- Home
- Search Results
RESULTS:
1 - 7 of 7 for "Doudna, Jennifer A"
Run a new search
CRISPR–Cas9 Structures and Mechanisms
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA Cas9–DNA interactions and associated conformational changes. The use of CRISPR–Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function guide RNA specificity and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
Structural Insights Into the Signal Recognition Particle
The signal recognition particle (SRP) directs integral membrane and secretory proteins to the cellular protein translocation machinery during translation. The SRP is an evolutionarily conserved RNA-protein complex whose activities are regulated by GTP hydrolysis. Recent structural investigations of SRP functional domains and interactions provide new insights into the mechanisms of SRP activity in all cells leading toward a comprehensive understanding of protein trafficking by this elegant pathway.
Molecular Mechanisms of RNA Interference
Small RNA molecules regulate eukaryotic gene expression during development and in response to stresses including viral infection. Specialized ribonucleases and RNA-binding proteins govern the production and action of small regulatory RNAs. After initial processing in the nucleus by Drosha precursor microRNAs (pre-miRNAs) are transported to the cytoplasm where Dicer cleavage generates mature microRNAs (miRNAs) and short interfering RNAs (siRNAs). These double-stranded products assemble with Argonaute proteins such that one strand is preferentially selected and used to guide sequence-specific silencing of complementary target mRNAs by endonucleolytic cleavage or translational repression. Molecular structures of Dicer and Argonaute proteins and of RNA-bound complexes have offered exciting insights into the mechanisms operating at the heart of RNA-silencing pathways.
Ribozyme Structures and Mechanisms
The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural phylogenetic and biochemical data. However many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead hairpin hepatitis delta virus and in vitro–selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes such as the group I intron.
Ribozyme Structures and Mechanisms
The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural phylogenetic and biochemical data. However many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead hairpin hepatitis delta virus and in vitro–selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes such as the group I intron.
Erratum: Molecular Mechanisms of RNA Interference
RNA Folds: Insights from Recent Crystal Structures
An RNA fold is the result of packing together two or more coaxial helical stacks. To date four RNA folds have been determined at near-atomic resolution by X-ray crystallography: transfer RNA the hammerhead ribozyme the P4–P6 domain of the Tetrahymena group I intron and the hepatitis delta virus ribozyme. All four folds result in RNAs that are considerably more compact than isolated A-form duplexes. These structures illustrate to varying degrees three modes of fold stabilization: association of complementary molecular surfaces stabilization of close RNA packing by binding of cations and stabilization through pseudoknotting.