scholarly journals Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease

Science ◽  
2010 ◽  
Vol 329 (5997) ◽  
pp. 1355-1358 ◽  
Author(s):  
Rachel E. Haurwitz ◽  
Martin Jinek ◽  
Blake Wiedenheft ◽  
Kaihong Zhou ◽  
Jennifer A. Doudna
Keyword(s):  
Rna Processing ◽  
Active Site ◽  
Large Family ◽  
Rna Recognition ◽  
Specific Interactions ◽  
Major Groove ◽  
Recognition Mechanism ◽  
Stem Loop ◽  
Crispr Locus ◽  
Phosphate Backbone

Many bacteria and archaea contain clustered regularly interspaced short palindromic repeats (CRISPRs) that confer resistance to invasive genetic elements. Central to this immune system is the production of CRISPR-derived RNAs (crRNAs) after transcription of the CRISPR locus. Here, we identify the endoribonuclease (Csy4) responsible for CRISPR transcript (pre-crRNA) processing in Pseudomonas aeruginosa. A 1.8 angstrom crystal structure of Csy4 bound to its cognate RNA reveals that Csy4 makes sequence-specific interactions in the major groove of the crRNA repeat stem-loop. Together with electrostatic contacts to the phosphate backbone, these enable Csy4 to bind selectively and cleave pre-crRNAs using phylogenetically conserved serine and histidine residues in the active site. The RNA recognition mechanism identified here explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases.

Structural and mechanistic insight into stem‐loop RNA processing by yeast Pichia stipitis Dicer

2021 ◽  
Author(s):  
Chan Jing Ru ◽  
Fu Qinqin ◽  
Li Jianwei ◽  
Chen Ying ◽  
Satoru Machida ◽  
...  
Keyword(s):  
Rna Processing ◽  
Pichia Stipitis ◽  
Stem Loop ◽  
Mechanistic Insight ◽  
Insight Into

scholarly journals Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomáš Kouba ◽  
Tomáš Koval’ ◽  
Petra Sudzinová ◽  
Jiří Pospíšil ◽  
Barbora Brezovská ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Rna Synthesis ◽  
Environmental Changes ◽  
Transcription Elongation ◽  
Specific Interactions ◽  
Inactive State ◽  
Transcription Machinery

AbstractRNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.

scholarly journals Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Charlotte A. Softley ◽  
Krzysztof M. Zak ◽  
Mark J. Bostock ◽  
Roberto Fino ◽  
Richard Xu Zhou ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Rational Drug Design ◽  
Tryptophan Residue ◽  
Resonance Spectroscopy ◽  
Global Public Health ◽  
Recognition Mechanism ◽  
Gram Negative ◽  
Content Type ◽  
Locking Mechanism

ABSTRACT Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely used antibiotic class, the β-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.

scholarly journals Sequence Specific RNA Recognition Mechanism of MazF

2015 ◽  
Vol 55 (1) ◽  
pp. 011-014
Author(s):  
Yoshihiro YAMAGUCHI
Keyword(s):  
Rna Recognition ◽  
Recognition Mechanism

Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation

Science ◽  
2019 ◽  
Vol 365 (6455) ◽  
pp. 825-829 ◽  
Author(s):  
Tae Hun Kim ◽  
Brian Tsang ◽  
Robert M. Vernon ◽  
Nahum Sonenberg ◽  
Lewis E. Kay ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Processing ◽  
Intrinsically Disordered Protein ◽  
Resonance Spectroscopy ◽  
Specific Interactions ◽  
Intrinsically Disordered ◽  
Functional Consequences ◽  
Translational Regulators

Membraneless organelles involved in RNA processing are biomolecular condensates assembled by phase separation. Despite the important role of intrinsically disordered protein regions (IDRs), the specific interactions underlying IDR phase separation and its functional consequences remain elusive. To address these questions, we used minimal condensates formed from the C-terminal disordered regions of two interacting translational regulators, FMRP and CAPRIN1. Nuclear magnetic resonance spectroscopy of FMRP-CAPRIN1 condensates revealed interactions involving arginine-rich and aromatic-rich regions. We found that different FMRP serine/threonine and CAPRIN1 tyrosine phosphorylation patterns control phase separation propensity with RNA, including subcompartmentalization, and tune deadenylation and translation rates in vitro. The resulting evidence for residue-specific interactions underlying co–phase separation, phosphorylation-modulated condensate architecture, and enzymatic activity within condensates has implications for how the integration of signaling pathways controls RNA processing and translation.

scholarly journals The Cotranscriptional Assembly of snoRNPs Controls the Biosynthesis of H/ACA snoRNAs in Saccharomyces cerevisiae

2005 ◽  
Vol 25 (13) ◽  
pp. 5396-5403 ◽  
Author(s):  
Monica Ballarino ◽  
Mariangela Morlando ◽  
Francesca Pagano ◽  
Alessandro Fatica ◽  
Irene Bozzoni
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Large Subunit ◽  
Large Family ◽  
The Core ◽  
Carboxy Terminal Domain ◽  
Assembly Factor ◽  
Core Components ◽  
Carboxy Terminal ◽  
Nucleolar Rna

ABSTRACT The carboxy-terminal domain (CTD) of RNA polymerase II large subunit acts as a platform to assemble the RNA processing machinery in a controlled way throughout the transcription cycle. In yeast, recent findings revealed a physical connection between phospho-CTD, generated by the Ctk1p kinase, and protein factors having a function in small nucleolar RNA (snoRNA) biogenesis. The snoRNAs represent a large family of polymerase II noncoding transcripts that are associated with highly conserved polypeptides to form stable ribonucleoprotein particles (snoRNPs). In this work, we have studied the biogenesis of the snoRNPs belonging to the box H/ACA class. We report that the assembly factor Naf1p and the core components Cbf5p and Nhp2p are recruited on H/ACA snoRNA genes very early during transcription. We also show that the cotranscriptional recruitment of Naf1p and Cbf5p is Ctk1p dependent and that Ctk1p and Cbf5p are required for preventing the readthrough into the snoRNA downstream genes. All these data suggest that proper cotranscriptional snoRNP assembly controls 3′-end formation of snoRNAs and, consequently, the release of a functional particle.

scholarly journals U2 small nuclear RNA 3' end formation is directed by a critical internal structure distinct from the processing site.

1993 ◽  
Vol 13 (2) ◽  
pp. 1119-1129 ◽  
Author(s):  
M R Jacobson ◽  
M Rhoadhouse ◽  
T Pederson
Keyword(s):  
Rna Processing ◽  
Minor Effect ◽  
Small Nuclear Rna ◽  
Recognition Sequence ◽  
Stem Loop ◽  
Branch Site ◽  
Nuclear Rna ◽  
A Minor ◽  
Site Recognition

Mature U2 small nuclear RNA is generated by the removal of 11 to 12 nucleotides from the 3' end of the primary transcript. This pre-U2 RNA processing reaction takes place in the cytoplasm. In this study, the sequences and/or structures of pre-U2 RNA that are important for 3' processing have been examined in an in vitro system. The 7-methylguanosine cap, stem-loops I and II, the lariat branch site recognition sequence, the conserved Sm domain, and several other regions throughout the 5' end of U2 RNA have no apparent role in the 3' processing reaction. In fact, deletion of the entire first 104 nucleotides resulted in mini-pre-U2 RNAs which were efficiently processed. Similarly, deletion of the top two-thirds of stem-loop III or mutation of nucleotides in the loop of stem-loop IV had little effect on 3' processing. Most surprisingly, the precursor's 11- to 12-nucleotide 3' extension itself was of relatively little importance, since this sequence could be replaced with completely different sequences with only a minor effect on the 3' processing reaction. In contrast, we have defined a critical structure consisting of the bottom of stem III and the stem of stem-loop IV that is essential for 3' processing of pre-U2 RNA. Compensatory mutations which restore base pairing in this region resulted in normal 3' processing. Thus, although the U2 RNA processing activity recognizes the bottom of stem III and stem IV, the sequence of this critical region is much less important than its structure. These results, together with the surprising observation that the reaction is relatively indifferent to the sequence of the 11- to 12-nucleotide 3' extension itself, point to a 3' processing reaction of pre-U2 RNA that has sequence and structure requirements significantly different from those previously identified for pre-mRNA 3' processing.

scholarly journals Recognition of the bcd mRNA Localization Signal in Drosophila Embryos and Ovaries

2005 ◽  
Vol 25 (4) ◽  
pp. 1501-1510 ◽  
Author(s):  
Mark J. Snee ◽  
Eric A. Arn ◽  
Simon L. Bullock ◽  
Paul M. Macdonald
Keyword(s):  
Protein Complexes ◽  
Regulation Of Gene Expression ◽  
Mrna Localization ◽  
Signal Recognition ◽  
Recognition Mechanism ◽  
Stem Loop ◽  
Multiple Contexts ◽  
Polarized Cells ◽  
Localization Signal ◽  
Drosophila Embryos

ABSTRACT The process of mRNA localization, often used for regulation of gene expression in polarized cells, requires recognition of cis-acting signals by components of the localization machinery. Many known RNA signals are active in the contexts of both the Drosophila ovary and the blastoderm embryo, suggesting a conserved recognition mechanism. We used variants of the bicoid mRNA localization signal to explore recognition requirements in the embryo. We found that bicoid stem-loop IV/V, which is sufficient for ovarian localization, was necessary but not sufficient for full embryonic localization. RNAs containing bicoid stem-loops III/IV/V did localize within the embryo, demonstrating a requirement for dimerization and other activities supplied by stem-loop III. Protein complexes that bound specifically to III/IV/V and fushi tarazu localization signals copurified through multiple fractionation steps, suggesting that they are related. Binding to these two signals was competitive but not equivalent. Thus, the binding complexes are not identical but appear to have some components in common. We have proposed a model for a conserved mechanism of localization signal recognition in multiple contexts.

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