Functional identification of ligands for a catalytic metal ion in group I introns.
Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A. We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M A, verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.
Department of Biochemistry and Chemistry, Stanford University, Stanford, California 94305, USA.
Marcello
M
Forconi
Jihee
J
Lee
Jungjoon K
JK
Lee
Joseph A
JA
Piccirilli
Daniel
D
Herschlag
D000818:Animals; D002384:Catalysis; D018360:Crystallography, X-Ray; D007438:Introns / Q000235:genetics*; D007477:Ions / Q000737:chemistry; D008024:Ligands; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013768:Tetrahymena / Q000201:enzymology
D007477:Ions; D008024:Ligands; D008670:Metals; D016337:RNA, Catalytic