scholarly journals Structural mechanism for HIV-1 TAR loop recognition by Tat and the super elongation complex

2018 ◽  
Vol 115 (51) ◽  
pp. 12973-12978 ◽  
Author(s):  
Ursula Schulze-Gahmen ◽  
James H. Hurley
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Affinity ◽  
Electrostatic Interactions ◽  
Mutational Analysis ◽  
Side Chains ◽  
Elongation Complex ◽  
Pol Ii ◽  
Structural Mechanism ◽  
Super Elongation Complex ◽  
Hiv 1

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key regulatory step in human immunodeficiency virus-1 (HIV-1) transcription and thus in the reversal of HIV latency. By binding to the nascent transactivating response region (TAR) RNA, HIV-1 Tat recruits the human super elongation complex (SEC) to the promoter and releases paused Pol II. Structural studies of TAR interactions have been largely focused on interactions between the TAR bulge and the arginine-rich motif (ARM) of Tat. Here, the crystal structure of the TAR loop in complex with Tat and the SEC core was determined at a 3.5-Å resolution. The bound TAR loop is stabilized by cross-loop hydrogen bonds. It makes structure-specific contacts with the side chains of the Cyclin T1 Tat-TAR recognition motif (TRM) and the zinc-coordinating loop of Tat. The TAR loop phosphate backbone forms electrostatic and VDW interactions with positively charged side chains of the CycT1 TRM. Mutational analysis showed that these interactions contribute importantly to binding affinity. The Tat ARM was present in the crystallized construct; however, it was not visualized in the electron density, and the TAR bulge was not formed in the RNA construct used in crystallization. Binding assays showed that TAR bulge-Tat ARM interactions contribute less to TAR binding affinity than TAR loop interactions with the CycT1 TRM and Tat core. Thus, the TAR loop evolved to make high-affinity interactions with the TRM while Tat has three roles: scaffolding and stabilizing the TRM, making specific interactions through its zinc-coordinating loop, and making electrostatic interactions through its ARM.

scholarly journals DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii287-iii287
Author(s):  
Hiroaki Katagi ◽  
Nozomu Takata ◽  
Yuki Aoi ◽  
Yongzhan Zhang ◽  
Emily J Rendleman ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Transcriptional Elongation ◽  
Pontine Glioma ◽  
Elongation Complex ◽  
Pol Ii ◽  
Repair Genes ◽  
Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

Allosteric transcription stimulation by RNA polymerase II super elongation complex

2021 ◽  
Author(s):  
Ying Chen ◽  
Seychelle M. Vos ◽  
Christian Dienemann ◽  
Momchil Ninov ◽  
Henning Urlaub ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Complex ◽  
Super Elongation Complex

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

scholarly journals The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ursula Schulze-Gahmen ◽  
Heather Upton ◽  
Andrew Birnberg ◽  
Katherine Bao ◽  
Seemay Chou ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Regulatory Proteins ◽  
Tat Protein ◽  
Elongation Complex ◽  
Cyclin T1 ◽  
Subunit Interface ◽  
Hiv Tat ◽  
Gene Transcripts ◽  
Hiv 1

Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS.

scholarly journals A Minor Subset of Super Elongation Complexes Plays a Predominant Role in Reversing HIV-1 Latency

2016 ◽  
Vol 36 (7) ◽  
pp. 1194-1205 ◽  
Author(s):  
Zichong Li ◽  
Huasong Lu ◽  
Qiang Zhou
Keyword(s):  
Rna Polymerase Ii ◽  
Cellular Level ◽  
Transcriptional Elongation ◽  
Predominant Role ◽  
Drug Induced ◽  
Pol Ii ◽  
Knock Out ◽  
Future Design ◽  
Latent Hiv ◽  
Hiv 1

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key rate-limiting step in HIV-1 transcription and latency reversal. The viral Tat protein recruits human super elongation complexes (SECs) to paused Pol II to overcome this restriction. Despite the recent progress in understanding the functions of different subsets of SECs in controlling cellular and Tat-activated HIV transcription, little is known about the SEC subtypes that help reverse viral latency in CD4+T cells. Here, we used the CRISPR-Cas9 genome-editing tool to knock out the gene encoding the SEC subunit ELL2, AFF1, or AFF4 in Jurkat/2D10 cells, a well-characterized HIV-1 latency model. Depletion of these proteins drastically reduced spontaneous and drug-induced latency reversal by suppressing HIV-1 transcriptional elongation. Surprisingly, a low-abundance subset of SECs containing ELL2 and AFF1 was found to play a predominant role in cooperating with Tat to reverse latency. By increasing the cellular level/activity of these Tat-friendly SECs, we could potently activate latent HIV-1 without using any drugs. These results implicate the ELL2/AFF1-SECs as an important target in the future design of a combinatorial therapeutic approach to purge latent HIV-1.

scholarly journals The Role of RNA Polymerase II Elongation Control in HIV-1 Gene Expression, Replication, and Latency

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Kyle A. Nilson ◽  
David H. Price
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Health Concern ◽  
Pol Ii ◽  
Positive Transcription Elongation Factor ◽  
Elongation Control ◽  
Hiv 1 ◽  
Latent Phase

HIV-1 usurps the RNA polymerase II elongation control machinery to regulate the expression of its genome during lytic and latent viral stages. After integration into the host genome, the HIV promoter within the long terminal repeat (LTR) is subject to potent downregulation in a postinitiation step of transcription. Once produced, the viral protein Tat commandeers the positive transcription elongation factor, P-TEFb, and brings it to the engaged RNA polymerase II (Pol II), leading to the production of viral proteins and genomic RNA. HIV can also enter a latent phase during which factors that regulate Pol II elongation may play a role in keeping the virus silent. HIV, the causative agent of AIDS, is a worldwide health concern. It is hoped that knowledge of the mechanisms regulating the expression of the HIV genome will lead to treatments and ultimately a cure.

scholarly journals Combining Molecular Dynamic Information and an Aspherical-Atom Data Bank in the Evaluation of the Electrostatic Interaction Energy in Multimeric Protein-Ligand Complex: A Case Study for HIV-1 Protease

2021 ◽  
Author(s):  
Prashant Kumar ◽  
Paulina Dominiak
Keyword(s):  
Interaction Energy ◽  
Molecular Dynamic ◽  
Binding Affinity ◽  
Electrostatic Interaction ◽  
Electrostatic Interactions ◽  
Data Bank ◽  
Scoring Functions ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Hiv 1

<div> <div> <div> <p>Computational analysis of protein-ligand interactions is of crucial importance for drug discovery. Assessment of ligand binding energy allows us to have a glimpse on the potential of a small organic molecule to be a ligand to the binding site of a protein target. Available scoring functions such as in docking programs, we could say that they all rely on equations that sum each type of protein-ligand interactions to model the binding affinity. Most of the scoring functions consider electrostatic interactions involving the protein and the ligand. Electrostatic interactions contribute one of the most important part of total interaction energies between macromolecules, unlike dispersion forces they are highly directional and therefore dominate the nature of molecular packing in crystals and in biological complexes and contribute significantly to differences in inhibition strength among related enzyme inhibitors. In this paper, complexes of HIV-1 protease with inhibitor molecules (JE-2147 and Darunavir) have been analysed using charge densities from a transferable aspherical-atom data bank. Moreover, we analyse the electrostatic interaction energy for an ensemble of structures using molecular dynamic simulation to highlight the main features related to the importance of this interaction for binding affinity. </p> </div> </div> </div>

scholarly journals Combining Molecular Dynamic Information and an Aspherical-Atom Data Bank in the Evaluation of the Electrostatic Interaction Energy in Multimeric Protein-Ligand Complex: A Case Study for HIV-1 Protease

2021 ◽  
Author(s):  
Prashant Kumar ◽  
Paulina Dominiak
Keyword(s):  
Interaction Energy ◽  
Molecular Dynamic ◽  
Binding Affinity ◽  
Electrostatic Interaction ◽  
Electrostatic Interactions ◽  
Data Bank ◽  
Scoring Functions ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Hiv 1

<div> <div> <div> <p>Computational analysis of protein-ligand interactions is of crucial importance for drug discovery. Assessment of ligand binding energy allows us to have a glimpse on the potential of a small organic molecule to be a ligand to the binding site of a protein target. Available scoring functions such as in docking programs, we could say that they all rely on equations that sum each type of protein-ligand interactions to model the binding affinity. Most of the scoring functions consider electrostatic interactions involving the protein and the ligand. Electrostatic interactions contribute one of the most important part of total interaction energies between macromolecules, unlike dispersion forces they are highly directional and therefore dominate the nature of molecular packing in crystals and in biological complexes and contribute significantly to differences in inhibition strength among related enzyme inhibitors. In this paper, complexes of HIV-1 protease with inhibitor molecules (JE-2147 and Darunavir) have been analysed using charge densities from a transferable aspherical-atom data bank. Moreover, we analyse the electrostatic interaction energy for an ensemble of structures using molecular dynamic simulation to highlight the main features related to the importance of this interaction for binding affinity. </p> </div> </div> </div>

scholarly journals Super elongation complex as a targetable dependency in diffuse midline glioma

2020 ◽  
Author(s):  
Nathan A. Dahl ◽  
Etienne Danis ◽  
Ilango Balakrishnan ◽  
Dong Wang ◽  
Angela Pierce ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Complex ◽  
Curative Therapy ◽  
Super Elongation Complex ◽  
Small Molecule Inhibition ◽  
Promising Therapeutic Approach ◽  
Clonogenic Potential ◽  
Diffuse Intrinsic Pontine Gliomas ◽  
Shrna Screen

AbstractMutations in the histone 3 gene (H3K27M) are the eponymous drivers in diffuse intrinsic pontine gliomas (DIPGs) and other diffuse midline gliomas (DMGs), aggressive pediatric brain cancers for which no curative therapy currently exists. The salient molecular consequence of these recurrent oncohistones is a global loss of repressive H3K27me3 residues and broad epigenetic dysregulation. In order to identify specific, therapeutically targetable epigenetic dependencies within this disease context, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cultures. This approach identified AFF4, the scaffold protein of the super elongation complex (SEC), as a previously-undescribed dependency in DMG. Interrogation of SEC function demonstrated a key role for maintaining DMG cell viability and clonogenic potential while promoting self-renewal of DMG tumor stem cells. Small-molecule inhibition of the SEC with the highly-specific, clinically relevant CDK9 inhibitors atuveciclib and AZD4573 restores regulatory RNA polymerase II pausing, promotes cellular differentiation, and leads to potent anti-tumor effect both in vitro and in patient-derived xenograft models. These studies present a biologic rationale for translational exploration of CDK9 inhibition as a promising therapeutic approach in a disease which currently has no effective medical therapies.

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