title: Distinct kinetic determinants for the stepwise CCA addition to tRNA.
abstract: The universally conserved CCA sequence is present at the 3' terminal 74-76 positions of all active tRNA molecules as a functional tag to participate in ribosome protein synthesis. The CCA enzyme catalyzes CCA synthesis in three sequential steps of nucleotide addition at rapid and identical rates. However, the kinetic determinant of each addition is unknown, thus limiting the insights into the kinetic basis of CCA addition. Using our recently developed single turnover kinetics of Escherichia coli CCA enzyme as a model, we show here that the identical rate of the stepwise CCA addition is determined by distinct kinetic parameters. Specifically, the kinetics of C74 and C75 addition is controlled by the chemistry of nucleotidyl transfer, whereas the kinetics of A76 addition is controlled by a prechemistry conformational transition of the active site. In multiple turnover condition, all three steps are controlled by slow product release, indicating enzyme processivity from one addition to the next. However, the processivity decreases as the enzyme progresses to complete the CCA synthesis. Together, these results suggest the existence of a network of diverse kinetic parameters that determines the overall rate of CCA addition for tRNA maturation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Sangbumn
authors.initials: S
authors.lastname: Kim
authors.forename: Cuiping
authors.initials: C
authors.lastname: Liu
authors.forename: Konstantine
authors.initials: K
authors.lastname: Halkidis
authors.forename: Howard B
authors.initials: HB
authors.lastname: Gamper
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry*
chemical_list: D012343:RNA, Transfer
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