Structural insights into amino acid binding and gene control by a lysine riboswitch

Abstract

In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1–4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids1–3. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 ångström (Å)) and free (3.1 Å) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligandbinding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

RNA–Amino Acid Binding: A Stereochemical Era for the Genetic Code

“…The aliphatic inner side chain of lysine is sandwiched between the base planes of nearby purines (Garst et al 2008;Serganov et al 2008)-thus extended; its length can be measured by contacts with the polar groups at both ends. This suffices to distinguish lysine from similar amino acids like ornithine, whose side chain is one methylene shorter, consequently binding markedly more weakly than lysine (Sudarsan et al 2003).…”

“…(Orgel 1968) methionines in three different riboswitches specific for Sadenosyl methionine (Gilbert et al 2008;Lu et al 2008;Montange and Batey 2006). Moreover, structures for the aptamer domain for the lysine riboswitch (Garst et al 2008;Serganov et al 2008) and an aptamer for citrulline and arginine (Yang et al 1996), and a natural arginine binding site (Pugilisi et al 1993) can also be consulted. Comparison and moderate extrapolation from these structures suggest that it will be possible for RNA sites to exist that bind most of the 20 major amino acids, though the abundances of RNA structures containing sites will likely vary, as will their affinity and discrimination.…”

Folding of the SAM-I riboswitch

“…17,24 Intriguingly, some crystal structures of ligand-free aptamers are highly similar to those obtained in their bound form, suggesting that free aptamers may sample various conformations resembling those adopted in presence of the ligand. [25][26][27] Because riboswitches have been identified as promising targets for antibacterial drug development, 28,29 it is imperative to get more information about how riboswitch folding processes are used for recognition of cellular metabolites, and how this is used to modulate gene expression.…”

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

“…Two of these, the lysine and glycine riboswitches, can directly detect amino acids. While the structure of several SAM and lysine riboswitches has been determined (Fuchs et al 2006;Montange and Batey 2006;Gilbert et al 2008;Serganov et al 2008), the structural basis of glycine riboswitch function is still not known. The glycine riboswitch was identified in a bioinformatic search for conserved sequences and secondary structural elements.…”