3 research outputs found
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
Designed DNA Crystal Habit Modifiers
DNA
is now one of the most widely used molecules for programmed
self-assembly of discrete nanostructures. One of the long-standing
goals of the DNA nanotechnology field has been the assembly of periodic,
macroscopic 3D DNA crystals for controlled positioning of guest molecules
to be used in a variety of applications. With continuing successes
in assembling DNA crystals, there is an enhanced need to tailor macroscopic
crystal propertiesî—¸including morphologyî—¸to enable their
integration into more complex systems. Here we describe the ability
to alter and control crystal habits of a 3D DNA crystal formed by
self-assembly of a DNA 13-mer. The introduction of “poison”
oligonucleotides that specifically disrupt critical noncanonical base-pairing
interactions in the crystal lattice leads to predictably modified
crystal habits. We demonstrate that the poison oligomers can act as
habit modifiers both during the initial crystallization and during
growth of shell layers on a crystal macroseed
Three-Dimensional DNA Crystals with pH-Responsive Noncanonical Junctions
Three-dimensional (3D) DNA crystals have been envisioned
as programmable
biomaterial scaffolds for creating ordered arrays of biological and
nonbiological molecules. Despite having excellent programmable properties,
the linearity of the Watson–Crick B-form duplex imposes limitations
on 3D crystal design. Predictable noncanonical base pairing motifs
have the potential to serve as junctions to connect linear DNA segments
into complex 3D lattices. Here, we designed crystals based on a template
structure with parallel-stranded noncanonical base pairs. Depending
on pH, the structures we determined contained all but one or two of
the designed secondary structure interactions. Surprisingly, a conformational
change of the designed Watson–Crick duplex region resulted
in crystal packing differences between the predicted and observed
structures. However, the designed noncanonical motif was virtually
identical to the template when crystals were grown at pH 5.5, highlighting
the motif’s predictability. At pH 7.0 we observed a structurally
similar variation on this motif that contains a previously unobserved
C–G•G–C quadruple base pair. We demonstrate that
these two variants can interconvert <i>in crystallo</i> in
response to pH perturbations. This study spotlights several important
considerations in DNA crystal design, describes the first 3D DNA lattice
composed of A-DNA helical sheets, and reveals a noncanonical DNA motif
that has adaptive features that may be useful for designing dynamic
crystals or biomaterial assemblies