Distinct kinetic determinants for the stepwise CCA addition to tRNA.
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.
Kim
Sangbumn
S
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Liu
Cuiping
C
Halkidis
Konstantine
K
Gamper
Howard B
HB
Hou
Ya-Ming
YM
D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry*
D012343:RNA, Transfer
EMBO J. 1987 Aug;6(8):2473-7
3311729
Biochemistry. 1985 Jul 16;24(15):4010-8
3902078
BMC Mol Biol. 2007 Oct 18;8:92
17949481
J Biol Chem. 1990 Sep 25;265(27):16216-20
2204621
Annu Rev Microbiol. 2004;58:303-28
15487940
Biochemistry. 2002 Aug 27;41(34):10571-6
12186540
Cell. 2002 Dec 13;111(6):815-24
12526808
RNA. 1996 Sep;2(9):895-908
8809016
EMBO J. 2008 Jul 23;27(14):1944-52
18583961
RNA. 2000 Jul;6(7):1031-43
10917598
J Biol Chem. 1972 Feb 10;247(3):824-31
4550762
Nucleic Acids Res. 2006 Feb 01;34(2):659-66
16452296
Annu Rev Biochem. 1993;62:685-713
7688945
Biochemistry. 2004 Nov 16;43(45):14317-24
15533035
Science. 2001 Nov 9;294(5545):1334-6
11701927
Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6498-502
8341661
Cell. 2006 Sep 22;126(6):1065-77
16962654
RNA. 2005 Jun;11(6):976-84
15872186
RNA. 2008 Mar;14(3):514-23
18230766
Nature. 2006 Oct 26;443(7114):956-60
17051158
Biochemistry. 2007 May 8;46(18):5463-72
17419590
Biochemistry. 2006 Aug 15;45(32):9675-87
16893169
Nature. 2004 Aug 5;430(7000):700-4
15295603
Biochemistry. 2005 Apr 5;44(13):5177-87
15794655
Biochemistry. 2005 Sep 27;44(38):12849-59
16171400
Nature. 2004 Aug 5;430(7000):640-5
15295590
Biochemistry. 2002 Apr 9;41(14):4521-32
11926813
J Mol Biol. 2008 Jun 6;379(3):579-88
18466919
Nature. 1998 Jan 15;391(6664):231-2
9440683
Curr Opin Microbiol. 2004 Apr;7(2):140-4
15063850
Mol Cell. 2003 Nov;12(5):1165-72
14636575
J Biol Chem. 2006 Nov 24;281(47):35677-85
17005565