scholarly journals Flexibility of telomerase in binding the RNA template and DNA telomeric repeat

2021 ◽  
Vol 119 (1) ◽  
pp. e2116159118
Author(s):  
Woo Suk Choi ◽  
Peter J. Weng ◽  
Wei Yang
Keyword(s):  
Reverse Transcriptase ◽  
Conformational Changes ◽  
Complex Structure ◽  
Model System ◽  
Telomeric Repeat ◽  
Cavity Size ◽  
Reverse Transcriptase Domain ◽  
Linear Chromosomes ◽  
Dna Substrate ◽  
Rna And Dna

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3′ end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5′ template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA–DNA binding likely underlies the processivity of telomeric repeat addition.

Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding

2015 ◽  
Vol 465 (2) ◽  
pp. 347-357 ◽  
Author(s):  
Christopher G. Tomlinson ◽  
Aaron L. Moye ◽  
Jessica K. Holien ◽  
Michael W. Parker ◽  
Scott B. Cohen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Reverse Transcriptase ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Reverse Transcriptase Domain ◽  
Human Telomerase ◽  
Initial Binding ◽  
Dna Substrate ◽  
Step Mechanism

Initial binding of the enzyme telomerase to its DNA substrate proceeds by a two-step mechanism involving enzyme conformational change. A protein loop in the reverse transcriptase domain is involved in these conformational changes.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Complexes of HIV-1 Reverse Transcriptase with Inhibitors of the HEPT Series Reveal Conformational Changes Relevant to the Design of Potent Non-Nucleoside Inhibitors

1996 ◽  
Vol 39 (8) ◽  
pp. 1589-1600 ◽  
Author(s):  
Andrew L. Hopkins ◽  
Jingshan Ren ◽  
Robert M. Esnouf ◽  
Benjamin E. Willcox ◽  
E. Yvonne Jones ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Conformational Changes ◽  
Nucleoside Inhibitors ◽  
Hiv 1

scholarly journals POT1-TPP1 differentially regulates telomerase via POT1 His266 and as a function of single-stranded telomere DNA length

2019 ◽  
Vol 116 (47) ◽  
pp. 23527-23533 ◽  
Author(s):  
Mengyuan Xu ◽  
Janna Kiselar ◽  
Tawna L. Whited ◽  
Wilnelly Hernandez-Sanchez ◽  
Derek J. Taylor
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Environmental Changes ◽  
Lymphocytic Leukemia ◽  
Protein Protein Interactions ◽  
Cellular Environment ◽  
Multiple Protein ◽  
Linear Chromosomes ◽  
Hydroxyl Radical Footprinting ◽  
Regulate Telomere Length

Telomeres cap the ends of linear chromosomes and terminate in a single-stranded DNA (ssDNA) overhang recognized by POT1-TPP1 heterodimers to help regulate telomere length homeostasis. Here hydroxyl radical footprinting coupled with mass spectrometry was employed to probe protein–protein interactions and conformational changes involved in the assembly of telomere ssDNA substrates of differing lengths bound by POT1-TPP1 heterodimers. Our data identified environmental changes surrounding residue histidine 266 of POT1 that were dependent on telomere ssDNA substrate length. We further determined that the chronic lymphocytic leukemia-associated H266L substitution significantly reduced POT1-TPP1 binding to short ssDNA substrates; however, it only moderately impaired the heterodimer binding to long ssDNA substrates containing multiple protein binding sites. Additionally, we identified a telomerase inhibitory role when several native POT1-TPP1 proteins coat physiologically relevant lengths of telomere ssDNA. This POT1-TPP1 complex-mediated inhibition of telomerase is abrogated in the context of the POT1 H266L mutation, which leads to telomere overextension in a malignant cellular environment.

scholarly journals Development of Aptamer-Based TID Assays Using Thermophoresis and Microarrays

Biosensors ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 124 ◽  
Author(s):  
Kurth ◽  
Witt ◽  
Bolten ◽  
Waniek ◽  
Kortmann ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Detection ◽  
Model System ◽  
Vascular Endothelial ◽  
Protein Biomarkers ◽  
High Affinity ◽  
Model Protein ◽  
Recognition Elements ◽  
Systematic Development ◽  
Endothelial Growth Factor

Aptamers are single-stranded oligonucleotides which can be used as alternative recognition elements for protein detection, because aptamers bind their targets with a high affinity similar to antibodies. Due to the targetinduced conformational changes of aptamers, these oligonucleotides can be applied in various biosensing platforms. In this work, aptamers directed against the vascular endothelial growth factor (VEGF) were used as a model system. VEGF plays a key role in physiological angiogenesis and vasculogenesis. Furthermore, VEGF is involved in the development and growth of cancer and other diseases like agerelated macular degeneration, rheumatoid arthritis, diabetes mellitus, and neurodegenerative disorders. Detecting the protein biomarker VEGF is therefore of great importance for medical research and diagnostics. In this research, VEGFbinding aptamers were investigated for the systematic development of a targetinduced dissociation (TID) assay utilizing thermophoresis and microarrays. The established aptamer-microarray allowed for the detection of 0.1 nM of VEGF. Furthermore, the systematic development of the TID method using the VEGF model protein could help to develop further TID assays for the detection of various protein biomarkers.

Development of Single-Domain Recombinant Antibodies to Reverse Transcriptase Domain of Human hTERT

2004 ◽  
Vol 23 (4) ◽  
pp. 244-249
Author(s):  
Minyan Huang ◽  
Bo Zhang ◽  
Junmei Wang ◽  
Fang Mei ◽  
Lin Hou
Keyword(s):  
Reverse Transcriptase ◽  
Recombinant Antibodies ◽  
Single Domain ◽  
Reverse Transcriptase Domain

scholarly journals Mode of inhibition of HIV reverse transcriptase by 2-hexaprenylhydroquinone, a novel general inhibitor of RNA-and DNA-directed DNA polymerases

1997 ◽  
Vol 324 (3) ◽  
pp. 721-727 ◽  
Author(s):  
Shoshana LOYA ◽  
Amira RUDI ◽  
Yoel KASHMAN ◽  
Amnon HIZI
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerases ◽  
Competitive Inhibitor ◽  
Polymerization Process ◽  
Hiv Reverse Transcriptase ◽  
Allosteric Site ◽  
Leukaemia Virus ◽  
General Inhibitor ◽  
Hiv 1 ◽  
Rna And Dna

A natural compound from the Red Sea sponge Ircinia sp., 2-hexaprenylhydroquinone (HPH), has been shown to be a general inhibitor of retroviral reverse transcriptases (from HIV-1, HIV-2 and murine leukaemia virus) as well as of cellular DNA polymerases (Escherichia coli DNA polymerase I, and DNA polymerases α and β). The pattern of inhibition was found to be similar for all DNA polymerases tested. Thus the mode of inhibition was studied in detail for HIV-1 reverse transcriptase. HPH is a non-competitive inhibitor and binds the enzyme irreversibly with high affinity (Ki = 0.62 μM). The polar hydroxy groups have been shown to be of key importance. A methylated derivative, mHPH, which is devoid of these polar moieties, showed a significantly decreased capacity to inhibit all DNA polymerases tested. Like the natural product, mHPH binds the enzyme independently at an allosteric site, but with reduced affinity (Ki = 7.4 μM). We show that HPH does not interfere with the first step of the polymerization process, i.e. the physical formation of the reverse-transcriptase–DNA complex. Consequently, we suggest that the natural inhibitor interferes with the subsequent steps of the overall reaction. Since HPH seems not to affect the affinity of dNTP for the enzyme (the Km is unchanged under conditions where the HPH concentration is increased), we speculate that its inhibitory capacity is derived from its effect on the nucleotidyl-transfer catalytic reaction. We suggest that such a mechanism of inhibition is typical of an inhibitor whose mode of inhibition should be common to all RNA- and DNA-directed polymerases.

Sign in / Sign up

Export Citation Format

Share Document