scholarly journals Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

2017 ◽  
Vol 136 (3) ◽  
Author(s):  
Katarzyna Świderek ◽  
Sergio Marti ◽  
Iñaki Tuñón ◽  
Vicent Moliner ◽  
Juan Bertran
Keyword(s):  
Molecular Mechanism ◽  
Site Specific ◽  
Phosphodiester Backbone

scholarly journals Hatchet ribozyme structure and implications for cleavage mechanism

2019 ◽  
Vol 116 (22) ◽  
pp. 10783-10791 ◽  
Author(s):  
Luqian Zheng ◽  
Christoph Falschlunger ◽  
Kaiyi Huang ◽  
Elisabeth Mairhofer ◽  
Shuguang Yuan ◽  
...  
Keyword(s):  
Viral Genome ◽  
Mrna Processing ◽  
Genome Replication ◽  
Site Specific ◽  
Phosphodiester Backbone ◽  
Close Proximity ◽  
Long Range Interactions ◽  
Viral Genome Replication ◽  
Functional Relevance ◽  
The Impact

Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5′ nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.

scholarly journals Insights into DNA Platination within Unusual Structural Settings

2011 ◽  
Vol 2011 ◽  
pp. 1-17
Author(s):  
Stephanie Harvie ◽  
Owen Wilson ◽  
John A. Parkinson
Keyword(s):  
Nucleic Acid ◽  
Nmr Spectroscopy ◽  
Nucleic Acids ◽  
Product Formation ◽  
Loop Formation ◽  
Hairpin Loop ◽  
Cross Link ◽  
Site Specific ◽  
Phosphodiester Backbone ◽  
Dna Platination

2D HSQC NMR spectroscopy has been used to monitor reaction and product formation between and nucleic acids possessing irregular topologies and containing site-specific phosphorothioate substitution in the phosphodiester backbone. Comparison of the reaction profiles of dimer nucleic acids with and without phosphorothioate substitution is made with their short nucleic acid counterparts containing the key dimer components. Whereas d(GpA) is relatively unreactive towards , NMR evidence suggests that the tandem sheared mismatch duplex d(GCG3pAGC)2 reacts to form the head-to-tail interstrand G3-N7-Pt-G3-N7 cross-link. The equivalent phosphorothioate R,S-d(GsA) reacts to form a monoiodo, monosulphur adduct, whereas the tandem sheared mismatch phosphorothioate duplex d(GCGsAG5C)2 (VIs) reacts to form the unusual intrastrand macrochelate , in which platinum is attached at both sulphur and G5-N7. Experimental evidence supports the formation of a stabilized mismatch duplex in which platinum is attached to two nitrogen centres in the sequence d(CGCGpTGCG) in contrast to R,S-d(CGCGsT5GCG) for which NMR evidence supports macrochelate-stabilized hairpin loop formation cross-linked at both phosphorothioate sulphur and T5-N3.

Transpositional Recombination and Site-Specific Recombination May Be Initiated by Copy Choice during DNA Synthesis Rather Than Break/Join Mechanism

2018 ◽  
Author(s):  
Lei Jia ◽  
Jingyun Li
Keyword(s):  
Dna Synthesis ◽  
Gene Conversion ◽  
Molecular Mechanism ◽  
Dna Recombination ◽  
Meiotic Segregation ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Nonhomologous Recombination ◽  
Novel Theory ◽  
Dna Template

Types of DNA recombination include homologous recombination and nonhomologous recombination. Homologous DNA recombination is a general term that includes exchange of information between chromatids: (reciprocal) crossing-over, gene conversion, and post-meiotic segregation. Gene conversion is now thought to be a type of non-Mendelian segregation of heterozygous markers near the recombination initiation site. Thus, it includes both gene conversion and post-meiotic segregation previously described. DNA non-HR including transpositional recombination and site-specific recombination. Our understanding of the molecular mechanism by which DNA recombination occurs has significantly increased in the past decades. Currently The synthesis-dependent strand annealing model is now thought to give rise to most or all noncrossovers, with the double-strand-break repair model forming mainly crossovers. The Shapiro model proposed by Dr. J. Shapiro explains the molecular mechanism of transpositional recombination. Site-specific recombination results from another distinct model. We previously proposed a novel theory which can provide a more reasonable and simpler explanation accounting for DNA HR including the 3 classes of recombinogenic events described above. In the new supplementedly molecular model, DNA meiotic recombination can be initiated by a copy choice mechanism, that is, copying part of 1 single-stranded DNA template, followed by DNA polymerase switching to another single-stranded DNA template, and then resuming the following DNA synthesis along the new template. The current review suggests that transpositional recombination and site-specific recombination should be initiated by copy choice during DNA synthesis rather than break/join mechanism. The work indicates that review of DNA nonhomologous recombination are very necessary. The novel theory would challenge earlier models accounting for transpositional recombination and site-specific recombination and would be critical to the understanding of the mechanisms. We hope copy choice initiating DNA nonhomologous recombination will be one of the concepts that are explored. Proper and specific experiments are required to reconstruct the detailed mechanism described here.

Molecular mechanism of negative cooperativity of ferredoxin-NADP+ reductase by ferredoxin and NADP(H): involvement of a salt bridge between Asp60 of ferredoxin and Lys33 of FNR

2021 ◽  
Author(s):  
Yutaro Chikuma ◽  
Masayuki Miyata ◽  
Young-Ho Lee ◽  
Toshiharu Hase ◽  
Yoko Kimata-Ariga
Keyword(s):  
Molecular Mechanism ◽  
Three Dimensional ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Chemical Shift Perturbation ◽  
Negative Cooperativity ◽  
Site Specific ◽  
A Site ◽  
Transfer Chain ◽  
Photosynthetic Electron Transfer

ABSTRACT Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) and converts NADP+ to NADPH at the end of the photosynthetic electron transfer chain. We previously showed that the interaction between FNR and Fd was weakened by the allosteric binding of NADP(H) on FNR, which was considered as a part of negative cooperativity. In this study, we investigated the molecular mechanism of this phenomenon using maize FNR and Fd, as the three-dimensional structure of this Fd:FNR complex is available. NMR chemical shift perturbation analysis identified a site (Asp60) on Fd molecule which was selectively affected by NADP(H) binding on FNR. Asp60 of Fd forms a salt bridge with Lys33 of FNR in the complex. Site-specific mutants of FdD60 and FNRK33 suppressed the negative cooperativity (downregulation of the interaction between FNR and Fd by NADPH), indicating that a salt bridge between FdD60 and FNRK33 is involved in this negative cooperativity.

scholarly journals Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

2015 ◽  
Vol 89 (13) ◽  
pp. 6952-6959 ◽  
Author(s):  
Laura K. Pritchard ◽  
Daniel I. R. Spencer ◽  
Louise Royle ◽  
Snezana Vasiljevic ◽  
Stefanie A. Krumm ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Neutralizing Antibodies ◽  
Vaccine Design ◽  
Broadly Neutralizing Antibodies ◽  
Site Specific ◽  
Antibody Recognition ◽  
Glycosylation Sites ◽  
Minor Population ◽  
A Minor ◽  
Hiv 1

Broadly neutralizing antibodies have been isolated that bind the glycan shield of the HIV-1 envelope spike. One such antibody, PGT135, contacts the intrinsic mannose patch of gp120 at the Asn332, Asn392, and Asn386 glycosylation sites. Here, site-specific glycosylation analysis of recombinant gp120 revealed glycan microheterogeneity sufficient to explain the existence of a minor population of virions resistant to PGT135 neutralization. Target microheterogeneity and antibody glycan specificity are therefore important parameters in HIV-1 vaccine design.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

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