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 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.

Elongation factor G(EF-G) and initiation factor 2 (IF2) are involved in the translocation of ribosomes on mRNA and in the binding of initiator tRNA to the 30 S ribosomal subunit, respectively. Here we report that the Escherichia coli EF-G and IF2 interact with unfolded and denatured proteins, as do molecular chaperones that are involved in protein folding and protein renaturation after stress. EF-G and IF2 promote the functional folding of citrate synthase and ␣-glucosidase after urea denaturation. They prevent the aggregation of citrate synthase under heat shock conditions, and they form stable complexes with unfolded proteins such as reduced carboxymethyl ␣-lactalbumin. Furthermore, the EF-G and IF2-dependent renaturations of citrate synthase are stimulated by GTP, and the GTPase activity of EF-G and IF2 is stimulated by the permanently unfolded protein, reduced carboxymethyl ␣-lactalbumin. The concentrations at which these chaperone-like functions occur are lower than the cellular concentrations of EF-G and IF2. These results suggest that EF-G and IF2, in addition to their role in translation, might be implicated in protein folding and protection from stress.The elongation phase of protein synthesis is promoted by two G proteins, elongation factor EF-Tu, 1 which delivers aminoacyl tRNAs to the ribosome, and EF-G, which catalyzes the translocation step, during which the A-and P-site tRNAs move to the P and E sites of the elongating ribosome, respectively, and mRNA is advanced by one codon (1-3). EF-G binds to the ribosome in its GTP form, hydrolyzes GTP to drive tRNA movement on the ribosome (4), and is released in its GDP form. The functional cycle is completed upon GDP release and reactivation of the empty factor by binding of a GTP molecule (1-3).IF2 is the only G protein among the three translation initiation factors in Escherichia coli. It promotes the binding of fMet-tRNA f Met to the 30 S ribosomal subunit leading to the formation of the 30 S initiation complex (for review see Refs. 5 and 6). IF2, together with IF3, helps the 30 S subunit to select the initiator tRNA over other tRNAs (7). Assembly of the 70 S initiation complex is accompanied by IF2-dependent hydrolysis of GTP and release of IF2 from the 70 S complex, a step that is essential for the first peptide bond formation (8).In addition to their role in translation, several ribosomal proteins and translational factors are involved in other mechanisms, including replication, transcription, RNA processing, DNA repair, regulation of translation, malignant transformation, and regulation of development (reviewed in Ref. 9). IF2 is suspected to participate in the activation of transcription of the rrnB operon (10) and displays DNA binding activity (11). EF-Tu interacts with the Q␤ replicative complex, the transcriptional apparatus, and membranes (reviewed in Ref. 12); EF-1␣ (the eukaryotic counterpart of EF-Tu) and EF-2 (the eukaryotic counterpart of EF-G) bind to actin filaments and influence the assembly of cytoskeletal polymers (13-15). Both EF-1␣ ...

We have recently shown that the Escherichia coli initiation factor 2 (IF2) G-domain mutants V400G and H448E do not support cell survival and have a strong negative effect on growth even in the presence of wildtype IF2. We have isolated both mutant proteins and performed an in vitro study of their main functions. The affinity of both mutant proteins for GTP is almost unchanged compared with wild-type IF2. However, the uncoupled GTPase activity of the V400G and H448E mutants is severely impaired, the V max values being 11-and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNA f Met binding to 70 S ribosomes with similar efficiencies and were as sensitive to competitive inhibition by GDP as wild-type IF2. Formation of the first peptide bond, as measured by the puromycin reaction, was completely inhibited in the presence of the H448E mutant but still significant in the case of the V400G mutant. Sucrose density gradient centrifugation revealed that, in contrast to wild-type IF2, both mutant proteins stay blocked on the ribosome after formation of the 70 S initiation complex. This probably explains their dominant negative effect in vivo. Our results underline the importance of GTP hydrolysis for the recycling of IF2.The first step of prokaryotic mRNA translation requires at least 3 proteins known as the initiation factors IF1, 1 IF2, IF3, and a complexed GTP molecule, in addition to fMet-tRNA f Met (1). Of these three proteins only IF2 has a GTP-binding domain. During 30 S initiation complex formation, the initiation factors, GTP, fMet-tRNA f Met , and mRNA are bound to the small ribosomal subunit. In this context IF2 binds at least three different ligands: GTP, the 30 S ribosomal subunit, and the initiator tRNA (1-3). The functional 70 S initiation complex ready for elongation contains only the initiator tRNA and mRNA. During its formation the initiation factors are released and GTP is hydrolyzed. The presence and hydrolysis of GTP which are directly linked to the presence of IF2 have been shown to be essential for initiation of protein biosynthesis (4). However, the precise role of GTP is still not clear. IF2 is one of the largest G-proteins known, and, in contrast to other proteins of this class which carry the G-domain in their N-terminal region, the G-domain of IF2 is centrally located in the protein. IF2 has been shown to bind GTP with a 10-fold lower affinity than GDP, in contrast to EF-Tu which binds GTP 100 times less efficiently than GDP (5, 6). Its lower overall affinity for guanine nucleotides (1000-fold for GDP, 10-fold for GTP) might enable IF2 to self-cycle from the GDP to the GTP state. Free IF2 does not show any GTPase activity; this is induced upon binding of the 50 S subunit during 70 S complex formation. The IF2 GTPase depends completely on the presence of the ribosome (7,8). However, in Bacillus stearothermophilus, both IF2 and its isolated G-domain were shown to be capable of hydrolyzing GTP in the absence of ribosomes when 20% ethanol was included in the reaction (9).In order to d...

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.

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.

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