Riboswitches-based expression systems

Each cell must regulate the expression of hundreds of different genes in response to changing environmental or cellular conditions. The majority of these sophisticated genetic control factors are proteins, which monitor metabolites and other chemical cues by selectively binding to targets. It has been discovered that RNA can also form precise genetic switches and that these elements can control fundamental biochemical processes. Riboswitches are a type of natural genetic control element that uses untranslated sequence in the 5’ region of mRNAs to form a binding pocket for a metabolite that regulates expression of that gene.

During the last years the great importance of RNA for regulating gene expression in all organisms has become obvious. Consequently, several recent approaches aim to utilize the outstanding chemical properties of RNA to develop artificial RNA regulators for conditional gene expression systems. A combination of rational design, in vitro selection and in vivo screening systems has been used to create a versatile set of RNA-based molecular switches.

These tools rely on diverse mechanisms and exhibit activity in several organisms, so they have been developmed recently in the application of engineered riboswitches for gene regulation in vivo [6]

1.1.3.1 General characteristics of riboswitches

As mentioned, riboswitches are metabolite binding domains located within the 5' untranslated regions (UTR) of some mRNAs which are involved in gene regulation [159, 166]. Allosteric rearrangement of mRNA structures is mediated by metabolite binding resulting in modulation of gene expression, and a change in expression with increasing ligand concentration, ranging from between 7-fold and 1,200-fold has been observed [50, 91, 95, 167] (Fig. 1.1).

Riboswitches are conceptually divided into two parts:the aptamer and the expression platform (Fig. 1.1B). The aptamer directly binds the metabolite, and undergoes structural changes in response. These structural changes affect the expression platform, which is the mechanism by which gene expression is regulated. Expression platforms typically turn off gene expression in response to the metabolite, but some turn it on [37, 86, 159].

In the past year, three newly confirmed riboswitch classes have been reported (Fig. 1.2) [148, 159]. The first of these, the regulation of transcription termination, is utilized by nearly every riboswitch class and typically involves metabolite-dependent formation of a terminator stem, which prevents transcription elongation and inhibits gene expression (Fig. 1.2A). Two exceptions are the adenine and glycine riboswitch, wherein metabolite binding prevents terminator stem formation and activates gene expression [91-93, 148]. Second, the regulation of translation initiation is less widely utilized and involves altering the accessibility of the SD sequence (Fig. 1.2B). In this case, metabolite binding masks the SD sequence within a secondary structure to prevent ribosome binding and thereby inhibit gene expression.

Interestingly, riboswitches in Gram-negative bacteria seemingly prefer regulation of translation initiation, whereas Gram-positive bacteria favour transcription termination, a correlation that probably reflects the higher frequency of polycistronic genes in Gram-positive

Fig. 1.1. Model of gene regulation by a typical riboswitch. (A) When the cellular concentration of metabolite is too low to occupy the riboswitch binding site, the transcription is completed, the biosynthetic and/or transport proteins are expressed; (B) when the cellular concentration of metabolite is high, the metabolite binds to the riboswitch and leads to formation of an intrinsic terminator, the metabolite biosynthetic or transport protein is not produced [41].

bacteria [108, 167]. For example, the TPP sensing riboswitch can terminate transcription of downstream genes in Gram-positive bacteria, suppress translation initiation in Gram-negative bacteria [135]. A third expression platform that can be utilized by riboswitches to affect gene expression is the regulation of RNA processing events. A conceptually simplistic manifestation of this expression platform is represented by the GlcN6P riboswitch, for which ligand binding induces catalytic self-cleavage of the mRNA and inhibition of gene expression (Fig. 1.2C) [168]. However, it seems unlikely that the aptamer and expression platform (ribozyme) are separable functionalities, as they are for other riboswitches. Interestingly, the discovery of TPP-dependent riboswitches in eukaryotic genes has unveiled other possibilities for riboswitch control of RNA processing [83, 152]. For instance, the presence of TPP aptamers within introns or 3’ untranslated regions (UTRs) suggests that riboswitches might regulate splicing or 3’ end formation, respectively [135, 148, 159].

1.1.3.2 The glycine riboswitch

It has been suggested that about 2% of the B. subtilis genes are regulated via riboswitches [93], and three riboswitches have been studied so far. One of these riboswitches precedes the lysC gene [24]. The second is the gcvT operon involved in the degradation of L-glycine if the concentration is high within the cell [5, 93]. Both of these riboswitches operate by opposite mechanisms. The third are the members of the GlcN6P class of riboswitch which are self-cleaving ribozymes; they are activated when they are bound with the sugar-phosphate compound [148].

Fig. 1.2. Mechanisms of riboswitch function. (A) Transcription termination induced by metabolite (M) binding to nascent RNA, as observed for a guanine riboswitch; (B) translation initiation modulated by metabolite-dependent sequestration of a SD sequence, as observed for a TPP riboswitch; (C) RNA processing regulated by metabolite-dependent self-cleavage, as observed for a GlcN6P riboswitch [148].

The tricistronic gcvT-gcvPA-gcvPB operon codes for enzymes involved in the degradation of L-glycine if its concentration is high within the cell. The gcvT operon will be transcribed when the L-glycine concentration within the cell is high, and the metabolite will bind to a tandem riboswitch. The glycine riboswitch consisting of two strikingly similar aptamers, connected by a short linker region present upstream of glycine catabolism and efflux genes in a wide variety of bacteria. The glycine riboswitch binds L-glycine to regulate three glycine metabolism genes by activation via inhibition of premature termination of transcription, to use L-glycine as an energy source (type of regulation as Fig. 1.2A) [5, 93, 148, 159].

1.1.3.3 The lysine riboswitch

The lysC gene of B. subtilis encodes the inphase overlapping genes for the α- and β-subunits of a lysine-responsive aspartokinase II [24]. The lysine riboswitch (also called L-box) binds L-lysine to regulate lysine biosynthesis, catabolism and transport. The lysC gene is induced when the L-lysine concentration is low within the cell and the metabolite-free riboswitch favors formation of an anti-terminator structure. If the concentration of L-lysine is high in the cell, transcription of the lysine operon is initiated but terminated after a transcript of about 270 nucleotides has been synthesized. This 5’ region of the lysine transcript is not translated, but forms a complicated secondary structure which is stabilized by L-lysine. This in turn leads to the formation of a terminator structure which causes the RNAP to dissociate from the DNA template and to release the transcript into the cytoplasm. This regulatory principle has been designated as riboswitch-mediated control of gene expression (type of regulation as Fig.

1.2A) [51, 153].