Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release
Open accessAbstract
In two-metal catalysis, metal ion A has been proposed to activate the nucleophile and metal ion B to stabilize the transition state. We recently reported crystal structures of RNase H-RNA/DNA substrate complexes obtained at 1.5-2.2 Angstroms. We have now determined and report here structures of reaction intermediate and product complexes of RNase H at 1.65-1.85 Angstroms. The movement of the two metal ions suggests how they may facilitate RNA hydrolysis during the catalytic process. Firstly, metal ion A may assist nucleophilic attack by moving towards metal ion B and bringing the nucleophile close to the scissile phosphate. Secondly, metal ion B transforms from an irregular coordination in the substrate complex to a more regular geometry in the product complex. The exquisite sensitivity of Mg(2+) to the coordination environment likely destabilizes the enzyme-substrate complex and reduces the energy barrier to form product. Lastly, product release probably requires dissociation of metal ion A, which is inhibited by either high concentrations of divalent cations or mutation of an assisting protein residue.
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Non-directed or internal cleavages occur within larger RNA/DNA duplex segments and may depend in part on the RNA sequence.
“Non-directed or internal cleavages take place within larger segments of RNA/DNA duplex and might depend, in part, on the sequence of the RNA [28,52,55].”
From Retroviral RNase H: structure, mechanism, and inhibition (2021) · cites this paper as [55]
The Introduction (page unavailable) describes RNase H as an endonuclease that cleaves RNA in RNA/DNA hybrids, consistent with internal cleavage. But it explicitly says cleavage occurs in a “sequence nonspecific manner,” contrary to the claim that it might depend in part on RNA sequence. The crystallographic-design section discusses duplex lengths and nick positions, not sequence-dependent cleavage. (LLM, conf=0.82)
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Experimental — accuracy not yet measuredThe side chains of the D,D-35-E residues coordinate a pair of Mg2+ ions that facilitate 3'-processing and strand transfer.
“The side chains of these residues serve to coordinate the essential pair of Mg2+ ions, which facilitate 3’-processing and strand transfer reactions.”
From Structure and function of retroviral integrase (2022) · cites this paper as [48]
The Introduction (page unavailable) describes active-site carboxylates coordinating two magnesium ions in RNase H catalysis, and the concluding remarks suggest that a two-metal mechanism may extend to recombinases. The paper does not identify a D,D-35-E motif or establish that its residues coordinate the ions for 3′-processing and strand transfer; those reaction-specific parts of the attributed claim are unsupported. (LLM, conf=0.86)
Experimental — accuracy not yet measuredIn the pre-reaction state, both active site metal ions interact with the scissile phosphodiester bond; metal A has near-perfect coordination geometry while metal B deviates significantly, potentially destabilizing the scissile phosphodiester.
“In this pre-reaction state, both active site metal ions (designated A and B, Fig. 2B) interact with the scissile phosphodiester bond. While Mn2+ ion A, which interacts with the water nucleophile, exists in a near perfect coordination geometry, the coordination of metal B deviates significantly from the ideal, potentially promoting destabilization of the scissile phosphodiester28,47,48.”
From Structure and function of retroviral integrase (2022) · cites this paper as [48]
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Experimental — accuracy not yet measuredRNase HI and RNase HIII are functionally similar, exhibiting the same metal preferences and contributing to Okazaki fragment maturation and R-loop resolution.
“biochemical studies show that RNase HI and RNase HIII are functionally similar, exhibiting the same metal preferences and contributing to Okazaki fragment maturation during lagging strand replication and resolution of R-loops during transcription”
From Ribonucleotide errors, RNase HII, and ribonucleotide excision repair (2026) · cites this paper as Nowotny and Yang 2006
The Introduction identifies RNase H as RNase HI and says its main function is removing RNA primers from Okazaki fragments during DNA replication. It discusses metal preferences for RNase H, but does not compare RNase HI with RNase HIII or establish that they share metal preferences. The passages also do not address RNase HIII’s role in Okazaki fragment maturation or either enzyme’s role in R-loop resolution. (LLM, conf=0.86)
Experimental — accuracy not yet measuredDuring DNA polymerization, the 3'-end of the elongating DNA in the polymerase active site orients the RNA strand in the ribonuclease H active site, allowing cleavage 15-20 nucleotides from the 3'-DNA terminus.
“During DNA polymerization from the RNA template, the 3'-end of the elongating DNA is positioned in the polymerase active site and orients the RNA strand in the RNH active site, which allows RNH to cleave the RNA at a position 15-20 nucleotides away from the 3’-DNA terminus [27,28,55,56].”
From Retroviral RNase H: structure, mechanism, and inhibition (2021) · cites this paper as [55]
The Introduction (page unavailable) says RNase H cleaves RNA in RNA/DNA hybrids and notes that retroviral reverse transcriptase has an RNase H domain. The crystallographic-design section (page unavailable) examines how RNA/DNA hybrids occupy the RNase H active site, but neither section describes the elongating DNA end orienting RNA during polymerization or cleavage 15–20 nucleotides from that end. Searches for the cleavage distance and polymerase/RNase H positioning found no passage addressing that specific mechanism. (LLM, conf=0.75)
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References 37 · Cited by 250
- Authors
- Nowotny, M., & Yang, W.
- Affiliations
- National Institutes of Health (US)
- Corresponding author
- Yang, Wei
- Venue
- The EMBO Journal
- Published
- 2006-04-06
- Volume
- 25
- Issue
- 9
- Pages
- 1924-1933
- ISSN
- 0261-4189 (ISSN-L)
- 1460-2075
- PMC
- PMC1456928
- OpenAlex
- W2137633625
- Cited by
- 250
- References
- 37
- Citations by year
- 2026: 8 · 2025: 5 · 2024: 8 · 2023: 8 · 2022: 13 · 2021: 21 · 2020: 3 · 2019: 6 · 2018: 10 · 2017: 11 · 2016: 11 · 2015: 14 · 2014: 15 · 2013: 18 · 2012: 18
- FWCI
- 11.13
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- The EMBO Journal (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Institute of Diabetes and Digestive and Kidney Diseases
- Topics
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Physical Sciences › Chemistry › Organic Chemistry › Chemical Reaction Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- RNase H, metal coordination, RNA hydrolysis, enzyme-substrate complex, reaction intermediates, product launch, magnesium ions, nucleophilic attack, transition state stabilization, divalent cations, crystal structure
- MeSH terms
- Bacillus, Catalysis, Cations, Divalent, Crystallography, Magnesium, Manganese, Protein Conformation, Ribonuclease H
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