37 research outputs found
Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots
A pseudoknot forms in an RNA when nucleotides in a loop pair with a region outside the helices that close the loop. Pseudoknots occur relatively rarely in RNA but are highly overrepresented in functionally critical motifs in large catalytic RNAs, in riboswitches, and in regulatory elements of viruses. Pseudoknots are usually excluded from RNA structure prediction algorithms. When included, these pairings are difficult to model accurately, especially in large RNAs, because allowing this structure dramatically increases the number of possible incorrect folds and because it is difficult to search the fold space for an optimal structure. We have developed a concise secondary structure modeling approach that combines SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) experimental chemical probing information and a simple, but robust, energy model for the entropic cost of single pseudoknot formation. Structures are predicted with iterative refinement, using a dynamic programming algorithm. This melded experimental and thermodynamic energy function predicted the secondary structures and the pseudoknots for a set of 21 challenging RNAs of known structure ranging in size from 34 to 530 nt. On average, 93% of known base pairs were predicted, and all pseudoknots in well-folded RNAs were identified
Understanding Flavin-Dependent Halogenase Reactivity via Substrate Activity Profiling
The
activity of four native FDHs and four engineered FDH variants on 93
low-molecular-weight arenes was used to generate FDH substrate activity
profiles. These profiles provided insights into how substrate class,
functional group substitution, electronic activation, and binding
affect FDH activity and selectivity. The enzymes studied could halogenate
a far greater range of substrates than have been previously recognized,
but significant differences in their substrate specificity and selectivity
were observed. Trends between the electronic activation of each site
on a substrate and halogenation conversion at that site were established,
and these data, combined with docking simulations, suggest that substrate
binding can override electronic activation even on compounds differing
appreciably from native substrates. These findings provide a useful
framework for understanding and exploiting FDH reactivity for organic
synthesis
Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots
A pseudoknot forms in an RNA when nucleotides in a loop pair with a region outside the helices that close the loop. Pseudoknots occur relatively rarely in RNA but are highly overrepresented in functionally critical motifs in large catalytic RNAs, in riboswitches, and in regulatory elements of viruses. Pseudoknots are usually excluded from RNA structure prediction algorithms. When included, these pairings are difficult to model accurately, especially in large RNAs, because allowing this structure dramatically increases the number of possible incorrect folds and because it is difficult to search the fold space for an optimal structure. We have developed a concise secondary structure modeling approach that combines SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) experimental chemical probing information and a simple, but robust, energy model for the entropic cost of single pseudoknot formation. Structures are predicted with iterative refinement, using a dynamic programming algorithm. This melded experimental and thermodynamic energy function predicted the secondary structures and the pseudoknots for a set of 21 challenging RNAs of known structure ranging in size from 34 to 530 nt. On average, 93% of known base pairs were predicted, and all pseudoknots in well-folded RNAs were identified
RNA Tertiary Structure Analysis by 2′-Hydroxyl Molecular Interference
We introduce a melded chemical and computational approach for probing and modeling higher-order intramolecular tertiary interactions in RNA. 2'-Hydroxyl molecular interference (HMX) identifies nucleotides in highly packed regions of an RNA by exploiting the ability of bulky adducts at the 2'-hydroxyl position to disrupt overall RNA structure. HMX was found to be exceptionally selective for quantitative detection of higher-order and tertiary interactions. When incorporated as experimental constraints in discrete molecular dynamics (DMD) simulations, HMX information yielded accurate three-dimensional models, emphasizing the power of molecular interference to guide RNA tertiary structure analysis and fold refinement. In the case of a large, multi-domain RNA, the Tetrahymena group I intron, HMX identified multiple distinct sets of tertiary structure interaction groups in a single, concise experiment
Structure and Dynamics of the HIV‑1 Frameshift Element RNA
The
HIV-1 ribosomal frameshift element is highly structured, regulates
translation of all virally encoded enzymes, and is a promising therapeutic
target. The prior model for this motif contains two helices separated
by a three-nucleotide bulge. Modifications to this model were suggested
by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features
of the SHAPE-directed model include alternate helical conformations
and a larger, more complex structure. These structural elements also
support the presence of a secondary frameshift site within the frameshift
domain. Here, we use oligonucleotide-directed structure perturbation,
probing in the presence of formamide, and in-virion experiments to
examine these models. Our data support a model in which the frameshift
domain is anchored by a stable helix outside the conventional domain.
Less stable helices within the domain can switch from the SHAPE-predicted
to the two-helix conformation. Translational frameshifting assays
with frameshift domain mutants support a functional role for the interactions
predicted by and specific to the SHAPE-directed model. These results
reveal that the HIV-1 frameshift domain is a complex, dynamic structure
and underscore the importance of analyzing folding in the context
of full-length RNAs
Long-Range Architecture in a Viral RNA Genome
We have developed a model for the secondary structure of the 1058-nucleotide plus-strand RNA genome of the icosahedral satellite tobacco mosaic virus (STMV) using nucleotide-resolution SHAPE chemical probing of the viral RNA isolated from virions and within the virion, perturbation of interactions distant in the primary sequence, and atomic force microscopy. These data are consistent with long-range base pairing interactions and a three-domain genome architecture. The compact domains of the STMV RNA have dimensions of 10 to 45 nm. Each of the three domains corresponds to a specific functional component of the virus: The central domain corresponds to the coding sequence of the single (capsid) protein encoded by the virus, whereas the 5′ and 3′ untranslated domains span signals essential for translation and replication, respectively. This three-domain architecture is compatible with interactions between the capsid protein and short RNA helices previously visualized by crystallography. STMV is among the simplest of the icosahedral viruses but, nonetheless, has an RNA genome with a complex higher-order structure that likely reflects high information content and an evolutionary relationship between RNA domain structure and essential replicative functions