Thermal activation of a group II intron ribozyme reveals multiple conformational states.
Conformational changes often accompany biological catalysis. Group II introns promote a variety of reactions in vitro that show an unusually sharp temperature dependence. This suggests that the chemical steps are accompanied by the conversion of a folded-but-inactive form to a differently folded active state. We report here the kinetic analysis of 5'-splice-junction hydrolysis (SJH) by E1:12345, a transcript containing the 5'-exon plus the first five of six intron secondary structure domains. The pseudo-first-order SJH reaction shows (1) activation by added KCl to 1.5 M; (2) cooperative activation by added MgCl2, nHill = 4.1-4.3, and [MgCl2]vmax/2 approximately 0.040 M; and (3) a rather high apparent activation energy, Ea approximately 50 kcal mol-l. In contrast, the 5'-terminal phosphodiester bond of a domain 5 transcript (GGD5) was hydrolyzed with Ea approximately 30 kcal mol-1 under SJH conditions; the 5'-GG leader dinucleotide presumably lacks secondary structure constraints. The effect of adding the chaotropic salt tetraethylammonium chloride (TEA) was also investigated. TEA reduced the melting temperatures of GGD5 and E1:12345. TEA also shifted the profile of rate versus temperature for SJH by E1:12345 toward lower temperatures without affecting the maximum rate. TEA had little effect on the rate of hydrolysis of the 5'-phosphodiester bond of GGD5. The high apparent activation enthalpy and entropy for SJH along with the effect of TEA on these parameters imply that conversion of an inactive form of E1:12345 to an active conformation accompanies enhanced occupation of the transition state as the temperature is raised to that for maximum SJH. Analytical modeling indicates that either a two-state model (open and disordered, with open being active) or a three-state model (compact, open, and disordered) could account for the temperature dependence of kSJH. However, the three-state model is clearly preferable, since it does not require that the activation parameters for phosphodiester bond hydrolysis exhibit exceptional values or that the rates for the chemical steps of SJH respond directly to TEA addition.
Franzen
J S
JS
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
Zhang
M
M
Chay
T R
TR
Peebles
C L
CL
D002151:Calorimetry; D006358:Hot Temperature; D007438:Introns; D007700:Kinetics; D008961:Models, Structural; D008962:Models, Theoretical; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011189:Potassium Chloride / Q000494:pharmacology; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D013816:Thermodynamics; D013997:Time Factors
D016337:RNA, Catalytic; D013757:Tetraethylammonium Compounds; D019789:Tetraethylammonium; D011189:Potassium Chloride