A U1 snRNA binding site improves the efficiency of in vitro pre-mRNA splicing

ABSTRACT Modulation of the interaction between U1 snRNP and the 5′ splice site (5′ss) is a key event that governs 5′ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5′ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5′ss during early stage of splicing. However, dissociation of the U1 from the 5′ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5′ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

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Yeast Pre-mRNA Splicing Requires a Pair of U1 snRNP-Associated Tetratricopeptide Repeat Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.353 ◽

1998 ◽

Vol 18 (1) ◽

pp. 353-360 ◽

Cited By ~ 24

Author(s):

Mitch R. McLean ◽

Brian C. Rymond

Keyword(s):

Transcriptional Repression ◽

Fusion Gene ◽

Mrna Splicing ◽

Tetratricopeptide Repeat ◽

Yeast Strains ◽

Wide Range ◽

U1 Snrna ◽

U1 Snrnp ◽

Snrnp Protein

ABSTRACT The U1 snRNP functions to nucleate spliceosome assembly on newly transcribed pre-mRNA. Saccharomyces cerevisiae is unusual among eukaryotes in the greatly extended length of its U1 snRNA and the apparent increased polypeptide complexity of the corresponding U1 snRNP. In this paper, we report the identification of a novel U1 snRNP protein, Prp42p, with unexpected properties. Prp42p was identified by its surprising structural similarity to the essential U1 snRNP protein, Prp39p. Both Prp39p and Prp42p possess multiple copies of a variant tetratricopeptide repeat, an element implicated in a wide range of protein assembly events. Yeast strains depleted of Prp42p by transcriptional repression of a GAL1::PRP42fusion gene arrest for splicing prior to pre-mRNA 5′ splice site cleavage. Prp42p was not observed in a recent biochemical analysis of purified U1 snRNPs from S. cerevisiae (28). Nevertheless, antibodies directed against an epitope-tagged version of Prp42p specifically precipitate U1 snRNA from yeast extracts. Furthermore, Prp42p is required for U1 snRNP biogenesis, because yeast strains depleted of Prp42p formed incomplete U1 snRNPs that failed to produce stable complexes with pre-mRNA in vitro. The evidence shows that Prp39p and Prp42p are both required to configure the atypical yeast U1 snRNP into a structure compatible with its evolutionarily conserved role in pre-mRNA splicing.

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Localization of a Vitronectin Binding Region of Plasminogen Activator Inhibitor-1

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653876 ◽

1995 ◽

Vol 73 (05) ◽

pp. 829-834 ◽

Cited By ~ 11

Author(s):

Jaya Padmanabhan ◽

David C Sane

Keyword(s):

Binding Site ◽

Plasminogen Activator ◽

Inhibitory Activity ◽

Plasminogen Activator Inhibitor ◽

Structural Homology ◽

Binding Region ◽

Plasminogen Activator Inhibitor 1 ◽

Activator Inhibitor ◽

Pai 1

SummaryThe PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70,1-105,1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

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Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190613152030 ◽

2020 ◽

Vol 21 (2) ◽

pp. 117-130 ◽

Cited By ~ 1

Author(s):

Mohammad J. Hosen ◽

Mahmudul Hasan ◽

Sourav Chakraborty ◽

Ruhshan A. Abir ◽

Abdullah Zubaer ◽

...

Keyword(s):

Virtual Screening ◽

Binding Site ◽

Glucose Transporter ◽

Substrate Binding ◽

Mutation Screening ◽

Homology Model ◽

Connective Tissue Disorder ◽

Crystallographic Structure ◽

Substrate Binding Site

Objectives: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. Methods: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. Results and Discussion: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. Conclusion: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

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New Mechanistic Insight on the PIM-1 Kinase Inhibitor AZD1208 Using Multidrug Resistant Human Erythroleukemia Cell Lines and Molecular Docking Simulations

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190509121606 ◽

2019 ◽

Vol 19 (11) ◽

pp. 914-926 ◽

Cited By ~ 2

Author(s):

Maiara Bernardes Marques ◽

Michael González-Durruthy ◽

Bruna Félix da Silva Nornberg ◽

Bruno Rodrigues Oliveira ◽

Daniela Volcan Almeida ◽

...

Keyword(s):

Molecular Docking ◽

Binding Site ◽

Cell Lines ◽

Chemotherapeutic Agents ◽

Atp Binding ◽

Human Leukemia ◽

Atp Binding Site ◽

Mechanistic Insight ◽

Multiple Drugs

Background:PIM-1 is a kinase which has been related to the oncogenic processes like cell survival, proliferation, and multidrug resistance (MDR). This kinase is known for its ability to phosphorylate the main extrusion pump (ABCB1) related to the MDR phenotype.Objective:In the present work, we tested a new mechanistic insight on the AZD1208 (PIM-1 specific inhibitor) under interaction with chemotherapy agents such as Daunorubicin (DNR) and Vincristine (VCR).Materials and Methods:In order to verify a potential cytotoxic effect based on pharmacological synergism, two MDR cell lines were used: Lucena (resistant to VCR) and FEPS (resistant to DNR), both derived from the K562 non-MDR cell line, by MTT analyses. The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123. Furthermore, we performed a molecular docking simulation to delve into the molecular mechanism of PIM-1 alone, and combined with chemotherapeutic agents (VCR and DNR).Results:Our in vitro results have shown that AZD1208 alone decreases cell viability of MDR cells. However, co-exposure of AZD1208 and DNR or VCR reverses this effect. When we analyzed the ABCB1 activity AZD1208 alone was not able to affect the pump extrusion. Differently, co-exposure of AZD1208 and DNR or VCR impaired ABCB1 activity, which could be explained by compensatory expression of abcb1 or other extrusion pumps not analyzed here. Docking analysis showed that AZD1208 is capable of performing hydrophobic interactions with PIM-1 ATP- binding-site residues with stronger interaction-based negative free energy (FEB, kcal/mol) than the ATP itself, mimicking an ATP-competitive inhibitory pattern of interaction. On the same way, VCR and DNR may theoretically interact at the same biophysical environment of AZD1208 and also compete with ATP by the PIM-1 active site. These evidences suggest that AZD1208 may induce pharmacodynamic interaction with VCR and DNR, weakening its cytotoxic potential in the ATP-binding site from PIM-1 observed in the in vitro experiments.Conclusion:Finally, the current results could have a pre-clinical relevance potential in the rational polypharmacology strategies to prevent multiple-drugs resistance in human leukemia cancer therapy.

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Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein

Journal of Virology ◽

10.1128/jvi.74.5.2084-2093.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2084-2093 ◽

Cited By ~ 23

Author(s):

Joel Schaley ◽

Robert J. O'Connor ◽

Laura J. Taylor ◽

Dafna Bar-Sagi ◽

Patrick Hearing

Keyword(s):

Dna Binding ◽

Binding Site ◽

Transient Expression ◽

Binding Sites ◽

Promoter Region ◽

Fluorescent Protein ◽

Protein Accumulation ◽

Promoter Regions ◽

Single Binding Site

ABSTRACT The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

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Synthesis, Characterization, and Preliminary In Vitro Cytotoxic Evaluation of a Series of 2-Substituted Benzo [d] [1,3] Azoles

Molecules ◽

10.3390/molecules26092780 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2780

Author(s):

Ozvaldo Linares-Anaya ◽

Alcives Avila-Sorrosa ◽

Francisco Díaz-Cedillo ◽

Luis Ángel Gil-Ruiz ◽

José Correa-Basurto ◽

...

Keyword(s):

Binding Site ◽

In Silico ◽

Cytotoxic Effect ◽

Human Cancer ◽

Cytotoxic Activities ◽

Human Cancer Cell ◽

Inhibitory Effects ◽

Reference Drug ◽

Human Cancer Cell Lines

A series of benzo [d] [1,3] azoles 2-substituted with benzyl- and allyl-sulfanyl groups were synthesized, and their cytotoxic activities were in vitro evaluated against a panel of six human cancer cell lines. The results showed that compounds BTA-1 and BMZ-2 have the best inhibitory effects, compound BMZ-2 being comparable in some cases with the reference drug tamoxifen and exhibiting a low cytotoxic effect against healthy cells. In silico molecular coupling studies at the tamoxifen binding site of ERα and GPER receptors revealed affinity and the possible mode of interaction of both compounds BTA-1 and BMZ-2.

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Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the Escherichia coli translation machinery

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013587 ◽

2020 ◽

Vol 295 (38) ◽

pp. 13314-13325

Author(s):

Yanyu Zhu ◽

James C. Weisshaar ◽

Mainak Mustafi

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptides ◽

Binding Site ◽

Elongation Factor ◽

Microscopy Study ◽

Single Step ◽

Long Term Effects ◽

Bacterial Membranes

Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro. Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.

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Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6866 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6866-6875 ◽

Cited By ~ 20

Author(s):

D C Hagen ◽

L Bruhn ◽

C A Westby ◽

G F Sprague

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Site ◽

Dna Sequences ◽

Point Mutations ◽

Transcription Activation ◽

Specific Gene ◽

Binding Assays ◽

Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

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A nucleosome-positioning sequence is required for GCN4 to activate transcription in the absence of a TATA element.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.4256 ◽

1990 ◽

Vol 10 (8) ◽

pp. 4256-4265 ◽

Cited By ~ 24

Author(s):

C J Brandl ◽

K Struhl

Keyword(s):

Binding Site ◽

Transcriptional Activation ◽

Nucleosome Positioning ◽

Alternative Mechanism ◽

Tata Element ◽

Binding Factor ◽

Positioning Analysis ◽

Binding Sequence

In the gal-his3 hybrid promoter his3-GG1, the yeast upstream activator protein GCN4 stimulates transcription when bound at the position normally occupied by the TATA element. This TATA-independent activation by GCN4 requires two additional elements in the gal enhancer region that are distinct from those involved in normal galactose induction. Both additional elements appear to be functionally distinct from a classical TATA element because they cannot be replaced by the TFIID-binding sequence TATAAA. One of these elements, termed Q, is essential for GCN4-activated transcription and contains the sequence GTCAC CCG, which overlaps (but is distinct from) a GAL4 binding site. Surprisingly, relatively small increases in the distance between Q and the GCN4 binding site significantly reduce the level of transcription. The Q element specifically interacts with a yeast protein (Q-binding protein [QBP]) that may be equivalent to Y, a protein that binds at a sequence that forms a constraint to nucleosome positioning. Analysis of various deletion mutants indicates that the sequence requirements for binding by QBP in vitro are indistinguishable from those necessary for Q activity in vivo, strongly suggesting that QBP is required for the function of this TATA-independent promoter. These results support the view that transcriptional activation can occur by an alternative mechanism in which the TATA-binding factor TFIID either is not required or is not directly bound to DNA. In addition, they suggest a potential role of nucleosome positioning for the activity of a promoter.

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