Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site.

ABSTRACT: Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5' UTR of the HIV genomic RNA (vRNA) and a host primer tRNA^Lys₃ Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription.

SUBMITTER: Coey A

PROVIDER: S-EPMC5066621 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site.

Coey Aaron A Larsen Kevin K Puglisi Joseph D JD Viani Puglisi Elisabetta E

RNA (New York, N.Y.) 20160909 11

Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5' UTR of the HIV genomic RNA (vRNA) and a host primer tRNA<sup>Lys</sup><sub>3</sub> Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activati ...[more]

PMID: 27613581

Similar Datasets

Conserved structures formed by heterogeneous RNA sequences drive silencing of an inflammation responsive post-transcriptional operon.

Project description:RNA-protein interactions with physiological outcomes usually rely on conserved sequences within the RNA element. By contrast, activity of the diverse gamma-interferon-activated inhibitor of translation (GAIT)-elements relies on the conserved RNA folding motifs rather than the conserved sequence motifs. These elements drive the translational silencing of a group of chemokine (CC/CXC) and chemokine receptor (CCR) mRNAs, thereby helping to resolve physiological inflammation. Despite sequence dissimilarity, these RNA elements adopt common secondary structures (as revealed by 2D-1H NMR spectroscopy), providing a basis for their interaction with the RNA-binding GAIT complex. However, many of these elements (e.g. those derived from CCL22, CXCL13, CCR4 and ceruloplasmin (Cp) mRNAs) have substantially different affinities for GAIT complex binding. Toeprinting analysis shows that different positions within the overall conserved GAIT element structure contribute to differential affinities of the GAIT protein complex towards the elements. Thus, heterogeneity of GAIT elements may provide hierarchical fine-tuning of the resolution of inflammation.

| S-EPMC5727460 | biostudies-literature

Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA.

Project description:Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5'-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5'-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5'-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5'-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5'-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription.

| S-EPMC4668388 | biostudies-literature

A novel translational control mechanism involving RNA structures within coding sequences

Project description:The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site, second, they are directly bound by Dhh1 with a specific binding distribution and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.

2016-11-04 | GSE87888 | GEO

Origin-dependent initiation of DNA replication within telomeric sequences.

Project description:Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.

| S-EPMC2811021 | biostudies-literature

Structures illustrate step-by-step mitochondrial transcription initiation.

Project description:Transcription initiation is a key regulatory step in gene expression during which RNA polymerase (RNAP) initiates RNA synthesis de novo, and the synthesized RNA at a specific length triggers the transition to the elongation phase. Mitochondria recruit a single-subunit RNAP and one or two auxiliary factors to initiate transcription. Previous studies have revealed the molecular architectures of yeast1 and human2 mitochondrial RNAP initiation complexes (ICs). Here we provide a comprehensive, stepwise mechanism of transcription initiation by solving high-resolution cryogenic electron microscopy (cryo-EM) structures of yeast mitochondrial RNAP and the transcription factor Mtf1 catalysing two- to eight-nucleotide RNA synthesis at single-nucleotide addition steps. The growing RNA-DNA is accommodated in the polymerase cleft by template scrunching and non-template reorganization, creating stressed intermediates. During early initiation, non-template strand scrunching and unscrunching destabilize the short two- and three-nucleotide RNAs, triggering abortive synthesis. Subsequently, the non-template reorganizes into a base-stacked staircase-like structure supporting processive five- to eight-nucleotide RNA synthesis. The expanded non-template staircase and highly scrunched template in IC8 destabilize the promoter interactions with Mtf1 to facilitate initiation bubble collapse and promoter escape for the transition from initiation to the elongation complex (EC). The series of transcription initiation steps, each guided by the interplay of multiple structural components, reveal a finely tuned mechanism for potential regulatory control.

| S-EPMC10600007 | biostudies-literature

Advances in understanding the initiation of HIV-1 reverse transcription.

Project description:Many viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Human Immunodeficiency Virus (HIV), use RNA as their genetic material. How viruses harness RNA structure and RNA-protein interactions to control their replication remains obscure. Recent advances in the characterization of HIV-1 reverse transcriptase, the enzyme that converts its single-stranded RNA genome into a double-stranded DNA copy, reveal how the reverse transcription complex evolves during initiation. Here we highlight these advances in HIV-1 structural biology and discuss how they are furthering our understanding of HIV and related ribonucleoprotein complexes implicated in viral disease.

| S-EPMC9973426 | biostudies-literature

The phage-type PclpP-53 plastid promoter comprises sequences downstream of the transcription initiation site.

Project description:The existence of a phage-type plastid transcription machinery (NEP), related to the mitochondrial RNA polymerase, has been recognized only recently. Here we report the cis sequences required for transcription initiation by the phage-type enzyme. The promoter chosen for the study, PclpP-53, is well expressed in tobacco leaves, unlike most NEP promoters. Promoter definition was carried out in vivo , in transplastomic tobacco plants expressing a uidA reporter gene from PclpP-53 promoter derivatives. We report here that sequences from -5 to +25 (relative to the transcription initiation site) are sufficient to support specific transcription initiation. Requirement of sequences downstream of the transcription initiation site contrasts with mitochondrial promoters, which have conserved sequences predominantly upstream. The promoter defined here is conserved in liverworts and conifers, indicating that the phage-type transcription machinery appeared in plastids early on during the evolution of land plants. The PclpP-53 promoter sequences are present in rice but do not function, suggesting that PclpP-53 recognition specificity is absent in some monocots.

| S-EPMC147934 | biostudies-other

A Region of Bdp1 Necessary for Transcription Initiation That Is Located within the RNA Polymerase III Active Site Cleft.

Project description:The RNA polymerase III (Pol III)-specific transcription factor Bdp1 is crucial to Pol III recruitment and promoter opening in transcription initiation, yet structural information is sparse. To examine its protein-binding targets within the preinitiation complex at the residue level, photoreactive amino acids were introduced into Saccharomyces cerevisiae Bdp1. Mutations within the highly conserved SANT domain cross-linked to the transcription factor IIB (TFIIB)-related transcription factor Brf1, consistent with the findings of previous studies. In addition, we identified an essential N-terminal region that cross-linked with the Pol III catalytic subunit C128 as well as Brf1. Closer examination revealed that this region interacted with the C128 N-terminal region, the N-terminal half of Brf1, and the C-terminal domain of the C37 subunit, together positioning this region within the active site cleft of the preinitiation complex. With our functional data, our analyses identified an essential region of Bdp1 that is positioned within the active site cleft of Pol III and necessary for transcription initiation.

| S-EPMC4508317 | biostudies-literature

Structures of transcription pre-initiation complex with TFIIH and Mediator.

Project description:For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC-core Mediator complex at nominal resolutions of 4.7?Å and 5.8?Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the 'E-bridge' helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC-core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II.

| S-EPMC6078178 | biostudies-literature

Structures and mechanism of transcription initiation by bacterial ECF factors.

Project description:Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.

| S-EPMC6648896 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data