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An in Vitro Peptide Complementation Assay for CYT-18-Dependent Group I Intron Splicing Reveals a New Role for the N‑Terminus
The
mitochondrial tyrosyl tRNA synthetase from <i>Neurospora crassa</i> (CYT-18 protein) is a bifunctional group I intron splicing cofactor.
CYT-18 is capable of splicing multiple group I introns from a wide
variety of sources by stabilizing the catalytically active intron
structures. CYT-18 and mt TyrRSs from related fungal species have
evolved to assist in group I intron splicing in part by the accumulation
of three N-terminal domain insertions. Biochemical and structural
analysis indicate that the N-terminal insertions serve primarily to
create a structure-stabilizing scaffold for critical tertiary interactions
between the two major RNA domains of group I introns. Previous studies
concluded that the primarily α-helical N-terminal insertion,
H0, contributes to protein stability and is necessary for splicing
the <i>N. crassa</i> ND1 intron but is dispensable for splicing
the <i>N. crassa</i> mitochondrial LSU intron. Here, we
show that CYT-18 with a complete H0 deletion retains residual ND1
intron splicing activity and that addition of the missing N-terminus
<i>in trans</i> is capable of restoring a significant portion of its splicing
activity. The development of this peptide complementation assay has
allowed us to explore important characteristics of the CYT-18/group
I intron interaction including the stoichiometry of H0 in intron splicing
and the importance of specific H0 residues. Evaluation of truncated
H0 peptides in this assay and a re-examination of the CYT-18 crystal
structure suggest a previously unknown structural role of the first
five N-terminal residues of CYT-18. These residues interact directly
with another splicing insertion, making H0 a central structural element
responsible for connecting all three N-terminal splicing insertions