Group I intron self-splicing with adenosine: evidence for a single nucleoside-binding site

Area of Science:

• Molecular Biology
• RNA Splicing
• Biochemistry

Background:

• Self-splicing of Tetrahymena ribosomal RNA precursor involves distinct catalytic steps.
• Guanosine binding is crucial for both 5' splice-site cleavage and exon ligation.
• The precise nature of the guanosine-binding site(s) for these reactions has been a subject of debate.

Purpose of the Study:

• To investigate whether the guanosine-binding sites for 5' splice-site cleavage and exon ligation are identical or distinct.
• To elucidate the role of specific nucleotide binding in the catalytic mechanism of Tetrahymena ribozyme.

Main Methods:

• Site-directed mutagenesis was employed to alter a putative guanosine-binding site within the intron.
• Assays were performed to assess the substrate preference for 5' splice-site cleavage.
• The effect of mutations on exon ligation efficiency was evaluated.
• Modifications at the 3' splice site were introduced to examine rescue effects.

Main Results:

• A double mutation within the intron's guanosine-binding site shifted substrate preference towards adenosine or adenosine triphosphate for 5' splice-site cleavage.
• The same mutation resulted in a complete blockage of the exon ligation step.
• Reversal of the splicing blockage was achieved by altering the 3' splice site from guanine to adenine.
• These findings indicate a single molecular determinant governs nucleoside binding for both catalytic steps.

Conclusions:

• A single binding site is responsible for specifying nucleoside interaction in both the cleavage and ligation steps of Tetrahymena self-splicing.
• The results suggest the potential for RNA to form an active site specifically recognizing adenosine triphosphate.
• This study provides critical insights into the catalytic mechanism and substrate specificity of ribozymes.