Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes Journal Article
| Authors: | Sheng, G.; Gogakos, T.; Wang, J.; Zhao, H.; Serganov, A.; Juranek, S.; Tuschl, T.; Patel, D. J.; Wang, Y. |
| Article Title: | Structure/cleavage-based insights into helical perturbations at bulge sites within T. thermophilus Argonaute silencing complexes |
| Abstract: | We have undertaken a systematic structural study of Thermus thermophilus Argonaute (TtAgo) ternary complexes containing single-base bulges positioned either within the seed segment of the guide or target strands and at the cleavage site. Our studies establish that single-base bulges 7T8, 5A6 and 4A5 on the guide strand are stacked-into the duplex, with conformational changes localized to the bulge site, thereby having minimal impact on the cleavage site. By contrast, single-base bulges 6'U7' and 6'A7' on the target strand are looped-out of the duplex, with the resulting conformational transitions shifting the cleavable phosphate by one step. We observe a stable alignment for the looped-out 6'N7' bulge base, which stacks on the unpaired first base of the guide strand, with the looped-out alignment facilitated by weakened Watson-Crick and reversed non-canonical flanking pairs. These structural studies are complemented by cleavage assays that independently monitor the impact of bulges on TtAgo-mediated cleavage reaction. © The Author(s) 2017. |
| Keywords: | genetics; mutation; protein domain; protein motif; metabolism; gene expression; protein binding; enzymology; bacterial protein; chemistry; bacterial proteins; kinetics; nucleotide sequence; recombinant proteins; substrate specificity; recombinant protein; base sequence; binding site; base pairing; models, molecular; thermodynamics; crystallography, x-ray; binding sites; conformation; nucleic acid conformation; x ray crystallography; enzyme specificity; bacterial dna; argonaute protein; dna, bacterial; protein interaction domains and motifs; molecular model; amino acid motifs; dna cleavage; oligodeoxyribonucleotides; thermus thermophilus; alpha helix; beta sheet; argonaute proteins; oligoribonucleotide; oligoribonucleotides; oligodeoxyribonucleotide; protein conformation, alpha-helical; protein conformation, beta-strand |
| Journal Title: | Nucleic Acids Research |
| Volume: | 45 |
| Issue: | 15 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2017-09-06 |
| Start Page: | 9149 |
| End Page: | 9163 |
| Language: | English |
| DOI: | 10.1093/nar/gkx547 |
| PUBMED: | 28911094 |
| PROVIDER: | scopus |
| PMCID: | PMC5587774 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 2 November 2017 -- Source: Scopus |
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