Importance of specific adenosine N3-nitrogens for efficient cleavage by a hammerhead ribozyme.
Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N3-nitrogens have been excised and replaced with carbon. The modified complexes were chemically synthesized with the substitution of a single 3-deazzaadenosine (c3A) base analogue for residues A6, A9, A13, A14, or A15.1. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/K(M), for the c3A6, c3A9, and c3A14 complexes were only marginally reduced (< or = 5-fold) relative to the native complex. By comparison, the cleavage efficiencies for the c3A13 and c315.1 complexes were reduced by 9-fold and 55-fold, respectively. these reductions in cleavage efficiency are primarily a result of lower kcat values. Profiles of pH and cleavage rate suggest that the chemical cleavage step is the rate-limiting reaction for these complexes. These results suggest that the N3-nitrogen of the A13 residue and particularly the A15.1 residue in the hammerhead ribozyme/substrate complex are critical for transition state stabilization and efficient cleavage activity. We have additionally compared the locations of these critical functional groups, as well as those identified from other studies, with recent crystallographic analyses. In some cases, the critical functional groups are clustered around proposed metal binding sites and may reflect functional groups critical for binding the metal cofactor. In other cases, clusters of functional groups may form a network of hydrogen bonds necessary for transition state stabilization.
Bevers
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S
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Xiang
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G
McLaughlin
L W
LW
D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009584:Nitrogen / Q000737:chemistry; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014372:Tubercidin / Q000737:chemistry* / Q000378:metabolism*
D008670:Metals; D016337:RNA, Catalytic; C018258:3-deazaadenosine; D014372:Tubercidin; D009584:Nitrogen