FC RNA aptamer

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Timeline

1997

Isolated RNA molecules that selectively bind to yeast Pol II[1]

2006

Report the crystal structure of FC RNA (aptamer) bound to Pol II[2]

2018

RNA sequences with high affinity to RNA Pol by performing genomic SELEX in Escherichia coli genome[3]

2020

Identified a variety of Pol II-binding aptamers, the nascent RNA binds Pol II to silence its own expression[4]

Description

 In 1997, Thomas, M. et al. isolated RNA aptamers targeted yeast RNA polymerase II (Pol II), one selected RNA ligand was a specific inhibitor of Saccharomyces cerevisiae Pol II. In 2006, Kettenberger, H. et al. determined the crystal structure of FC RNA aptamer bound to Pol II. The noncoding RNA B2 and the RNA aptamer FC bind RNA polymerase (Pol) II and inhibit messenger RNA transcription initiation[1,2].

SELEX

RNA aptamers that bind to S. cerevisiae Pol II were selected by the Selex strategy. The starting RNA library consisted of a randomized region of 40 nucleotides flanked by defined sequences at the 5′- and 3′- ends. An excess of RNAs (40 pmol) was incubated with a highly purified preparation of Pol II (5 pmol). The RNA·protein complexes were retained on a nitrocellulose filter by filtration, whereas free RNAs passed through the membrane. After extensive washing of the membrane, the protein·RNA complexes were dissociated by phenol extraction, and the RNA was amplified. This selection/amplification procedure was repeated nine times and resulted in the new RNA pool[1].
Detailed information are accessible on SELEX page.



Structure

2D representation

Here we used ribodraw to complete the figure, through the 3D structure information[2].

5'-ACAGCACUGAUUGCGGUCGAGGUAGCUUGAUGG-3'

 drawing

3D visualisation

FC RNA was cocrystallized with the complete 12-subunit Pol II, and the X-ray structure was determined at 3.8-Å resolution. The molecular architecture reveals that the FC RNA molecule is strategically positioned within the polymerase's active center cleft, which is situated superior to the bridge helix. The RNA establishes contact with both the clamp and fork domains, which are located on opposing sides of the cleft, aligning with the cross-linking evidence that FC RNA interacts with the two major subunits of Pol II. The PDB ID of this structure is 2B63[2].
Additional available structures that have been solved and detailed information are accessible on Structures page.

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Binding pocket

Left: Surface representation of the binding pocket of the aptamer generated from PDB ID: 2B63 at 3.8 Å resolution. RNA polymerase (Pol) II (shown in vacuumm electrostatics), blue is positive charge, red is negative charge. Right: The hydrogen bonds of binding sites of the aptamer bound with RNA polymerase (Pol) II. The hydrogen bond interaction refers to pymol prediction and articles.

drawing drawing


Ligand information

SELEX ligand

Affinity determined by in vitro binding assays. For filter-binding competition assays, 5′ 32P-labeled RNA was incubated with 100 nM protein under the same conditions, adding increasing amounts (1–1,000 nM) of unlabeled RNA competitor and then incubating at 30 °C for 30 min[2].

Name Sequence Ligand Affinity
FC RNA aptamer 5'-ACAGCACUGAUUGCGGUCGAGGUAGCUUGAUGG-3' Pol II 33±2 nM
Mutant 1 5'-ACAGGACUGAUUGCGGUCGAGGUAGCUUGAUGG-3' Pol II 43±1 nM
Mutant 2 5'-ACAGCACUAAUUGCGGUCGAGGUAGCUUGAUGG-3' Pol II 45±2 nM
Mutant 3 5'-ACAGCACUGGUUGCGGUCGAGGUAGCUUGAUGG-3' Pol II 62±7 nM
Mutant 4 5'-ACAGCACUGAUUCCGGUCGAGGUAGCUUGAUGG-3' Pol II 35±4 nM
Mutant 5 5'-ACAGGACUGAUUCCGGUCGAGGUAGCUUGAUGG-3' Pol II 45±6 nM
Mutant 6 5'-ACAGCCACUGAUUGGCGGUCGAGGUAGCUUGAUGG-3' Pol II 37±3 nM
Mutant 7 5'-ACAGCGCACUGAUUGCGCGGUCGAGGUAGCUUGAUGG-3' Pol II 53±4 nM
Mutant 8 5'-ACAGCACUGAUUGCGGUCGAUGGUAGCGUUGAUGG-3' Pol II 25±3 nM
Mutant 9 5'-AGCUUGAUGAGCACUGAUUGCCGUCGAGGU-3' Pol II No binding

Structure ligand

DNA-directed RNA polymerases (also known as DNA-dependent RNA polymerases) are responsible for the polymerisation of ribonucleotides into a sequence complementary to the template DNA. In eukaryotes, there are three different forms of DNA-directed RNA polymerases transcribing different sets of genes. Eukaryotic nuclei contain three distinct types of RNA polymerases that differ in the RNA they synthesise: RNA polymerase I: located in the nucleoli, synthesises precursors of most ribosomal RNAs. RNA polymerase II: occurs in the nucleoplasm, synthesises mRNA precursors. RNA polymerase III: also occurs in the nucleoplasm, synthesises the precursors of 5S ribosomal RNA, the tRNAs, and a variety of other small nuclear and cytosolic RNAs.
Uniprot ID Pfam MW Amino acids sequences PDB ID GenBank
P04050 IPR045867 191.64 kDa MVGQQYSSAPLRTVKEVQFGLFSPEEVRAISVAKIRFPETMDETQTRAKIGGLNDPRLGSIDRNLKCQTCQEGMNECPGHFGHIDLAKPVFHVGFIAKIKKVCECVCMHCGKLLLDEHNELMRQALAIKDSKKRFAAIWTLCKTKMVCETDVPSEDDPTQLVSRGGCGNTQPTIRKDGLKLVGSWKKDRATGDADEPELRVLSTEEILNIFKHISVKDFTSLGFNEVFSRPEWMILTCLPVPPPPVRPSISFNESQRGEDDLTFKLADILKANISLETLEHNGAPHHAIEEAESLLQFHVATYMDNDIAGQPQALQKSGRPVKSIRARLKGKEGRIRGNLMGKRVDFSARTVISGDPNLELDQVGVPKSIAKTLTYPEVVTPYNIDRLTQLVRNGPNEHPGAKYVIRDSGDRIDLRYSKRAGDIQLQYGWKVERHIMDNDPVLFNRQPSLHKMSMMAHRVKVIPYSTFRLNLSVTSPYNADFDGDEMNLHVPQSEETRAELSQLCAVPLQIVSPQSNKPCMGIVQDTLCGIRKLTLRDTFIELDQVLNMLYWVPDWDGVIPTPAIIKPKPLWSGKQILSVAIPNGIHLQRFDEGTTLLSPKDNGMLIIDGQIIFGVVEKKTVGSSNGGLIHVVTREKGPQVCAKLFGNIQKVVNFWLLHNGFSTGIGDTIADGPTMREITETIAEAKKKVLDVTKEAQANLLTAKHGMTLRESFEDNVVRFLNEARDKAGRLAEVNLKDLNNVKQMVMAGSKGSFINIAQMSACVGQQSVEGKRIAFGFVDRTLPHFSKDDYSPESKGFVENSYLRGLTPQEFFFHAMGGREGLIDTAVKTAETGYIQRRLVKALEDIMVHYDNTTRNSLGNVIQFIYGEDGMDAAHIEKQSLDTIGGSDAAFEKRYRVDLLNTDHTLDPSLLESGSEILGDLKLQVLLDEEYKQLVKDRKFLREVFVDGEANWPLPVNIRRIIQNAQQTFHIDHTKPSDLTIKDIVLGVKDLQENLLVLRGKNEIIQNAQRDAVTLFCCLLRSRLATRRVLQEYRLTKQAFDWVLSNIEAQFLRSVVHPGEMVGVLAAQSIGEPATQMTLNTFHFAGVASKKVTSGVPRLKEILNVAKNMKTPSLTVYLEPGHAADQEQAKLIRSAIEHTTLKSVTIASEIYYDPDPRSTVIPEDEEIIQLHFSLLDEEAEQSFDQQSPWLLRLELDRAAMNDKDLTMGQVGERIKQTFKNDLFVIWSEDNDEKLIIRCRVVRPKSLDAETEAEEDHMLKKIENTMLENITLRGVENIERVVMMKYDRKVPSPTGEYVKEPEWVLETDGVNLSEVMTVPGIDPTRIYTNSFIDIMEVLGIEAGRAALYKEVYNVIASDGSYVNYRHMALLVDVMTTQGGLTSVTRHGFNRSNTGALMRCSFEETVEILFEAGASAELDDCRGVSENVILGQMAPIGTGAFDVMIDEESLVKYMPEQKITEIEDGQDGGVTPYSNESGLVNADLDVKDELMFSPLVDSGSNDAMAGGFTAYGGADYGEATSPFGAYGEAPTSPGFGVSSPGFSPTSPTYSPTSPAYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPAYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSPNYSPTSPSYSPTSPGYSPGSPAYSPKQDEQKHNENENSR (RNA polymerase II largest subunit) 1I50 559292
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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.
PDB Z-score RMSD Description
2JA7-A 47.0 0.8 DNA-directed rna polymerase II largest subunit
2JA7-M 46 0.8 DNA-directed rna polymerase II largest subunit
5FMF-A 37.9 1.0 Dna repair helicase rad25, ssl2
3HOY-A 45.9 0.9 DNA-directed rna polymerase II subunit rpb1
3K1F-A 45.9 1.2 DNA-directed rna polymerase II subunit rpb1
3S1M-A 45.5 1.2 DNA-directed rna polymerase II subunit rpb1
1WCM-A 40.0 0.6 DNA-directed rna polymerase II largest subunit
3J0K-A 45.1 1.4 DNA-directed rna polymerase II largest subunit
7O4J-A 45.1 1.1 General transcription and DNA repair factor II
3HOV-A 44.7 0.8 DNA-directed rna polymerase II subunit rpb1


References

[1] Selective targeting and inhibition of yeast RNA polymerase II by RNA aptamers.
Thomas, M., Chédin, S., Carles, C., Riva, M., Famulok, M., & Sentenac, A.
The Journal of biological chemistry, 272(44), 27980–27986. (1997)
[2] Structure of an RNA polymerase II-RNA inhibitor complex elucidates transcription regulation by noncoding RNAs.
Kettenberger, H., Eisenführ, A., Brueckner, F., Theis, M., Famulok, M., & Cramer, P.
Nature structural & molecular biology, 13(1), 44–48. (2006)
[3] Nascent RNA signaling to yeast RNA Pol II during transcription elongation.
Klopf, E., Moes, M., Amman, F., Zimmermann, B., von Pelchrzim, F., Wagner, C., & Schroeder, R.
PloS one, 13(3), e0194438. (2018)
[4] RNA polymerase II-binding aptamers in human ACRO1 satellites disrupt transcription in cis.
Boots, J. L., von Pelchrzim, F., Weiss, A., Zimmermann, B., Friesacher, T., Radtke, M., Żywicki, M., Chen, D., Matylla-Kulińska, K., Zagrovic, B., & Schroeder, R.
Transcription, 11(5), 217–229. (2020)