functional copy of the same gene on a thermosensitive lysogenic lambda phage, we could cure the lambda phage when the plasmids expressing only one form of IF2 were supplied in trans. We found that each one of the two forms of IF2, at near physiological levels, can support growth of Escherichia coli, but that growth is retarded at 37 degrees C. This result shows that both forms of IF2 are required for maximal growth of the cell and suggests that they have acquired some specialized but not essential function.

We have constructed strains carrying null mutations in the chromosomal copy of the gene for translational initiation factor (IF) 2 (infB). A functional copy of the infB gene is supplied in trans by a thermosensitive lysogenic lambda phage integrated at att lambda. These strains enabled us to test in vivo the importance of different structural elements of IF2 expressed from genetically engineered plasmid constructs. We found that, as expected, the gene for IF2 is essential. However, a protein consisting of the C-terminal 55,000 Mr fragment of the wild-type IF2 protein is sufficient to allow growth when supplied in excess. This result suggests that the catalytic properties are localized in the C-terminal half of the protein, which includes the G-domain, and that this fragment is sufficient to complement the IF2 deficiency in the infB deletion strain.