TetR aptamer

Apr 10, 2024 • Qiaozhen Liu, Zhijie Tan

Timeline

Description

In 2009, Anke Hunsicker and colleagues identified an RNA aptamer by means of in vitro screening for TetR binding and in vivo screening for TetR inducible binding, which induces tet-controlled gene expression in E. coli. In 2020, Florian C. Grau and co-workers presented the crystal structure of the TetR-RNA aptamer complex at a resolution of 2.7 Å^[1,5].

SELEX

In 2009, Anke Hunsicker et al. isolated sequences that bind TetR through 12 rounds of selection. They used a two-step approach to identify RNA aptamers that induce the transcriptional regulator TetR in vivo. First, an automated in vitro selection protocol was employed to isolate RNA aptamers that bind TetR. After 12 rounds of selection, a significant enrichment of TetR-binding RNA was detected and its specificity of binding was verified by a filter-retention assay^[1].

Detailed information are accessible on SELEX page.

Structure

2D representation

The TetR-binding aptamer is capable of inducing TetR-controlled gene expression. TetR-binding aptamers were isolated via in vitro selection along with an additional screening step, which was crucial for identifying aptamers that are active within the cell. Mutational analyses defined the minimal active sequence and highlighted the aptamer bases involved in induction. TetR binding RNA aptamer K1 was named by Beatrix Suess et al. in the article. The orange represents the bases that interact with the protein, while the grey indicates parts of the 3D structure that remain unresolved. Here we utilized Ribodraw to complete the figure based on the 3D structure information^[1,5].

5'-GGCCGGAGAAUGUUAUGGCGCGAAAGCGCAGAGAAAACCGGUC-3'

3D visualisation

Florian C. Grau1 et al. sovled the crystal structure, at 2.7 Å resolution, of an RNA aptamer bound to the transcription repressor TetR has been determined. The PDB ID of this structure is 6SY4^[5].

Additional available structures that have been solved and detailed information are accessible on Structures page.

Binding pocket

Left: A surface representation of the binding pocket of the aptamer originating from PDB ID: 6SY4 at a resolution of 2.7 Å is presented. In the case of TetR (displayed with vacuum electrostatics), blue is indicative of a positive charge, whereas red represents a negative charge. Right: The hydrogen bonds at the binding sites where the aptamer binds to TetR are shown. Arg28' and are symmetric unit amino acids.

Ligand information

SELEX ligand

Florian C. Grau et al. investigated the affinity of RNA aptamer K1 for TetR using EMSA and ITC experiments. EMSA gave a KD of 80 nM; ITC recorded a KD of 5.6 nM and confirmed a stoichiometry. ITC also showed the complex formation is enthalpy-driven (ΔH = –156.0 kJ mol⁻¹) with large entropy reduction (TΔS = –108.8 kJ mol⁻¹)^[5].

Structure ligand

TetR family regulators have been implicated in the transcriptional regulation of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, responses to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The TetR proteins identified within over 115 genera of bacteria and archaea possess a common helix-turn-helix (HTH) structure within their DNA-binding domain.Nevertheless, TetR proteins are capable of functioning in diverse ways. They can directly bind to a target operator to exert their effect. For instance, TetR binds to the Tet(A) gene to repress it in the absence of tetracycline. Alternatively, they can be involved in complex regulatory cascades where the TetR protein may either be modulated by another regulator or can trigger the cellular response.This entry relates to the tetracycline transcriptional repressor TetR, which binds to the Tet(A) gene to suppress its expression when tetracycline is absent.-----from Pfam

UniProt ID: uniquely identifies protein sequences in the UniProt database, a resource for protein information.

Pfam: a widely recognised database of protein families and domains.

GenBank: maintained by NCBI(National Center for Biotechnology Information), is a database of nucleotide sequences from various organisms, vital for genetic and molecular biology research.

Mass: an intrinsic property of a body.

Uniprot ID	Pfam	Mass	Protein sequence	PDB ID	GenBank
P04483	IPR003012	46.51 kDa	GGPYLQ ...... MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS	4AC0	912848

Similar compound

We used the Dail server website to compare the structural similarities of ligand proteins, and chose the top 10 in terms of similarity for presentation. The Dali server is a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB). Z-score is a standard score that is converted from an original score. The list of neighbours is sorted by Z-score. Similarities with a Z-score lower than 2 are spurious. RMSD(Root Mean Square Deviation) value is used to measure the degree to which atoms deviate from the alignment position.

Dail server website: a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB).

Z-score: a standard score that is converted from an original score. The list of neighbours is sorted by Z-score. Similarities with a Z-score lower than 2 are spurious.

RMSD: (Root Mean Square Deviation) is used to measure the degree to which atoms deviate from the alignment position.

PDB: PDB ID+ chain name.

PDB	Z-score	RMSD	Description
6YS4-A	8.6	0	Original chain
3ZQG-A	25	1.1	Tracycline repressor protein class b from trans
3BQY-A	19.6	2	Putative tetr family transcriptional regulator
1Z0X-A	18.1	2.3	Transcriptional regulator,tetr family
2HXO-A	15	3.1	Putative tetr-family transcriptional regulator
2Y31-A	15	3.1	Putative repressor simreg2
2HXI-B	14.9	3.3	Putative transcriptional regulator
1ZK8-A	14.6	2.8	Transcriptional regulator, tetr family
2NP5-A	13.1	2.9	Transcriptional regulator
2ZCX-A	12.9	3.5	Tetr-family transcriptional regulator

References

[1] An RNA aptamer that induces transcription.
Hunsicker, A., Steber, M., Mayer, G., Meitert, J., Klotzsche, M., Blind, M., Hillen, W., Berens, C., & Suess, B.
Chemistry & biology, 16(2), 173–180. (2009)
[2] Mechanistic basis for RNA aptamer-based induction of TetR.
Steber, M., Arora, A., Hofmann, J., Brutschy, B., & Suess, B.
Chembiochem : a European journal of chemical biology, 12(17), 2608–2614. (2011)
[3] Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals.
Atanasov, J., Groher, F., Weigand, J. E., & Suess, B.
Nucleic acids research, 45(22), e181. (2017)
[4] Robust gene expression control in human cells with a novel universal TetR aptamer splicing module.
Mol, A. A., Groher, F., Schreiber, B., Rühmkorff, C., & Suess, B.
Nucleic acids research, 47(20), e132. (2019)
[5] The complex formed between a synthetic RNA aptamer and the transcription repressor TetR is a structural and functional twin of the operator DNA-TetR regulator complex.
Grau, F. C., Jaeger, J., Groher, F., Suess, B., & Muller, Y. A.
Nucleic acids research, 48(6), 3366–3378. (2020)
[6] Inducible nuclear import by TetR aptamer-controlled 3' splice site selection.
Mol, A. A., Vogel, M., & Suess, B.
RNA (New York, N.Y.), 27(2), 234–241. (2021)