Abstract
Although group II intron ribozymes are intensively studied the question how structural dynamics affects splicing catalysis has remained elusive. We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine. Our study determined a functional sugar pucker equilibrium between the transesterification active C2′-endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3′-endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step. Our detailed NMR spectroscopic investigation identified magnesium (II) ions and the branching reaction as regulators of the equilibrium populations. The tuneable secondary structure/sugar pucker equilibrium supports a conformational selection mechanism to up- and downregulate catalytically active and inactive states of the branch site adenosine to orchestrate the multi-step splicing process. The conformational dynamics of group II intron domain 6 is also proposed to be a key aspect for the directionality selection in reversible splicing.
Highlights
Group identified magnesium (II) introns are catalytically active RNAs with selfsplicing activity [1,2]
We report for the first time that the group II intron domain 6 exists in a secondary structure equilibrium between a single- and a two-nucleotide bulge conformation, which is directly linked to a switch between sugar puckers of the branch site adenosine
Our study determined a functional sugar pucker equilibrium between the transesterification active C2 -endo conformation of the branch site adenosine in the 1nt bulge and an inactive C3 -endo state in the 2nt bulge fold, allowing the group II intron to switch its activity from the branching to the exon ligation step
Summary
RNA is formed, which resembles lariats formed during splicing of nuclear immature mRNA This similarity in the reaction pathway suggests that the spliceosome might originate from the older group II introns further supported by an akin structure of domain 5 (D5) in group II introns and the U6/U2 snRNA. In the recent crystal structure of Costa and co-workers, a basal 3-base pair (bp) stem with the branch point A in a 2nt bulge was consistent with the electron density maps (Figure 1C, fold 2B). Such a 2nt bulge arrangement in D6 was earlier reported in an X-ray crystal structure of a trans-active 70 nucleotide D56 construct of the ai5␥ group II intron [20]. The 2nt bulge is speculated to be the favoured conformational ar-
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