Riboswitches and their genetic and molecular significance in living organisms

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Riboswitches are defined as RNA domains at the 5’-ends of mRNA that recognize small molecules and respond by changing their three-dimensional structure. This change in turn affects the translation of the mRNA or, sometimes, it’s premature termination, especially during protein synthesis in the cell, including microbial cells. A riboswitch can be defined as an RNA domain, usually in an mRNA molecule, that can bind a specific small molecule and alter its secondary structure. And this alteration in turn controls translation of the mRNA in the affected cell. Riboswitching as explained above is an important process in molecular biology because it helps to control biosynthetic pathways for amino acids and other metabolites in the cell of an organism. Riboswitches are mostly used to control biosynthetic pathways for amino acids, purines, and other metabolites produced in the cells of microbes.

One of the most interesting findings in molecular biology has been the discovery that RNA molecules can carry out several roles once thought to be the purview of proteins only. RNA can specifically recognize and bind other molecules, including low-molecular-weight metabolites. It is important to emphasize that such binding does not involve complementary base pairing (as does antisense RNA) but occurs as a result of a folding of the RNA into a specific three-dimensional structure that recognizes the target molecule, much as a protein enzyme recognizes its substrate. Some of these RNA molecules are called ribozymes because they are catalytically active like enzymes. Theseother RNA molecules that resemble repressors and activators in binding metabolites such as amino acids or vitamins and regulating gene expression in the process are known as riboswitches.  

It is also important to note that certain mRNAs contain regions upstream of the coding sequences that can fold specific three-dimensional structures that bind small molecules. These recognition domains are riboswitches and they exist as two alternative structures, one with the small molecule bound and the other without. Alternation between the two forms of the riboswitch thus depends on the presence or absence of the small molecule and in turn controls the expression of the mRNA. Riboswitches have been found that controls the synthesis of enzymes in biosynthetic pathways for various enzymatic cofactors including vitamins thiamine, riboflavin and cobalamin (B12), for a few amino acids, for the purine bases adenine and guanine, and for glucosamine 6-phosphate, a precursor in peptidoglycan synthesis.

Molecular mechanisms of riboswitches and their importance

The riboswitch control mechanism is analogous to one we have seen before, and this is exemplified in the regulation of enzyme synthesis by negative control of transcription – which you may already be familiar with. In this case, the presence of a specific metabolite shuts down the transcription of genes encoding enzymes for the corresponding biosynthetic pathway. And this is usually achieved by the use of a protein repressor, such as the arginine repressor in the case of the arginine biosynthetic operon. In the case of a riboswitch, there is no regulatory protein molecule. Rather, the metabolite binds directly to the riboswitch domain at the 5’ end of the mRNA. Riboswitches usually exert their control after the mRNA has already been synthesized. Therefore, most riboswitches function to control translation of the mRNA, rather than transcription.

The metabolite that is bound by the riboswitch is typically the product of a biosynthetic pathway whose constituent enzymes are encoded by the mRNAs that carry the corresponding riboswitches. For example, the thiamine riboswitch that binds thiamine pyrophosphate is upstream of the coding sequences for enzymes that participate in the thiamine biosynthetic pathway. When the pool of thiamine pyrophosphate is sufficient in the cell, this metabolite bind to its specific riboswitch mRNA. The new secondary metabolite structure of the riboswitch blocks the Shine-Dalgarno ribosome-binding sequence on the mRNA and this prevents the message from binding to the ribosome. This activity or process prevents translation. In the regulation of the riboswitch, the binding of a specific metabolite alters the secondary structure of the riboswitch domain, which is located in the 5’-untranslated region of the mRNA, preventing translation. The Shine-Dalgarno site is where the ribosome binds the RNA.

If the concentration of thiamine pyrophosphate drops sufficiently low, this molecule can dissociate from its riboswitch mRNA. And this unfolds the message and exposes the Shine-Dalgarno site, this allowing the mRNA to bind to the ribosome and become translated. The thiamine analog pyrithiamine blocks the synthesis of thiamine and hence, inhibits bacterial growth. Until the discovery of riboswitches, the site action of pyrithiamine remained mysterious. It now appears that pyrithiamine is converted by cells to pyrithiamine pyrophosphate, which then binds to the thiamine riboswitch. Thus the biosynthetic pathway is shut off even when no thiamine is available. Bacterial mutants selected for resistance to pyrithiamine have alternations in the sequence of the riboswitch that result in failure to bind both pyrithiamine pyrophosphate and thiamine pyrophosphate.

In Bacillus subtilis (a soil-dwelling bacterium), where about 2% of the genes are under riboswitch control, the same riboswitch is present on several mRNAs that together encode the proteins for a particular pathway. For example, over a dozen genes in 6 operons are controlled by the thiamine riboswitch. Despite being part of the mRNA, some riboswitches nevertheless do control transcription. The mechanism is similar to that seen in attenuation – a conformational change in the riboswitch causes premature termination of the synthesis of the mRNA that carries it.

The link between evolution and riboswitches

Riboswitches have been found in some bacteria and a few other plants and fungi. Some scientists believe that riboswitches are remnants of the RNA world, a period eons ago before cells, DNA and protein, when it is hypothesized that catalytic RNAs (i.e., ribozymes) were the only self-replicating life forms. In such an environment, riboswitches may have been a primitive mechanism of metabolic control – a simple means by which RNA life forms could have controlled the synthesis of other RNAs. As proteins evolved, riboswitches might have also been the first control mechanisms for their synthesis, as well. If this is scientifically true, the riboswitches that remain today may be the last vestiges of this simple form of gene / biological control in genetic processes because it is generally known that metabolic regulations in the cells is almost exclusively carried out by way of the regulatory proteins saddled with that responsibility of gene expression control.

Further reading

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

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