Tina M. Henkin

Catabolite repression of α amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacl and galR repressors

Expression of the α‐amylase gene of Bacillus subtilis is controlled at the transcriptional level, and responds to the growth state of the cell as well as the availability of rapidly metabolizable carbon sources. Glucose‐mediated repression has previously been shown to involve a site near the transcriptional start‐point of the amyE gene. In this study, a transposon insertion mutation was characterized which resulted in loss of glucose repression of amyE gene expression. The gene affected by this mutation, which was localized near 263° on the B. subtilis chromosomal map, was isolated and its DNA sequence was determined. This gene, designated ccpA, exhibited striking homology to repressor genes of the lac and gal repressor family. The ccpA gene was found to be allelic to alsA, previously identified as a regulator of acetoin biosynthesis, and may be involved in catabolite regulation of other systems as well.

The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in Gram-positive bacteria

The molecular mechanisms for regulation of the genes involved in the biosynthesis of methionine and cysteine are poorly characterized in Bacillus subtilis. Analyses of the recently completed B. subtilis genome revealed 11 copies of a highly conserved motif. In all cases, this motif was located in the leader region of putative transcriptional units, upstream of coding sequences that included genes involved in methionine or cysteine biosynthesis. Additional copies were identified in Clostridium acetobutylicum and Staphylococcus aureus, indicating conservation in other Gram‐positive genera. The motif includes an element resembling an intrinsic transcriptional terminator, suggesting that regulation might be controlled at the level of premature termination of transcription. The 5′ portion of all of the leaders could fold into a conserved complex structure. Analysis of the yitJ gene, which is homologous to Escherichia coli metH and metF, revealed that expression was induced by starvation for methionine and that induction was independent of the promoter and dependent on the leader region terminator. Mutation of conserved primary sequence and structural elements supported a model in which the 5′ portion of the leader forms an anti‐antiterminator structure, which sequesters sequences required for the formation of an antiterminator, which, in turn, sequesters sequences required for the formation of the terminator; the anti‐antiterminator is postulated to be stabilized by the binding of some unknown factor when methionine is available. This set of genes is proposed to form a new regulon controlled by a global termination control system, which we designate the S box system, as most of the genes are involved in sulphur metabolism and biosynthesis of methionine and cysteine.

Transcription termination control of the S box system: Direct measurement of S-adenosylmethionine by the leader RNA

Modulation of the structure of a leader RNA to control formation of an intrinsic termination signal is a common mechanism for regulation of gene expression in bacteria. Expression of the S box genes in Gram-positive organisms is induced in response to limitation for methionine. We previously postulated that methionine availability is monitored by binding of a regulatory factor to the leader RNA and suggested that methionine or S-adenosylmethionine (SAM) could serve as the metabolic signal. In this study, we show that efficient termination of the S box leader region by bacterial RNA polymerase depends on SAM but not on methionine or other related compounds. We also show that SAM directly binds to and induces a conformational change in the leader RNA. Both binding of SAM and SAM-directed transcription termination were blocked by leader mutations that cause constitutive expression in vivo. Overproduction of SAM synthetase in Bacillus subtilis resulted in delay in induction of S box gene expression in response to methionine starvation, consistent with the hypothesis that SAM is the molecular effector in vivo. These results indicate that SAM concentration is sensed directly by the nascent transcript in the absence of a trans-acting factor.

tRNA as a positive regulator of transcription antitermination in B. subtilis

Most Bacillus tRNA synthetase genes are regulated by a common transcription antitermination mechanism but respond individually to limitation for the cognate amino acid. The mRNA leader regions of these genes exhibit extensive structural conservation, with a single codon specific for the appropriate amino acid at the identical position in each structure. Alteration of this sequence in the tyrS gene from UAC (tyrosine) to UUC (phenylalanine) resulted in loss of induction by tyrosine limitation and a switch to induction by phenylalanine limitation. Insertion of an extra base immediately upstream of the codon did not alter regulation, indicating a nontranslational mechanism. A nonsense codon resulted in an uninducible phenotype that was suppressible in a lysyl-tRNA nonsense suppressor mutant, indicating that tRNA acts as an effector.

Riboswitch RNAs: using RNA to sense cellular metabolism

Riboswitches are RNA elements that undergo a shift in structure in response to binding of a regulatory molecule. These elements are encoded within the transcript they regulate, and act in cis to control expression of the coding sequence(s) within that transcript; their function is therefore distinct from that of small regulatory RNAs (sRNAs) that act in trans to regulate the activity of other RNA transcripts. Riboswitch RNAs control a broad range of genes in bacterial species, including those involved in metabolism or uptake of amino acids, cofactors, nucleotides, and metal ions. Regulation occurs as a consequence of direct binding of an effector molecule, or through sensing of a physical parameter such as temperature. Here we review the global role of riboswitch RNAs in bacterial cell metabolism.

The L box regulon: Lysine sensing by leader RNAs of bacterial lysine biosynthesis genes

Expression of amino acid biosynthesis genes in bacteria is often repressed when abundant supplies of the cognate amino acid are available. Repression of the Bacillus subtilis lysC gene by lysine was previously shown to occur at the level of premature termination of transcription. In this study we show that lysine directly promotes transcription termination during in vitro transcription with B. subtilis RNA polymerase and causes a structural shift in the lysC leader RNA. We find that B. subtilis lysC is a member of a large family of bacterial lysine biosynthesis genes that contain similar leader RNA elements. By analogy with related regulatory systems, we designate this leader RNA pattern the “L box.” Genes in the L box family from Gram-negative bacteria appear to be regulated at the level of translation initiation rather than transcription termination. Mutations of B. subtilis lysC that disrupt conserved leader features result in loss of lysine repression in vivo and loss of lysine-dependent transcription termination in vitro. The identification of the L box pattern also provides an explanation for previously described mutations in both B. subtilis and Escherichia coli lysC that result in lysC overexpression and resistance to the lysine analog aminoethylcysteine. The L box regulatory system represents an example of gene regulation using an RNA element that directly senses the intracellular concentration of a small molecule.

The S(MK) box is a new SAM-binding RNA for translational regulation of SAM synthetase.

We have identified the SMK box as a conserved RNA motif in the 5′ untranslated leader region of metK (SAM synthetase) genes in lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species. This RNA element bound SAM in vitro, and binding of SAM caused an RNA structural rearrangement that resulted in sequestration of the Shine-Dalgarno (SD) sequence. Mutations that disrupted pairing between the SD region and a sequence complementary to the SD blocked SAM binding, whereas compensatory mutations that restored pairing restored SAM binding. The Enterococcus faecalis SMK box conferred translational repression of a lacZ reporter when cells were grown under conditions where SAM pools are elevated, and mutations that blocked SAM binding resulted in loss of repression, demonstrating that the SMK box is functional in vivo. The SMK box therefore represents a new SAM-binding riboswitch distinct from the previously identified S box RNAs.

Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis

The Bacillus subtilis ccpA gene has previously been shown to be involved in repression of amyE expression when cells are grown in excess glucose. The region of the B. subtilis chromosome downstream from ccpA was characterized to determine if additional genes involved in carbohydrate metabolism were present. Two open reading frames that exhibited sequence similarity to the Escherichia coli and B. subtilis motA and motB motility genes were found immediately downstream from ccpA; disruption of this region had no effect on growth, sporulation or motility. Two divergent transcriptional units containing the acsA and acuABC genes were also found in this region. The acsA gene encodes acetyl‐CoA synthetase, and inactivation of this gene resulted in loss of the ability to utilize acetate as a carbon source for growth or sporulation. Disruption of the acuABC genes resulted in poor growth or sporulation on acetoin or butanediol. The acsA and acuABC promoter sequences were identified by primer extension, and are in close proximity. Two sequences resembling the amyO regulatory target site necessary for glucose repression of amyE were identified in the acsA‐acuABC promoter regions.

From ribosome to riboswitch: control of gene expression in bacteria by RNA structural rearrangements.

ABSTRACT Structural elements in the 5′ region of a bacterial mRNA can have major effects on expression of downstream coding sequences. Folding of the nascent RNA into the helix of an intrinsic transcriptional terminator results in premature termination of transcription and in failure to synthesize the full-length transcript. Structure in the translation initiation region of an mRNA blocks access of the translation initiation complex to the ribosome binding site, thereby preventing protein synthesis. RNA structures can also affect the stability of an RNA by altering sensitivity to ribonucleases. A wide variety of mechanisms have been uncovered in which changes in mRNA structure in response to a regulatory signal are used to modulate gene expression in bacteria. These systems allow the cell to recognize an impressive array of signals, and to monitor those signals in many different ways.

tRNA-mediated transcription antitermination in vitro: Codon–anticodon pairing independent of the ribosome

Uncharged tRNA acts as the effector for transcription antitermination of genes in the T box family in Bacillus subtilis and other Gram-positive bacteria. Genetic studies suggested that expression of these genes is induced by stabilization of an antiterminator element in the leader RNA of each target gene by the cognate uncharged tRNA. The specificity of the tRNA response is dependent on a single codon in the leader, which was postulated to pair with the anticodon of the corresponding tRNA. It was not known whether the leader RNA–tRNA interaction requires additional factors. We show here that tRNA-dependent antitermination occurs in vitro in a purified transcription system, in the absence of ribosomes or accessory factors, demonstrating that the RNA–RNA interaction is sufficient to control gene expression by antitermination. The tRNA response exhibits similar specificity in vivo and in vitro, and the antitermination reaction in vitro is independent of NusA and functions with either B. subtilis or Escherichia coli RNA polymerase.