pH dependence of self-splicing by the group IA2 intron in a pre-mRNA derived from the nrdB gene of bacteriophage T4.

The nrdB gene of bacteriophage T4 contains a group IA2 intron. We have investigated the kinetics of self-splicing by a shortened variant of nrdB pre-mRNA in the presence of the co-substrates guanosine and 2'-amino-2'-deoxyguanosine. The pH dependence of the first transesterification step displayed parallel linear correlations for the two different co-substrates up to pH 7, above which the reaction with guanosine levels off to become pH independent. The plot for the 30-fold slower reaction with 2'-aminoguanosine is linear up to pH 8-8.5 and then levels off. The linear correlations with slopes close to unity suggest that a deprotonation event accelerates the transesterification reaction and that a change in rate limiting step occurs at a first order rate constant of approximately 1 min-1(i.e. for our system k cat/ K m approximately 10(5) M-1 min-1). The pH dependence of observed rate constants in different divalent metal ion mixtures, where the 2'-aminoguanosine-dependent reaction is enhanced 6- and 35-fold compared with that in magnesium, strongly supports this conclusion. This is, to our knowledge, the first report on an intact self-splicing group I intron where use of different co-substrates and divalent metal ions shows that a deprotonation enhances the rate and verifies that the transitions occurring during splicing of group I introns are all part of a common reaction sequence.

Metal ion interaction with cosubstrate in self-splicing of group I introns.

The catalytic mechanism for self-splicing of the group I intron in the pre-mRNA from the nrdB gene in bacteriophage T4 has been investigated using 2'-amino- 2'-deoxyguanosine or guanosine as cosubstrates in the presence of Mg2+, Mn2+and Zn2+. The results show that a divalent metal ion interacts with the cosubstrate and thereby influences the efficiency of catalysis in the first step of splicing. This suggests the existence of a metal ion that catalyses the nucleophilic attack of the cosubstrate. Of particular significance is that the transesterification reactions of the first step of splicing with 2'-amino-2'-deoxyguanosine as cosubstrate are more efficient in mixtures containing either Mn2+or Zn2+together with Mg2+than with only magnesium ions present. The experiments in metal ion mixtures show that two (or more) metal ions are crucial for the self-splicing of group I introns and suggest the possibility that more than one of these have a direct catalytic role. A working model for a two-metal-ion mechanism in the transesterification steps is suggested.

2'-Amino-2'-deoxyguanosine is a cofactor for self-splicing in group I catalytic RNA.

In investigations on self-splicing in the group I intron of the pre-mRNA from the nrdB gene of bacteriophage T4 it was found that 2'-amino-2'-deoxyguanosine can replace guanosine as cofactor. This is the first guanosine-analogue with a modification in the 2'-position and substantial activity in a group I self-splicing reaction. The results suggest that the 2'-amino and 2'-hydroxy groups of the cosubstrates have some properties in common, which are important for binding as well as for catalysis.

Three-dimensional model and molecular dynamics simulation of the active site of the self-splicing intervening sequence of the bacteriophage T4 nrdB messenger RNA.

The secondary and 3D structure of the active site of the self-splicing T4 nrdB RNA has been modeled on a graphics workstation by use of the suggested 3D arrangement of the active site of the Tetrahymena IVS [Kim, S.H., & Cech, T.R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8788-8792] as a guideline. The initially obtained crude structure was then subjected to molecular mechanics energy minimization and molecular dynamics simulation to relax tensions. In this process the energy decreased considerably and gave a final structure that deviated by 3 A [root mean square (rms)] from the initial structure. The cofactor guanosine (and the competitive inhibitor arginine) was docked to a proposed [Michel, F., Hanna, M., Green, R., Bartel, D.P., & Szostak, J.W. (1989) Nature 342, 391-395] binding site, where it was found to fit rather well. A minor modification of the binding mode easily brought the O3' end of the guanosine within 2 A of the phosphodiester bond where the primary cleavage occurs.