Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability.

We describe a method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability. This method utilizes a chloramphenicol-resistant plasmid containing a temperature-sensitive (ts) replication origin. In this method, the target gene is first cloned into the ts vector and the recombinant plasmid is used to transform wild-type B. subtilis. The second step involves transformation of the resulting strain with a linear DNA fragment containing a second antibiotic resistance marker (tet) that disrupts the gene of interest. Selection for tetracycline resistance forces a double crossover between the chromosomal and fragment-borne copies of the gene, thereby replacing the wild-type gene in the chromosome with the disrupted allele. Cells survive even if the gene is essential by virtue of the complementing plasmid. Transformants are then grown at the non-permissive temperature for plasmid replication under various growth conditions. Isolation of chloramphenicol-sensitive colonies indicates that the gene is dispensable, whereas the inability to isolate chloramphenicol-sensitive colonies indicates that the gene is essential. The general utility of this method is demonstrated by allowing disruption of mtrA and trpE under conditions that render each gene non-essential, but not under growth conditions in which each gene is essential.

Expression of the Bacillus subtilis trpEDCFBA operon is influenced by translational coupling and Rho termination factor.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translational control mechanisms. Both mechanisms require binding of tryptophan-activated TRAP to 11 (G/U)AG repeats in the trp leader transcript. trpE translational control involves formation of a TRAP-dependent RNA structure that sequesters the trpE Shine-Dalgarno (SD) sequence (the SD blocking hairpin). By comparing expression levels from trpE'-'lacZ translational fusions controlled by the wild-type leader or by a leader that cannot form the SD blocking hairpin, we found that translational control requires a tryptophan concentration higher than that required for transcription attenuation. We also found that inhibition of trpE translation by the SD blocking hairpin does not alter the stability of the downstream message. Since the coding sequences for trpE and trpD overlap by 29 nucleotides, we examined expression levels from trpED'-'lacZ translational fusions to determine if these two genes are translationally coupled. We found that introduction of a UAA stop codon in trpE resulted in a substantial reduction in expression. Since expression was partially restored in the presence of a tRNA suppressor, our results indicate that trpE and trpD are translationally coupled. We determined that the coupling mechanism is TRAP independent and that formation of the SD blocking hairpin regulates trpD translation via translational coupling. We also constructed a rho mutation to investigate the role of Rho-dependent termination in trp operon expression. We found that TRAP-dependent formation of the SD blocking hairpin allows Rho access to the nascent transcript, causing transcriptional polarity.

Regulatory interactions of Csr components: the RNA binding protein CsrA activates csrB transcription in Escherichia coli.

The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, forming a ribonucleoprotein complex, which antagonizes CsrA activity. We have further examined the regulatory interactions of CsrA and CsrB RNA. The 5' end of the CsrB transcript was mapped, and a csrB::cam null mutant was constructed. CsrA protein and CsrB RNA levels were estimated throughout the growth curves of wild-type and isogenic csrA, csrB, rpoS, or csrA rpoS mutant strains. CsrA levels exhibited modest or negligible effects of these mutations. The intracellular concentration of CsrA exceeded the total CsrA-binding capacity of intracellular CsrB RNA. In contrast, CsrB levels were drastically decreased (~10-fold) in the csrA mutants. CsrB transcript stability was unaffected by csrA. The expression of a csrB-lacZ transcriptional fusion containing the region from -242 to +4 bp of the csrB gene was decreased ~20-fold by a csrA::kanR mutation in vivo but was unaffected by CsrA protein in vitro. These results reveal a significant, though most likely indirect, role for CsrA in regulating csrB transcription. Furthermore, our findings suggest that CsrA mediates an intriguing form of autoregulation, whereby its activity, but not its levels, is modulated through effects on an RNA antagonist, CsrB.

Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli.

Many species of bacteria devote considerable metabolic resources and genetic information to the ability to sense the environment and move towards or away from specific stimuli using flagella. In Escherichia coli and related species, motility is regulated by several global regulatory circuits, which converge to modulate the overall expression of the master operon for flagellum biosynthesis, flhDC. We now show that the global regulator CsrA of E. coli K-12 is necessary for motility under a variety of growth conditions, as a result of its role as an activator of flhDC expression. A chromosomally encoded flhDC'-'lacZ translational fusion was expressed at three- to fourfold higher levels in csrA wild-type strains than in isogenic csrA mutants. Purified recombinant CsrA protein stimulated the coupled transcription-translation of flhDC'-' lacZ in S-30 extracts and bound to the 5' segment of flhDC mRNA in RNA mobility shift assays. The steady-state level of flhDC mRNA was higher and its half-life was approximately threefold greater in a csrA wild-type versus a csrA mutant strain. Thus, CsrA stimulates flhDC gene expression by a post-transcriptional mechanism reminiscent of its function in the repression of glycogen biosynthesis.

trp RNA-binding attenuation protein-5' stem-loop RNA interaction is required for proper transcription attenuation control of the Bacillus subtilis trpEDCFBA operon.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5' end of the trp leader transcript also plays a role in the transcription attenuation mechanism. The 5' stem-loop increases the affinity of TRAP for trp leader RNA. Results from RNA structure mapping experiments demonstrate that the 5' stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5' stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with trpE'-'lacZ translational fusions demonstrate that TRAP-5' stem-loop interaction is required for proper regulation of the trp operon. 3' RNA boundary experiments indicate that the 5' structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus, TRAP-5' stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.

Effects of mutations in the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis.

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.

A 5' RNA stem-loop participates in the transcription attenuation mechanism that controls expression of the Bacillus subtilis trpEDCFBA operon.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats. TRAP binding prevents formation of an antiterminator structure, thereby promoting formation of an overlapping terminator, and hence transcription is terminated before RNA polymerase can reach the trp structural genes. In addition to the antiterminator and terminator, a stem-loop structure is predicted to form at the 5' end of the trp leader transcript. Deletion of this structure resulted in a dramatic increase in expression of a trpE'-'lacZ translational fusion and a reduced ability to regulate expression in response to tryptophan. By introducing a series of point mutations in the 5' stem-loop, we found that both the sequence and the structure of the hairpin are important for its regulatory function and that compensatory changes that restored base pairing partially restored wild-type-like expression levels. Our results indicate that the 5' stem-loop functions primarily through the TRAP-dependent regulatory pathway. Gel shift results demonstrate that the 5' stem-loop increases the affinity of TRAP for trp leader RNA four- to fivefold, suggesting that the 5' structure interacts with TRAP. In vitro transcription results indicate that this 5' structure functions in the attenuation mechanism, since deletion of the stem-loop caused an increase in transcription readthrough. An oligonucleotide complementary to a segment of the 5' stem-loop was used to demonstrate that formation of the 5' structure is required for proper attenuation control of this operon.

trp RNA-binding attenuation protein-mediated long distance RNA refolding regulates translation of trpE in Bacillus subtilis.

Expression of the trpEDCFBA operon is regulated at both the transcriptional and translational levels by the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis. When cells contain sufficient levels of tryptophan to activate TRAP, the protein binds to trp operon transcripts as they are being synthesized, most often causing transcription termination. However, termination is never 100% efficient, and transcripts that escape termination are subject to translational control. We determined that TRAP-mediated translational control of trpE can occur via a novel RNA conformational switch mechanism. When TRAP binds to the 5'-untranslated leader segment of a trp operon read-through transcript, it can disrupt a large secondary structure containing a portion of the TRAP binding target. This promotes refolding of the RNA such that the trpE Shine-Dalgarno sequence, located more than 100 nucleotides downstream from the TRAP binding site, becomes sequestered in a stable RNA hairpin. Results from cell-free translation, ribosome toeprint, and RNA structure mapping experiments demonstrate that formation of this structure reduces TrpE synthesis by blocking ribosome access to the trpE ribosome binding site. The role of the Shine-Dalgarno blocking hairpin in controlling translation of trpE was confirmed by examining the effect of multiple nucleotide substitutions that abolish the structure without altering the Shine-Dalgarno sequence itself. The possibility of protein-mediated RNA refolding as a general mechanism in controlling gene expression is discussed.

Myotonic dystrophy: molecular windows on a complex etiology.

Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy, with an incidence of approximately 1 in 8500 adults. DM is caused by an expanded number of trinucleotide repeats in the 3'-untranslated region (UTR) of a cAMP-dependent protein kinase (DM protein kinase, DMPK). Although a large number of transgenic animals have been generated with different gene constructions and knock-outs, none of them faithfully recapitulates the multisystemic and often severe phenotype seen in human patients. The transgenic data suggest that myotonic dystrophy is not caused simply by a biochemical deficiency or abnormality in the DM kinase gene product. Emerging studies suggest that two novel pathogenetic mechanisms may play a role in the disease: the expanded repeats appear to cause haploinsufficiency of a neighboring homeobox gene and also abnormal DMPK RNA appears to have a detrimental effect on RNA homeostasis. The complex, multisystemic phenotype may reflect an underlying multifaceted molecular pathophysiology: the facial dysmorphology may be due to pattern defects caused by haploinsufficiency of the homeobox gene, while the muscle disease and endocrine abnormalities may be due to both altered RNA metabolism and deficiency of the cAMP DMPK protein.

The Escherichia coli mrsC gene is required for cell growth and mRNA decay.

We have identified a gene in Escherichia coli that is required for both the normal decay of mRNA and RNA synthesis. Originally designated mrsC (mRNA stability), the mrsC505 mutation described here is, in fact, an allele of the hflB/ftsH locus (R.-F. Wang et al., J. Bacteriol. 180:1929-1938, 1998). Strains carrying the thermosensitive mrsC505 allele stopped growing soon after the temperature was shifted to 44 degrees C but remained viable for several hours. Net RNA synthesis stopped within 20 min after the shift, while DNA and protein synthesis continued for over 60 min. At 44 degrees C, the half-life of total pulse-labeled RNA rose from 2.9 min in a wild-type strain to 5.9 min in the mrsC505 single mutant. In an rne-1 mrsC505 double mutant, the average half-life was 19.8 min. Inactivating mrsC significantly increased the half-lives of the trxA, cat, secG, and kan mRNAs, particularly in an mrsC505 pnp-7 rnb-500 rne-1 multiple mutant. In addition, Northern analysis showed dramatic stabilizations of full-length mRNAs in a variety of mrsC505 multiple mutants at 44 degrees C. These results suggest that MrsC, directly or indirectly, controls endonucleolytic processing of mRNAs that may be independent of the RNase E-PNPase-RhlB multiprotein complex.

The trp RNA-binding attenuation protein regulates TrpG synthesis by binding to the trpG ribosome binding site of Bacillus subtilis.

The trpG gene of Bacillus subtilis encodes a glutamine amidotransferase subunit which is involved in the biosynthesis of L-tryptophan and folic acid. The trp RNA-binding attenuation protein (TRAP) is involved in controlling expression of trpG at the level of translation in response to changes in the intracellular concentration of tryptophan. We performed in vitro experiments using purified TRAP to elucidate the mechanism of TRAP-dependent trpG regulation. A TRAP-trpG RNA footprint analysis showed that tryptophan-activated TRAP interacts with one UAG, one AAG, and seven GAG repeats present in the trpG transcript. Results from ribosome and TRAP toeprint experiments indicated that the ribosome and TRAP binding sites overlap. Experiments with a B. subtilis cell-free translation system demonstrated that TRAP inhibits TrpG synthesis. Thus, TRAP regulates translation of trpG by blocking ribosome access to the trpG ribosome binding site. Our results are consistent with a model in which each tryptophan-activated TRAP subunit interacts with one trinucleotide repeat in an RNA target, thereby wrapping the transcript around the periphery of the TRAP complex.

Regulation of tryptophan biosynthesis: Trp-ing the TRAP or how Bacillus subtilis reinvented the wheel.

The Bacillus subtilis tryptophan biosynthetic genes are regulated by TRAP. Radiographic crystallography indicates that the TRAP complex contains 11 identical subunits arranged in a doughnut-like structure termed the beta-wheel. The trpEDCFBA operon is regulated by an attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the trp leader transcript. TRAP binding blocks formation of an anti-terminator structure, thereby promoting the formation of an overlapping terminator, resulting in transcription termination preceding the structural genes. When TRAP is not activated, it is unable to bind to the transcript, which allows anti-terminator formation and, hence, transcription of the operon. TRAP is also responsible for regulating translation of trpEand trpG. TRAP binding to trp operon readthrough transcripts promotes refolding of the RNA such that the trpE Shine-Dalgarno sequence is sequestered in a hairpin, thus inhibiting TrpE synthesis. In the case of trpG, TRAP binds to nine repeats that overlap the ribosome-binding site, thereby blocking translation.

Interaction of the trp RNA-Binding attenuation protein (TRAP) of Bacillus subtilis with RNA: effects of the number of GAG repeats, the nucleotides separating adjacent repeats, and RNA secondary structure.

The 11-subunit trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates transcription and translation by binding to several (G/U)AG repeats present in the trp leader and trpG transcripts. Filter binding assays were used to study interactions between L-tryptophan-activated TRAP and synthetic RNAs. RNAs that contained GAG and/or UAG repeats were tested while the length and sequence of the nucleotides separating adjacent trinucleotide repeats were altered. TRAP-RNA complexes formed with transcripts containing GAG repeats were more stable than those with transcripts containing UAG repeats or alternating GAG and UAG repeats. The stability of TRAP-RNA complexes also increased substantially when the number of GAG repeats was increased from five to six and from six to seven. A gradual increase in complex stability was observed when the number of GAG repeats was increased from 7 to 11. The optimal spacer between adjacent trinucleotide repeats was found to be 2 nucleotides, with A and U residues preferred over G and C residues. TRAP binding was specific for single-stranded RNA; TRAP could not bind to RNA containing GAG repeats base paired in a stable RNA duplex. Overall, our findings suggest that each L-tryptophan-activated TRAP subunit can bind one (G/U)AG repeat and that multiple TRAP subunit-RNA binding site interactions are required for stable TRAP-RNA association.

trp RNA-binding attenuation protein (TRAP)-trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis trp operon.

Expression of the Bacillus subtilis trpEDCFBA operon has been shown to be regulated by transcription attenuation in response to the availability of L-tryptophan. Regulation is mediated by the tryptophan-activated trp RNA-binding attenuation protein, TRAP, the product of mtrB. Formation of mutually exclusive RNA anti-terminator and terminator structures within trp leader RNA determines whether transcription will terminate in the leader region of the operon. Previous studies suggested that transcripts that escape termination are subject to translational regulation via the formation of a secondary structure that blocks ribosome access to the trpE ribosome-binding site. To assess the relative importance of these postulated events in trp operon regulation, we used site-directed mutagenesis to alter the putative elements involved in transcriptional and translational control. Using a trpE'-'lacZ reporter, we measured translational yield and specific mRNA levels with various leader constructs, in both mtrB+ and mtrB strains, during growth in the presence and absence of excess tryptophan. To verify that the altered regulatory regions behaved as expected, we carried out in vitro transcription assays with the wild-type and altered leader region templates and performed oligonucleotide competition assays with an oligonucleotide complementary to a segment of the transcription terminator. Our results establish that binding of TRAP to leader RNA regulates of transcription termination in the trp operon over about an 88-fold range and regulates translation of trpE over about a 13-fold range. The roles played by different trp leader RNA segments in mediating transcriptional and translational regulation are documented by our findings.

TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a toroid-shaped molecule that binds transcripts containing GAG or UAG repeats separated by two nucleotides.

The trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates both transcription and translation by binding to specific transcript sequences. The optimal transcript sequences required for TRAP binding were determined by measuring complex formation between purified TRAP protein and synthetic RNAs. RNAs were tested that contained repeats of different trinucleotide sequences, with differing spacing between the repeats. A transcript containing GAG repeats separated by two-nucleotide spacers was bound most tightly. In addition, transmission electron microscopy was used to examine the structure of TRAP and the TRAP-transcript complex. TRAP was observed to be a toroid-shaped oligomer when free or when bound to either a natural or a synthetic RNA.

Structural features of L-tryptophan required for activation of TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis.

A filter binding assay was used to determine the structural features of L-tryptophan required for activation of TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis. We examined the ability of L-tryptophan and 26 of its analogs to activate TRAP. Our findings show that TRAP activation by L-tryptophan is highly cooperative. We also observed that TRAP activation is stereospecific; D-tryptophan did not activate. Our results further indicate that the alpha-amino group and the carbonyl moiety of the alpha-carboxyl group of the ligand are required for TRAP activation and that the heterocyclic amino nitrogen of L-tryptophan greatly enhances TRAP activation. We also found that changes at several positions of the indole ring of L-tryptophan resulted in reduced TRAP activation. In addition, indole and 5-methylindole were shown to be effective competitors of L-tryptophan activation of TRAP.

TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a multisubunit complex that appears to recognize G/UAG repeats in the trpEDCFBA and trpG transcripts.

A filter binding assay was developed to study interactions between purified TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, and trp specific transcripts. TRAP formed stable complexes with trpEDCFBA leader RNA; binding was L-tryptophan-dependent and was complete within 60 s. TRAP binds to a segment of the trp leader transcript that includes part of an RNA antiterminator structure. Binding to this segment allows formation of an RNA terminator structure, thereby promoting transcription termination. Using several trpEDCFBA leader deletion transcripts, we identified several closely spaced trinucleotide repeats (seven GAG and four UAG repeats) in the trp leader transcript that appeared to be required for TRAP binding. We also showed that TRAP binds to a segment of the trpG transcript that includes the trpG ribosome binding site; the nucleotide sequence of this segment contains several appropriately spaced trinucleotide repeats (seven GAG, one UAG, and one AAG). TRAP binding to the trpG transcript would block translation initiation. RNA footprint analysis confirmed interaction between TRAP and the trinucleotide repeats in the various transcripts. TRAP, in the presence or absence of L-tryptophan, appears to consist of 11 or 12 identical 8-kDa subunits. Our findings suggest that each tryptophan-activated TRAP subunit can bind one G/UAG repeat in a target transcript. Multiple protein-RNA interactions are required for stable association.

Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III.

RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed.

Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein.

We have reconstituted Bacillus subtilis trp attenuation in vitro. Purification of the mtrB gene product (TRAP) to near homogeneity allowed us to demonstrate that addition of this protein plus L-tryptophan to template, RNA polymerase, and nucleoside triphosphates caused transcription termination in the trpEDCFBA leader region. TRAP acts by binding to the nascent transcript and preventing formation of an RNA antiterminator structure, thereby allowing terminator formation and transcription termination. Oligonucleotides complementary to segments of the antiterminator were used to demonstrate that formation of this RNA hairpin was responsible for transcription read-through. TRAP was found to be a 60-kDa multimeric protein composed of identical 6- to 8-kDa subunits, and its elution profile on a chromatographic column did not change in the presence of tryptophan.