Nucleic Acids Research: Uncovering the structural mechanisms by which THF-II riboswitches recognize ligands and regulate gene translation

Tetrahydrofolate (THF) and its derivatives, collectively known as folic acid, are coenzymes of the one-carbon transferase system in the body, which can be used as a carrier of one carbon group to participate in the synthesis of a variety of biologically active substances, and are therefore essential
in the normal cellular metabolism of almost all life forms.
In most plants, most fungi, bacteria, and archaea, folic acid can be "synthesized de novo"
via a similar biosynthetic pathway.
Unfortunately, the human body cannot synthesize folic acid and must rely entirely on exogenous supplies
.
Lack of folate intake can lead to a variety of diseases, such as anemia, fetal malformations, cardiovascular diseases, neurological diseases, etc
.
Therefore, the biosynthesis and metabolic pathway of folic acid has become a popular target for the development of antibacterial and fungal drugs
.

In bacteria, the expression of genes associated with multiple key metabolic pathways is regulated
by a large class of structured RNA elements called Riboswitches.
Riboswitches are cis-acting RNA elements located primarily in the 5' untranslated region of bacterial mRNA, and their structure can generally be divided into aptamer domains and their downstream expression platform domains
.
The aptamer domain can induce conformational changes in the expression platform domain by binding ligands (such as amino acids/nucleotides and their derivatives, ions, tRNA, etc.
) or in response to environmental changes (temperature, pH, etc.
), thereby regulating
the expression of downstream genes at the transcription level or translation level.
At present, more than 40 kinds of ribose switches have been identified
.
Among them, ribose switches that recognize THF and its analogues can be divided into two categories (THF-I and THF-II).

THF-I was discovered in 2010 and is mainly present in gram-positive bacteria, and its crystal structure was resolved
in 2011.
THF-I contains four double helix structures and two THF binding sites (sites FA3WJ and FAPK), in which the double helix P2, P3, P4 form an inverted "three-branch" structure stabilized by a long-range pseudoknot interaction
.
By resolving the crystal structure, the molecular mechanism by which THF-1 recognizes ligands and regulates gene expression is
revealed.
In 2019, a new class of ribose switches that recognize THF and its analogues and regulate gene expression at the translation level were identified in gram-negative bacteria, named THF-II
.
Unlike THF-I, THF-II has a simpler structure, containing only 2 double helix structures, and has no clearly distinguishable aptamer domain and expression platform domain boundaries, and a ribosome binding site (RBS)
in the right arm of the double helix structure P1.
However, the mechanism by which THF-II recognizes ligands and regulates gene expression remains unclear
.

Fang Xianyang's research group in the School of Life Sciences of Tsinghua University published a research paper entitled "Structural insights into translation regulation by the THF-II riboswitch" online in the journal Nucleic Acids Research.
The three-dimensional structure of the second class of natural ribose switch THF-II, which specifically binds to THF and its analogues, is reported, revealing its structural mechanism
of recognizing ligands and regulating gene translation.

The researchers first applied isothermal titration calorimetry to study the important influence of magnesium ions on the binding of THF-II to ligands (such as THF), crystallized screening by optimizing RNA sequences, and analyzed the high-resolution structure
of THF-II in the ligand-free binding state of complex and C22G mutants of different ligands by X-ray crystallography 。 The crystal structure indicates that the two double helix P1, P2 and their junction regions of THF-II form a long rod-like structure by coaxial stacking, and the conserved pyrimidine located in the junction region (C22 of J12, U44 of J21) are recognized by forming 6 pairs of hydrogen bond interactions with the pterin ring fraction of tetrahydrofolate and its analogues, and their recognition patterns are similar to FAPK of THF-I (Figure 1A).

。 Next, the researchers combined small-angle X-ray scattering, site-directed mutations, and oligonucleotide-directed RNase H cleavage assay to investigate the effects of magnesium ion and ligand binding on THF-II folding, conformational dynamics, and accessibility of RBS located at P1 (Figure 1B
).
。 Based on the relevant experimental results, the researchers proposed a model of THF-II ribose switch regulating gene translation (Figure 1C): in the absence of magnesium ions, RNA is in a defolded state and cannot bind ligands; The binding of magnesium ions can greatly promote the folding of RNA to obtain ligand binding ability, but at this time, its coaxial stacked structure has not yet formed, RBS exposure is high, and ribosomes can bind to it and mediate the initiation of downstream gene translation.
When ligands are bound, a coaxial stacking structure similar to THF-II in the crystal structure is formed, the exposure of RBS is greatly reduced, the ribosome cannot bind to it, and downstream gene translation stops
.

Figure 1.
THF-II riboswitch recognizes ligands and regulates gene translation.

(A) the molecular mechanism of THF-II recognition of THF and the high-level structure of its complexes, (B) SAXS analysis reveals conformational dynamics of magnesium ion and ligand binding to regulate THF-II, and (C) a model
of THF-II regulating gene expression at the translation level.

In conclusion, by integrating a variety of research methods (X-ray crystallography, small-angle X-ray scattering, isothermal titration calorimetry, RNase H cleavage experiments, etc.
), this work deeply studies the high-level structure, conformational dynamics and interactions of a class of THF ribose switches with simple structures, and reveals the molecular basis
of their specific recognition of ligands and regulation of gene expression at the translation level.
The research results can also provide an important reference
for the de novo design of functional RNA, the development of RNA sensors based on ribose switches, and the design of small molecule drugs targeting RNA.

Fang Xianyang, associate professor at the School of Life Sciences, Tsinghua University, is the corresponding author
of this article.
Xu Lilei, a 2017 graduate of the School of Life Sciences of Tsinghua University, Xiao Yu, a postdoctoral fellow, is the joint first author of the paper, and Zhang Jie, a 2020 doctoral student, has made important contributions
to this research.
The X-ray crystallography platform of Tsinghua University, the activity screening platform of the Pharmaceutical Center, the BL18U1 and BL19U1 line stations of Shanghai synchrotron radiation light source, and the 12-ID-B line station of Argonne National Laboratory provided equipment and technical support for this study.
Professor Wang Jiawei from the School of Life Sciences of Tsinghua University guided the crystal model correction
.
The research was supported
by the National Natural Science Foundation of China and the Beijing Frontier Center for Biological Structures.