scholarly journals Microprocessor depends on hemin to recognize the apical loop of primary microRNA

2018 ◽  
Vol 46 (11) ◽  
pp. 5726-5736 ◽  
Author(s):  
Tuan Anh Nguyen ◽  
Joha Park ◽  
Thi Lieu Dang ◽  
Yeon-Gil Choi ◽  
V Narry Kim
Keyword(s):  
Apical Loop

scholarly journals Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

Crystals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 273
Author(s):  
Yoshita Srivastava ◽  
Rachel Bonn-Breach ◽  
Sai Shashank Chavali ◽  
Geoffrey M. Lippa ◽  
Jermaine L. Jenkins ◽  
...  
Keyword(s):  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Molecular Replacement ◽  
Hydrophobic Core ◽  
Anomalous Diffraction ◽  
Recognition Motif ◽  
Determination Process ◽  
Experimental Phasing ◽  
Crystal Contacts ◽  
Apical Loop

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

scholarly journals Effect of apical portion of T-, sloped L-, and reversed L-closing loops on their force systems

2016 ◽  
Vol 87 (1) ◽  
pp. 104-110 ◽  
Author(s):  
Paiboon Techalertpaisarn ◽  
Antheunis Versluis
Keyword(s):  
Three Dimensional ◽  
Vertical Force ◽  
Element Analysis ◽  
Force System ◽  
Apical Portion ◽  
Beam Elements ◽  
Force Ratio ◽  
Force Systems ◽  
Three Dimensional Models ◽  
Apical Loop

ABSTRACT Objective: To investigate the effect of the position of the apical portion of closing loops on the force system at both loop ends. Materials and Methods: T-loops were compared with backward-sloped L-loops (SL) and reversed L-loops (RL). SL-loops were directed toward the anterior side; RL-loops were directed toward the posterior side. Loop response to loop pulling was determined with finite element analysis at six positions of the apical loop portion for 12-mm interbracket distance and 8-mm loop length and height. Three-dimensional models of the closing loops were created using beam elements with the properties of stainless steel. Loop responses (horizontal load/deflection, vertical force, and moment-to-force ratio) at both loop ends were calculated as well as at 100 g and 200 g activation forces. Results: T-, SL-, and RL-loops with the same position of the apical portion showed approximately the same force system at both loop ends. This behavior was found across the investigated range through which the loops were moved (interbracket center to posterior bracket). Conclusions: The center of the apical portion determined the force system of the closing loops regardless of the position of the loop legs. The centers of the apical portion of the T-, SL-, and RL-loops acted like V-bend positions.

scholarly journals Structure and stabilization of the Hendra virus F glycoprotein in its prefusion form

2015 ◽  
Vol 113 (4) ◽  
pp. 1056-1061 ◽  
Author(s):  
Joyce J. W. Wong ◽  
Reay G. Paterson ◽  
Robert A. Lamb ◽  
Theodore S. Jardetzky
Keyword(s):  
Nipah Virus ◽  
Fusion Peptide ◽  
Hendra Virus ◽  
Cell Entry ◽  
Loss Of Function ◽  
Host Cell Membrane ◽  
Peptide Cleavage ◽  
Apical Loop ◽  
The Stability ◽  
Biosafety Level

Hendra virus (HeV) is one of the two prototypical members of theHenipavirusgenus of paramyxoviruses, which are designated biosafety level 4 (BSL-4) organisms due to the high mortality rate of Nipah virus (NiV) and HeV in humans. Paramyxovirus cell entry is mediated by the fusion protein, F, in response to binding of a host receptor by the attachment protein. During posttranslational processing, the fusion peptide of F is released and, upon receptor-induced triggering, inserts into the host cell membrane. As F undergoes a dramatic refolding from its prefusion to postfusion conformation, the fusion peptide brings the host and viral membranes together, allowing entry of the viral RNA. Here, we present the crystal structure of the prefusion form of the HeV F ectodomain. The structure shows very high similarity to the structure of prefusion parainfluenza virus 5 (PIV5) F, with the main structural differences in the membrane distal apical loops and the fusion peptide cleavage loop. Functional assays of mutants show that the apical loop can tolerate perturbation in length and surface residues without loss of function, except for residues involved in the stability and conservation of the F protein fold. Structure-based disulfide mutants were designed to anchor the fusion peptide to conformationally invariant residues of the F head. Two mutants were identified that inhibit F-mediated fusion by stabilizing F in its prefusion conformation.

scholarly journals Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome

2008 ◽  
Vol 37 (4) ◽  
pp. 1141-1151 ◽  
Author(s):  
E. Babaylova ◽  
D. Graifer ◽  
A. Malygin ◽  
J. Stahl ◽  
I. Shatsky ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Apical Loop

scholarly journals The Apical Loop of the HIV-1 TAR RNA Hairpin Is Stabilized by a Cross-loop Base Pair

2003 ◽  
Vol 278 (40) ◽  
pp. 38892-38901 ◽  
Author(s):  
Tadeusz Kulinski ◽  
Mikolaj Olejniczak ◽  
Hendrik Huthoff ◽  
Lukasz Bielecki ◽  
Katarzyna Pachulska-Wieczorek ◽  
...  
Keyword(s):  
Base Pair ◽  
Tar Rna ◽  
Apical Loop ◽  
Rna Hairpin ◽  
Hiv 1

scholarly journals Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit Are Critical for Its Fusion Activity

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Stephanie N. Hicks ◽  
Supranee Chaiwatpongsakorn ◽  
Heather M. Costello ◽  
Jason S. McLellan ◽  
William Ray ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Target Cell ◽  
Antiviral Drug ◽  
High Energy ◽  
Heptad Repeat ◽  
Alanine Scanning Mutagenesis ◽  
F Protein ◽  
Syncytial Virus ◽  
Apical Loop ◽  
Tract Infections

ABSTRACT The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein. IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545–1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577–587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749–1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110–128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.

scholarly journals Rules of RNA specificity of hnRNP A1 revealed by global and quantitative analysis of its affinity distribution

2017 ◽  
Vol 114 (9) ◽  
pp. 2206-2211 ◽  
Author(s):  
Niyati Jain ◽  
Hsuan-Chun Lin ◽  
Christopher E. Morgan ◽  
Michael E. Harris ◽  
Blanton S. Tolbert
Keyword(s):  
Rna Binding ◽  
Sequence Motif ◽  
Hnrnp A1 ◽  
Loop Analysis ◽  
Splicing Silencer ◽  
Rna Ligands ◽  
Apical Loop ◽  
Affinity Distribution

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a multipurpose RNA-binding protein (RBP) involved in normal and pathological RNA metabolism. Transcriptome-wide mapping and in vitro evolution identify consensus hnRNP A1 binding motifs; however, such data do not reveal how surrounding RNA sequence and structural context modulate affinity. We determined the affinity of hnRNP A1 for all possible sequence variants (n = 16,384) of the HIV exon splicing silencer 3 (ESS3) 7-nt apical loop. Analysis of the affinity distribution identifies the optimal motif 5′-YAG-3′ and shows how its copy number, position in the loop, and loop structure modulate affinity. For a subset of ESS3 variants, we show that specificity is determined by association rate constants and that variants lacking the minimal sequence motif bind competitively with consensus RNA. Thus, the results reveal general rules of specificity of hnRNP A1 and provide a quantitative framework for understanding how it discriminates between alternative competing RNA ligands in vivo.

scholarly journals Investigating the Properties of Bacillus thuringiensis Cry Proteins with Novel Loop Replacements Created Using Combinatorial Molecular Biology

2008 ◽  
Vol 74 (11) ◽  
pp. 3497-3511 ◽  
Author(s):  
Craig R. Pigott ◽  
Martin S. King ◽  
David J. Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Important Determinant ◽  
Large Family ◽  
Cry Proteins ◽  
Diverse Range ◽  
The Family ◽  
Complementarity Determining Regions ◽  
Apical Loop ◽  
Loop 2

ABSTRACTCry proteins are a large family of crystalline toxins produced byBacillus thuringiensis. Individually, the family members are highly specific, but collectively, they target a diverse range of insects and nematodes. Domain II of the toxins is important for target specificity, and three loops at its apex have been studied extensively. There is considerable interest in determining whether modifications in this region may lead to toxins with novel specificity or potency. In this work, we studied the effect of loop substitution on toxin stability and specificity. For this purpose, sequences derived from antibody complementarity-determining regions (CDR) were used to replace native domain II apical loops to create “Crybodies.” Each apical loop was substituted either individually or in combination with a library of third heavy-chain CDR (CDR-H3) sequences to create seven distinct Crybody types. An analysis of variants from each library indicated that the Cry1Aa framework can tolerate considerable sequence diversity at all loop positions but that some sequence combinations negatively affect structural stability and protease sensitivity. CDR-H3 substitution showed that loop position was an important determinant of insect toxicity: loop 2 was essential for activity, whereas the effects of substitutions at loop 1 and loop 3 were sequence dependent. Unexpectedly, differences in toxicity did not correlate with binding to cadherins—a major class of toxin receptors—since all Crybodies retained binding specificity. Collectively, these results serve to better define the role of the domain II apical loops as determinants of specificity and establish guidelines for their modification.

An Aptamer Targeting the Apical-Loop Domain Modulates pri-miRNA Processing

2010 ◽  
Vol 49 (27) ◽  
pp. 4674-4677 ◽  
Author(s):  
Christina E. Lünse ◽  
Gracjan Michlewski ◽  
Christine S. Hopp ◽  
Andrea Rentmeister ◽  
Javier F. Cáceres ◽  
...  
Keyword(s):  
Mirna Processing ◽  
Loop Domain ◽  
Apical Loop
Sign in / Sign up

Export Citation Format

Share Document