Structural basis for guanidine sensing by the ykkC family of riboswitches.

Regulation of gene expression by cis-encoded riboswitches is a prevalent theme in bacteria. Of the hundreds of riboswitch families identified, the majority of them remain as orphans, without a clear ligand assignment. The ykkC orphan family was recently characterized as guanidine-sensing riboswitches. Herein we present a 2.3 Å crystal structure of the guanidine-bound ykkC riboswitch from Dickeya dadantii The riboswitch folds into a boot-shaped structure, with a coaxially stacked P1/P2 stem forming the boot, and a 3'-P3 stem-loop forming the heel. Sophisticated base-pairing and cross-helix tertiary contacts give rise to the ligand-binding pocket between the boot and the heel. The guanidine is recognized in its positively charged guanidinium form, in its sp2 hybridization state, through a network of coplanar hydrogen bonds and by a cation-π stacking contact on top of a conserved guanosine residue. Disruption of these contacts resulted in severe guanidinium-binding defects. These results provide the structural basis for specific guanidine sensing by ykkC riboswitches and pave the way for a deeper understanding of guanidine detoxification-a previously unappreciated aspect of bacterial physiology.

Structural basis for tRNA decoding and aminoacylation sensing by T-box riboregulators.

T-box riboregulators are a class of cis-regulatory RNAs that govern the bacterial response to amino acid starvation by binding, decoding and reading the aminoacylation status of specific transfer RNAs. Here we provide a high-resolution crystal structure of a full-length T-box from Mycobacterium tuberculosis that explains tRNA decoding and aminoacylation sensing by this riboregulator. Overall, the T-box consists of decoding and aminoacylation sensing modules bridged by a rigid pseudoknot structure formed by the mid-region domains. Stem-I and the Stem-II S-turn assemble a claw-like decoding module, while the antiterminator, Stem-III, and the adjacent linker form a tightly interwoven aminoacylation sensing module. The uncharged tRNA is selectively recognized by an unexpected set of favorable contacts from the linker region in the aminoacylation sensing module. A complex structure with a charged tRNA mimic shows that the extra moiety dislodges the linker, which is indicative of the possible chain of events that lead to alternative base-pairing and altered expression output.

Guanidine-sensing riboswitches: How do they work and what do they regulate?

After remaining an orphan for over a decade, the ykkC riboswitch family (ykkC, mini-ykkC, and ykkC-III) was recently characterized as guanidine-specific genetic regulatory elements (guanidine-I, II, and III). They respond to increased levels of intracellular guanidine by turning on genes involved in guanidine export and breakdown. Their existence suggests that regulation of intracellular guanidine levels could be an important piece of bacterial physiology which was not appreciated previously. Structural biologists moved exceptionally fast to reveal the guanidine-sensing mechanisms of these riboswitches at the atomic level. The crystal structures of all three guanidine family members have been determined. They appear to represent three independently evolved RNA sensors, with distinct tertiary folds but surprisingly similar guanidine-binding cores. A few key questions remain to be addressed: It is not known which metabolic pathway(s) may lead to guanidine accumulation and the function of close relatives to the guanidine-I riboswitch that do not respond to guanidine remains unclear. The continued characterization of these and other orphan cis-regulatory elements represents an orthogonal approach to reveal new facets of bacterial physiology. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Riboswitches RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry.

Acting in tandem.

RNA structures called tandem riboswitches allow bacteria to employ complex logical operations in response to nutrient starvation.