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Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain
Janina Buck
1
2
Jonas Noeske
1
2
Jens W ohnert
0
1
Harald Schwalbe
1
2
0
Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University
, Max von Laue-Strasse 7 & 9,
60438 Frankfurt am Main, Germany
1
Present address: Jonas Noeske,
Department of Molecular and Cell Biology, University of California at Berkeley
,
Berkeley, CA 94720, USA
2
Institute for Organic Chemistry and Chemical Biology
Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated preformation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg2+ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gswloop) of the guanine-sensing riboswitch and their Mg2+induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gswloop or the increase in temperature for both Gsw and Gswloop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg2+. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.
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The complex three-dimensional structures of RNAs
include a large variety of secondary and tertiary structural
motifs. Structural motifs defining tertiary folds contribute
to local RNA structure formation but can also connect
sequentially distant nucleotides and globally constrain
RNA conformation. Interestingly, it has been shown
that tertiary contacts of structural elements remote from
the active center influence the biological functions for a
number of different RNAs (1). In addition to the intrinsic
properties of an RNA, cofactors including proteins, ions
or small ligands mediate RNA structure formation and
alter or promote cellular function. Mg2+ ions play a
particularly important role since they often enable the
formation and functional stabilization of compact RNA
structures (2).
Riboswitches represent a class of recently identified
RNA regulatory elements. They are generally found in
the 50-untranslated regions of mRNAs. Transcriptional
or translational regulation or RNA processing is
modulated by binding of a small ligand to an
evolutionarily highly conserved ligand-binding domain
(aptamer domain). The conformational switch between
alternate RNA conformations is supposed to be the
structural basis for the regulation of gene expression.
According to this model, ligand binding stabilizes one of
the alternative conformations of the aptamer domain that
further affects formation or destabilization of a
30-downstream structural element (3). Riboswitches sense
various small molecule metabolites, ranging from purine
nucleobases to amino acids (4). The high affinity and
specificity of riboswitch aptamer domains for their small
molecule effectors is coupled to complex tertiary
architectures, involving formation of intricate networks of
intraand intermolecular interactions (5). To gain function, a
remarkable variety of strategies for ligand recognition and
conformational adaptation of riboswitch elements has
been discovered. RNAligand complex formation can,
for example, be critically dependent on cations as e.g.
observed for FMN- (6) and TPP-sensing (7,8)
riboswitches. A single point mutation in the
ligand-binding region of the guanine-sensing riboswitch
element (C74U) converts the specificity of the aptamer
domain from the cognate ligand guanine to the originally
rejected ligand adenine (9). In the case of the metabolite
Sadenosylmethionine (SAM), even five structurally
different classes of riboswitches with different ligand
recognition modes could be identified (1016). In addition, not
only nucleotides that define the ligand-binding pocket but
also residues in remote regions are found to be important
for ligand binding and/or the regulatory function of
various riboswitch elements (17). These residues are
involved in the formation of a large number of different
long-range tertiary interactions and enable the formation
of compact RNA structures (18). Pre-formation of
peripheral structural elements already in the ligand-free state
could be observed in some riboswitch RNAs; the extent
to which secondary and (...truncated)