tetano
Editor, Senior Moderator
Nucleic Acids Res
. 2024 Oct 30:gkae939.
doi: 10.1093/nar/gkae939. Online ahead of print. Alternative substrate kinetics of SARS-CoV-2 Nsp15 endonuclease reveals a specificity landscape dominated by RNA structure
Nidhi Kalia[SUP] 1 [/SUP], Kimberly C Snell[SUP] 1 [/SUP], Michael E Harris[SUP] 1 [/SUP]
Affiliations
Coronavirus endoribonuclease Nsp15 contributes to the evasion of host innate immunity by suppressing levels of viral dsRNA. Nsp15 cleaves both ssRNA and dsRNA in vitro with a strong preference for unpaired or bulged U residues, and its activity is stimulated by divalent ions. Here, we systematically quantified effects of RNA sequence and structure context that define its specificity. The results show that sequence preference for U↓A/G, observed previously, contributes only ca. 2-fold to kcat/Km. In contrast, dsRNA structure flanking a bulged U residue increases kcat/Km by an order of magnitude compared to ssRNA while base pairing in dsRNA essentially blocks cleavage. Despite enormous differences in multiple turnover kinetics, the effect of RNA structure on the cleavage step is minimal. Surprisingly, although divalent ion activation of Nsp15 is widely considered to be important for its biological function, the effect on kcat/Km is only ∼2-fold and independent of RNA structure. These results reveal a specificity landscape dominated by RNA structure and provide a quantitative framework for identifying interactions that underlie specificity, determining mechanisms of inhibition and resistance and defining targets important for coronavirus biology.
. 2024 Oct 30:gkae939.
doi: 10.1093/nar/gkae939. Online ahead of print. Alternative substrate kinetics of SARS-CoV-2 Nsp15 endonuclease reveals a specificity landscape dominated by RNA structure
Nidhi Kalia[SUP] 1 [/SUP], Kimberly C Snell[SUP] 1 [/SUP], Michael E Harris[SUP] 1 [/SUP]
Affiliations
- PMID: 39475186
- DOI: 10.1093/nar/gkae939
Coronavirus endoribonuclease Nsp15 contributes to the evasion of host innate immunity by suppressing levels of viral dsRNA. Nsp15 cleaves both ssRNA and dsRNA in vitro with a strong preference for unpaired or bulged U residues, and its activity is stimulated by divalent ions. Here, we systematically quantified effects of RNA sequence and structure context that define its specificity. The results show that sequence preference for U↓A/G, observed previously, contributes only ca. 2-fold to kcat/Km. In contrast, dsRNA structure flanking a bulged U residue increases kcat/Km by an order of magnitude compared to ssRNA while base pairing in dsRNA essentially blocks cleavage. Despite enormous differences in multiple turnover kinetics, the effect of RNA structure on the cleavage step is minimal. Surprisingly, although divalent ion activation of Nsp15 is widely considered to be important for its biological function, the effect on kcat/Km is only ∼2-fold and independent of RNA structure. These results reveal a specificity landscape dominated by RNA structure and provide a quantitative framework for identifying interactions that underlie specificity, determining mechanisms of inhibition and resistance and defining targets important for coronavirus biology.