Energetic contribution of non-essential 5' sequence to catalysis in a hepatitis delta virus ribozyme.
Hepatitis delta virus (HDV) ribozymes employ multiple catalytic strategies to achieve overall rate enhancement of RNA cleavage. These strategies include general acid-base catalysis by a cytosine side chain and involvement of divalent metal ions. Here we used a trans-acting form of the antigenomic ribozyme to examine the contribution of the 5' sequence in the substrate to HDV ribozyme catalysis. The cleavage rate constants increased for substrates with 5' sequence alterations that reduced ground-state binding to the ribozyme. Quantitatively, a plot of activation free energy of chemical conversion versus Gibb's free energy of substrate binding revealed a linear relationship with a slope of -1. This relationship is consistent with a model in which components of the substrate immediately 5' to the cleavage site in the HDV ribozyme-substrate complex destabilize ground-state binding. The intrinsic binding energy derived from the ground-state destabilization could contribute up to 2 kcal/mol toward the total 8.5 kcal/mol reduction in activation free energy for RNA cleavage catalyzed by the HDV ribozyme.
Shih Ih
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Been
M D
MD
D020121:5' Untranslated Regions / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002122:Calcium Chloride / Q000378:metabolism; D002384:Catalysis; D003698:Hepatitis Delta Virus / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D020121:5' Untranslated Regions; D016337:RNA, Catalytic; D012367:RNA, Viral; D015636:Magnesium Chloride; D002122:Calcium Chloride