Posttranscriptional control of bacterial gene expression

All organisms must regulate the expression of their genes temporally, quantitatively, functionally, and oftentimes, spatially. Additionally, these regulatory processes must be responsive to a wide variety of chemical and physical cues. Exploration of the full range of regulatory possibilities and the biochemical mechanisms that are utilized will reveal the interconnectedness of metabolic pathways and elucidate life’s underlying genetic circuitry.

Regulation of gene expression can occur through an alteration in any one of the steps that occur along the information processing pathways. Although control of transcription initiation is likely to be the predominate mode of gene expression control, there is a growing appreciation for posttranscriptional regulatory mechanisms. These regulatory mechanisms typically involve an RNA element, located at the 5' UTR, which is capable of receiving intracellular signals. Upon binding of the appropriate signal, which may occur via protein, RNA, or small molecule intermediates (see Figure 1), conformational changes are induced within the 5' UTR RNA that in turn affect expression of the downstream genes. As a reflection of how important such RNA-mediated genetic control is for bacteria, greater than 4% of the Bacillus subtilis genome is believed to be regulated in this manner (Winkler WC. 2005. Arch. Microbiol. 183:151-9).

Fig. 1  Categories of RNA-mediated genetic control in Bacillus subtilis. a RNA-binding protein that binds to a structure in the 5' untranslated region and stabilizes antiterminator (AT) formation through interactions of (2) with (3). In the absence of protein, (3) pairs with (4), resulting in terminator formation (T). A survey of similar mechanisms in B. subtilis reveals that 21 transcriptional units (TUs) are controlled in this manner (each number constitutes a different TU and is defined further in Table 1 of Winkler, 2005. Arch. Microbiol. 183:151-9). Approximate genomic locations of the TUs are indicated. b Uncharged cognate tRNA interacts with T-box RNAs and promotes formation of an AT and synthesis of downstream genes. Absence of tRNA results in formation of a T. A survey of similar mechanisms reveals that 19 B. subtilis TUs are controlled in this manner. c A metabolite binds to a structure in the 5' untranslated region and stabilizes an anti-antiterminator (A-AT), which allows for T formation. In the absence of a metabolite, an AT is formed, and downstream genes are synthesized. A survey of metabolite-binding RNAs in B. subtilis reveals that 36 TUs are controlled in this manner. RBS Ribosome-binding site. Green color indicates genetic elements that are switched 'ON' in response to effector binding. Red color indicates genetic elements that are switched 'OFF' in response to effector binding.

Table 1.

Our laboratory is interested in the genetic and biochemical characterization of these regulatory strategies, as well as the discovery of novel regulatory mechanisms. We are using this data to develop biological engineering techniques that will be used for a variety of biomedical applications, including development of novel drugs and genetic tools.

Biology of riboswitch RNAs: molecular mechanisms and biological distribution

Recent data indicate that, in bacteria, posttranscriptional regulatory strategies often employ specific RNA structures termed riboswitches (Figure 1C), which are embedded within the 5' untranslated region of mRNAs. These RNA sequences fold into specific three-dimensional shapes that act as molecular receptors for certain intracellular metabolites. Upon binding of the metabolite there is a change in conformation that ultimately results in a change in gene expression. Switching between conformations can influence mRNA stability, efficiency of translation initiation, or in some cases, the processivity of RNA polymerase. Riboswitches are widespread throughout biology and are utilized for genetic control over many fundamental genes. For these and other reasons, we are interested in exploring the biochemistry, biological distribution, and biomedical applications of riboswitch RNAs. We are particularly interested in a riboswitch that harnesses the self-cleavage ability of a ribozyme in order to regulate expression of a bacterial gene, glmS (Figure 2; Winkler et al., 2004. Nature 428:281-6). We are uncovering the molecular details for the novel mechanism by which this cleavage event results in a change in gene expression. This particular riboswitch serves as a model for how a ribozyme can be utilized to regulate bacterial gene expression. Furthermore, the glmS riboswitch appears to control the initial step in aminosugar synthesis and may be the dominate regulatory step for this important metabolic pathway. Too much or too little GlmS is deleterious for the cell, resulting in death or developmental problems (e.g., see accompanying picture). Therefore, the glmS ribozyme also represents a novel potential target for drug development.

Fig 2 Metabolite-responsive cleavage by the B. subtilis glmS riboswitch. A conserved RNA structure is located in the 5'UTR of the glmS gene for many gram-positive bacteria. A metabolite produce by this enzyme, glucosamine-6-phosphate, directly binds to the leader RNA and stimulates a self-processing activity. This self-cleavage reaction releases an oligonucleotide sequence and results in downregulation of the glmS gene.

Fig. 3 Reduction in GlmS results in altered cell morphology for Bacillus subtilis. a The B. subtilis glmS gene was deleted and place under IPTG-inducible control on a self-replicating plasmid. Cells were grown in glucose minimal medium to an OD₆₀₀ of 0.1 whereupon IPTG was removed. Bacteria are shown after 6 hours of incubation in the absence of inducer (b) or presence of inducer.

Diverse roles for RNAs in biology

When considering the assorted range of functions accomplished by RNAs in biology one has to consider more than just cis-acting RNA structures like riboswitches. RNAs are also involved in modification of other nucleic acids, as unstructured regulatory oligonucleotides, and as structured, trans-acting regulatory RNAs. And this partial list may be just the beginning. Several billion years ago, organisms with surprisingly complex metabolisms were likely to have relied upon specific classes of RNAs for all of their genomic and catalytic requirements. Therefore, given the extensive precedence for present or past roles of RNA polymers, there is no reason not to expect that modern organisms are replete with biological RNAs enacting functions other than that of the ‘passive mRNA’ transcript. Our lab is interested in discovering just how often, and in what capacity, these interesting and important RNA sequences are utilized inside a given cell.

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