Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function.

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.

In vitro study of two dominant inhibitory GTPase mutants of Escherichia coli translation initiation factor IF2. Direct evidence that GTP hydrolysis is necessary for factor recycling.

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 wild-type 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 Vmax values being 11- and 40-fold lower, respectively. Both mutant forms promoted fMet-tRNAfMet 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.

In vitro and in vivo secondary structure probing of the thrS leader in Bacillus subtilis.

The Bacillus subtilis thrS gene is a member of the T-box gene family in Gram-positive organisms whose expression is regulated by a tRNA-mediated transcriptional antitermination mechanism involving a direct tRNA:mRNA interaction. The complex leader sequences of these genes share only short stretches of primary sequence homology, but a common secondary structure has been proposed by comparing the leaders of many genes of this family. The proposed mechanism forthe tRNA:mRNA interaction depends heavily on the secondary structure model, but is so far only supported by genetic evidence. We have studied the structure of the B.subtilis thrS leader in solution, in protection experiments using both chemical and enzymatic probes. The thrS leader structure was also probed in vivo using dimethylsulphate and the in vitro and in vivo data are in good accordance. We have organized the thrS leader into three major domains comprising six separate stem-loops. All but one of the short sequences conserved in this gene family are present in loop structures. The ACC specifier codon proposed to interact with the tRNAThrGGUisoacceptor is present in a bulge and probably exists in a stacking conformation. The proposed antiterminator structure is not visible in transcripts containing the terminator, but was probed using a transcript with the 3'-half of the terminator deleted and its folding appears consistent with the regulatory model. The leader sequences, and in particular the specifier domains, of the other genes of this family can be folded similarly to the experimentally solved thrS structure.

Processing of the Bacillus subtilis thrS leader mRNA is RNase E-dependent in Escherichia coli.

We have recently reported that processing occurs in the untranslated leader region of several members of a family of Gram-positive genes regulated by tRNA-mediated antitermination. We showed that cleavage at this site plays an important role in the induction of Bacillus subtilis thrS gene expression, following threonine starvation, by stabilising the downstream mRNA. Here we show that, when transferred on a plasmid, processing of the B. subtilis thrS leader can occur at the same site in Escherichia coli. Cleavage at this site is dependent on the E. coli endoribonuclease E, both in vivo and in vitro, suggesting that a functional homologue of RNase E is responsible for thrS processing in B. subtilis.

Structure and regulation of expression of the Bacillus subtilis valyl-tRNA synthetase gene.

We have sequenced the valyl-tRNA synthetase gene (valS) of Bacillus subtilis and found an open reading frame coding for a protein of 880 amino acids with a molar mass of 101,749. The predicted amino acid sequence shares strong similarity with the valyl-tRNA synthetases from Bacillus stearothermophilus, Lactobacillus casei, and Escherichia coli. Extracts of B. subtilis strains overexpressing the valS gene on a plasmid have increased valyl-tRNA aminoacylation activity. Northern analysis shows that valS is cotranscribed with the folC gene (encoding folyl-polyglutamate synthetase) lying downstream. The 300-bp 5' noncoding region of the gene contains the characteristic regulatory elements, T box, "specifier codon" (GUC), and rho-independant transcription terminator of a gene family in gram-positive bacteria that encodes many aminoacyl-tRNA synthetases and some amino acid biosynthetic enzymes and that is regulated by tRNA-mediated antitermination. We have shown that valS expression is induced by valine limitation and that the specificity of induction can be switched to threonine by changing the GUC (Val) specifier triplet to ACC (Thr). Overexpression of valS from a recombinant plasmid leads to autorepression of a valS-lacZ transcriptional fusion. Like induction by valine starvation, autoregulation of valS depends on the presence of the GUC specifier codon. Disruption of the valS gene was not lethal, suggesting the existence of a second gene, as is the case for both the thrS and the tyrS genes.

Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis.

The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.

Aminoacyl-tRNA synthetase gene regulation in Bacillus subtilis.

In this review, we summarize progress on the regulation of the aminoacyl-tRNA synthetase genes in Bacillus subtilis. Most of the genes encoding this set of enzymes in B subtilis are members of a large family of Gram-positive genes and operons controlled by a novel antitermination mechanism that uses their cognate uncharged tRNA as the effector. A subset of these genes is, in addition, likely to be controlled at the level of mRNA processing and degradation. We describe the key experiments leading to these conclusions.

Aminoacyl-tRNA synthetase gene regulation in Bacillus subtilis: induction, repression and growth-rate regulation.

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.

Clustering and co-transcription of the Bacillus subtilis genes encoding the aminoacyl-tRNA synthetases specific for glutamate and for cysteine and the first enzyme for cysteine biosynthesis.

The Bacillus subtilis cysE and cysS genes encoding, respectively, the serine acetyltransferase and the cysteinyl-tRNA synthetase were found downstream from the gltX gene encoding the glutamyl-tRNA synthetase. This gene organization is also conserved in Bacillus stearothermophilus where the cysE and cysS genes show high amino acid identity with those of B. subtilis. In both organisms the coding sequences of cysE and cysS overlap, suggesting a translational coupling. B. subtilis cysE and cysS were expressed in Escherichia coli using the inducible trc promoter; they functionally complement mutants of E. coli affected in those genes. Overproduction of B. subtilis CysRS in E. coli has a toxic effect on cell growth. Disruption of gltX and cysS by Campbell-type insertion is lethal for the cell, indicating that these genes code for an essential and unique function in B. subtilis. S1 mapping analysis shows that the transcription of gltX is under the control of a sigma A promoter located 43 base pairs upstream of the initiation codon. A T-box sequence and a rho-independent terminator known to regulate expression of other aminoacyl-tRNA synthetase genes and of some amino acid biosynthetic operons in Bacillus sp., were found between gltX and cysE. No sigma A promoter was detected upstream of cysE, which is consistent with the lethality of a Campbell-type insertion using a plasmid that interrupts transcription coming from the gltX promoter, and suggests that gltX, cysE, and cysS constitute an operon. This is the first case where genes implicated in the biosynthesis of an amino acid and its cognate aminoacyl-tRNA synthetase are shown to be co-transcribed.

In vivo study of engineered G-domain mutants of Escherichia coli translation initiation factor IF2.

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.

Expression of both Bacillus subtilis threonyl-tRNA synthetase genes is autogenously regulated.

The "housekeeping" threonyl-tRNA synthetase gene (thrS) of Bacillus subtilis is shown to be transcribed in vivo and in vitro from a single promoter. In vitro, 85% of all messages transcribed from the thrS promoter are terminated at a strong factor-independent terminator localized upstream of the thrS Shine-Dalgarno sequence, within the 305-nucleotide-long leader region. Overexpression of thrS represses transcriptional and translational thrS-lacZ fusions to a similar extent, suggesting that thrS is autoregulated at the transcriptional level. We show that autogenous control does not act at the level of transcription initiation but involves antitermination of the transcription mechanism. thrZ, the second threonyl-tRNA synthetase gene, is also autogenously regulated. However, the ability of the ThrS synthetase to repress thrS as well as thrZ expression is much greater than that of the ThrZ synthetase.

Co-ordinate expression of the two threonyl-tRNA synthetase genes in Bacillus subtilis: control by transcriptional antitermination involving a conserved regulatory sequence.

In Bacillus subtilis, two genes, thrS and thrZ, encode distinct threonyl-tRNA synthetase enzymes. Normally, only the thrS gene is expressed. Here we show that either gene, thrS or thrZ, is sufficient for normal cell growth and sporulation. Reducing the intracellular ThrS protein concentration induces thrZ expression in a dose-compensatory manner. Starvation for threonine simultaneously induces thrZ and stimulates thrS expression. The 5'-leader sequences of thrS and thrZ contain, respectively, one and three transcription terminators preceded by a conserved sequence. We show that this sequence is essential for the regulation of thrS via a transcriptional antitermination mechanism. We propose that both genes, thrS and thrZ, are regulated by the same mechanism such that the additional regulatory domains present before thrZ account for its non-expression. In contrast to Escherichia coli, structurally similar regulatory domains, i.e. the consensus sequence preceding a terminator structure, are found in the leader regions of most aminoacyl-tRNA synthetase genes of Gram-positive bacteria. This suggests that they are regulated by a common mechanism.

A severely truncated form of translational initiation factor 2 supports growth of Escherichia coli.

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.

Structure and nucleotide sequence of the Bacillus subtilis phenylalanyl-tRNA synthetase genes.

The nucleotide sequence of the Bacillus subtilis pheST genes coding for the 2 subunits of phenylalanyl-tRNA synthetase has been determined. The pheS gene corresponds to 1029 bp and the pheT gene to 2412 bp. The encoded proteins have Mrs of 38,947 (343 amino acids, alpha-subunit) and 87,916 (804 amino acids, beta-subunit), respectively. The genes are adjacent on the chromosome separated by only 15 nucleotides. The pheT gene is immediately followed by a hairpin structure typical of a rho-independent transcription terminator. S1 nuclease mapping and primer extension analysis of pheST mRNA revealed a major start of transcription 318 nucleotides upstream of the pheS gene, and 6 nucleotides downstream of a E sigma 43 promoter consensus sequence. Within the 5'-noncoding region several potential secondary structures have been noted.

Independent genes for two threonyl-tRNA synthetases in Bacillus subtilis.

With the exception of Escherichia coli lysyl-tRNA synthetase, the genes coding for the different aminoacyl-tRNA synthetases in procaryotes are always unique. Here we report on the occurrence and cloning of two genes (thrSv and thrS2), both encoding functional threonyl-tRNA synthetase in Bacillus subtilis. The two proteins share only 51.5% identical residues, which makes them almost as distinct from each other as each is from E. coli threonyl-tRNA synthetase (42 and 47%). Both proteins complement an E. coli thrS mutant and effectively charge E. coli threonyl tRNA in vitro. Their genes have been mapped to 250 degrees (thrSv) and 344 degrees (thrS2) on the B. subtilis chromosome. The regulatory regions of both genes are quite complex and show structural similarities. During vegetative growth, only the thrSv gene is expressed.

Bacillus subtilis phenylalanyl-tRNA synthetase genes: cloning and expression in Escherichia coli and B. subtilis.

The genes that encode the two subunits of Bacillus subtilis phenylalanyl-tRNA synthetase were cloned from alpha lambda library of chromosomal B. subtilis DNA by specific complementation of a thermosensitive Escherichia coli pheS mutation. Both genes (we named them pheS and pheT, analogous to the corresponding genes of E. coli) are carried by a 6.6-kilobase-pair PstI fragment which also complements E. coli pheT mutations. This fragment directs the synthesis of two proteins identical in size to the purified alpha and beta subunits of the phenylalanyl-tRNA synthetase of B. subtilis with Mrs of 42,000 and 97,000, respectively. A recombinant shuttle plasmid carrying the genes caused 10-fold overproduction of functional phenylalanyl-tRNA synthetase in B. subtilis.

Photomorphogenesis in Physarum: induction of tubulins and sporulation-specific proteins and of their mRNAs.

Sporangiophore formation in Physarum plasmodia starts about 10 hr after photoinduction. It is characterized by the induction of two tubulins and of at least 15 major sporangiophore morphogenetic proteins. In vitro translation of extracted mRNA revealed that differential gene expression is based on a highly synchronous temporal program of loss of plasmodial and induction of sporulation-specific mRNA species. Using a cloned cDNA encoding part of a sporangiophore morphogenetic protein from Physarum as a probe it was found that the induction of the complementary mRNA activity is due to the induction of the mRNA itself. The results suggest that light induces, with a lag phase of about 10 hr, the transient activation of sporulation-specific genes.

Blue light inhibits slime mold differentiation at the mRNA level.

The influence of blue light on protein synthesis in spherulating Physarum polycephalum microplasmodia was studied using two-dimensional protein separation techniques. The starvation-induced plasmodium-spherule transition proceeds in the dark and is accompanied by the synthesis of 20 major differentiation-specific proteins as revealed by in vivo labelling with [35S]methionine. Three of these proteins are identical with cell wall components with respect to their mol. wts. (35 K, 34 K and 14 K) and isoelectric points. Spherulation is also accompanied by the appearance of 26 prominent differentiation-specific mRNA species translatable in the rabbit reticulocyte cell-free system. Six of the proteins synthesized in vitro co-migrate on two-dimensional gels with proteins labelled in vivo, two of them being cell wall components. Blue light, which inhibits spherulation completely, inhibits also the synthesis of spherule proteins and of spherule-specific mRNA activity. Only three protein components are induced by blue light, indicating that illumination does not induce a novel differentiated plasmodial state.