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1.
Nat Methods ; 20(4): 523-535, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36973549

RESUMO

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas , Transferência Ressonante de Energia de Fluorescência/métodos , Reprodutibilidade dos Testes , Proteínas/química , Conformação Molecular , Laboratórios
2.
Biochem J ; 477(16): 2935-2947, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32706021

RESUMO

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the 'parental' DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.


Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , DNA Arqueal/metabolismo , DNA de Cadeia Simples/metabolismo , Methanobacteriaceae/enzimologia , Proteínas Arqueais/química , Proteínas Arqueais/genética , DNA Helicases/química , DNA Helicases/genética , DNA Arqueal/química , DNA Arqueal/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Methanobacteriaceae/genética , Conformação Proteica
3.
Biochem Soc Trans ; 47(1): 411-423, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30710057

RESUMO

In all domains of life, the regulation of transcription by DNA-dependent RNA polymerases (RNAPs) is achieved at the level of initiation to a large extent. Whereas bacterial promoters are recognized by a σ-factor bound to the RNAP, a complex set of transcription factors that recognize specific promoter elements is employed by archaeal and eukaryotic RNAPs. These initiation factors are of particular interest since the regulation of transcription critically relies on initiation rates and thus formation of pre-initiation complexes. The most conserved initiation factor is the TATA-binding protein (TBP), which is of crucial importance for all archaeal-eukaryotic transcription initiation complexes and the only factor required to achieve full rates of initiation in all three eukaryotic and the archaeal transcription systems. Recent structural, biochemical and genome-wide mapping data that focused on the archaeal and specialized RNAP I and III transcription system showed that the involvement and functional importance of TBP is divergent from the canonical role TBP plays in RNAP II transcription. Here, we review the role of TBP in the different transcription systems including a TBP-centric discussion of archaeal and eukaryotic initiation complexes. We furthermore highlight questions concerning the function of TBP that arise from these findings.


Assuntos
Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/metabolismo , Iniciação da Transcrição Genética , Proteínas Arqueais , RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular , Conformação Proteica
4.
Chembiochem ; 19(8): 780-783, 2018 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-29394002

RESUMO

Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single-molecule FRET measurements allow exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome, as a method for the site-specific coupling of organic dyes into native, non-recombinant mammalian proteins is lacking. We address this issue showing the site-specific engineering of fluorescent dyes into human proteins on the basis of bioorthogonal reactions. We show the applicability of the method to study functional and post-translationally modified proteins on the single-molecule level, among them the hitherto inaccessible human Argonaute 2.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/química , Cromatografia de Afinidade , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Proteínas/isolamento & purificação , Proteoma
5.
RNA ; 22(3): 383-96, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26769856

RESUMO

In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing.


Assuntos
Drosophila melanogaster/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Dimerização , Proteínas de Drosophila , Inativação Gênica , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência
6.
Methods ; 86: 10-8, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25912642

RESUMO

The transcriptional apparatus is one of the most complex cellular machineries and in order to fully appreciate the behavior of these protein-nucleic acid assemblies one has to understand the molecular details of the system. In addition to classical biochemical and structural studies, fluorescence-based techniques turned out as an important--and sometimes the critical--tool to obtain information about the molecular mechanisms of transcription. Fluorescence is not only a multi-modal parameter that can report on molecular interactions, environment and oligomerization status. Measured on the single-molecule level it also informs about the heterogeneity of the system and gives access to distances and distance changes in the molecular relevant nanometer regime. A pre-requisite for fluorescence-based measurements is the site-specific incorporation of one or multiple fluorescent dyes. In this respect, the archaeal transcription system is ideally suited as it is available in a fully recombinant form and thus allows for site-specific modification via sophisticated labeling schemes. The application of fluorescence based approaches to the archaeal transcription apparatus changed our understanding of the molecular mechanisms and dynamics that drive archaeal transcription and unraveled the architecture of transcriptional complexes not amenable to structural interrogation.


Assuntos
RNA Polimerases Dirigidas por DNA/química , Corantes Fluorescentes/química , Transcrição Gênica , Sequência de Aminoácidos , Archaea/enzimologia , Archaea/genética , RNA Polimerases Dirigidas por DNA/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Conformação Proteica
7.
Nat Commun ; 13(1): 3825, 2022 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-35780145

RESUMO

Human Argonaute 2 (hAgo2) constitutes the functional core of the RNA interference pathway. Guide RNAs direct hAgo2 to target mRNAs, which ultimately leads to hAgo2-mediated mRNA degradation or translational inhibition. Here, we combine site-specifically labeled hAgo2 with time-resolved single-molecule FRET measurements to monitor conformational states and dynamics of hAgo2 and hAgo2-RNA complexes in solution that remained elusive so far. We observe dynamic anchoring and release of the guide's 3'-end from the PAZ domain during the stepwise target loading process even with a fully complementary target. We find differences in structure and dynamic behavior between partially and fully paired canonical hAgo2-guide/target complexes and the miRNA processing complex formed by hAgo2 and pre-miRNA451. Furthermore, we detect a hitherto unknown conformation of hAgo2-guide/target complexes that poises them for target-directed miRNA degradation. Taken together, our results show how the conformational flexibility of hAgo2-RNA complexes determines function and the fate of the ribonucleoprotein particle.


Assuntos
Proteínas Argonautas , MicroRNAs , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação Molecular , Estabilidade de RNA , RNA Mensageiro
8.
Nat Commun ; 11(1): 2828, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504003

RESUMO

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.


Assuntos
DNA de Cadeia Simples/metabolismo , RNA Polimerase III/metabolismo , Imagem Individual de Molécula/métodos , Fator de Transcrição TFIIIB/metabolismo , Transcrição Gênica , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sondas Moleculares/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Estabilidade Proteica , RNA Polimerase III/química , Proteínas Recombinantes/metabolismo , Proteína de Ligação a TATA-Box/metabolismo
9.
J Mol Biol ; 431(20): 4116-4131, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31207238

RESUMO

The discovery of the archaeal domain of life is tightly connected to an in-depth analysis of the prokaryotic RNA world. In addition to Carl Woese's approach to use the sequence of the 16S rRNA gene as phylogenetic marker, the finding of Karl Stetter and Wolfram Zillig that archaeal RNA polymerases (RNAPs) were nothing like the bacterial RNAP but are more complex enzymes that resemble the eukaryotic RNAPII was one of the key findings supporting the idea that archaea constitute the third major branch on the tree of life. This breakthrough in transcriptional research 40years ago paved the way for in-depth studies of the transcription machinery in archaea. However, although the archaeal RNAP and the basal transcription factors that fine-tune the activity of the RNAP during the transcription cycle are long known, we still lack information concerning the architecture and dynamics of archaeal transcription complexes. In this context, single-molecule measurements were instrumental as they provided crucial insights into the process of transcription initiation, the architecture of the initiation complex and the dynamics of mobile elements of the RNAP. In this review, we discuss single-molecule approaches suitable to examine molecular mechanisms of transcription and highlight findings that shaped our understanding of the archaeal transcription apparatus. We furthermore explore the possibilities and challenges of next-generation single-molecule techniques, for example, super-resolution microscopy and single-molecule tracking, and ask whether these approaches will ultimately allow us to investigate archaeal transcription in vivo.


Assuntos
Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Imagem Individual de Molécula/métodos , Transcrição Gênica , Archaea/enzimologia , DNA Arqueal/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Transcrição/metabolismo
10.
Nat Commun ; 8(1): 130, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28743884

RESUMO

Initiation of gene transcription by RNA polymerase (Pol) III requires the activity of TFIIIB, a complex formed by Brf1 (or Brf2), TBP (TATA-binding protein), and Bdp1. TFIIIB is required for recruitment of Pol III and to promote the transition from a closed to an open Pol III pre-initiation complex, a process dependent on the activity of the Bdp1 subunit. Here, we present a crystal structure of a Brf2-TBP-Bdp1 complex bound to DNA at 2.7 Å resolution, integrated with single-molecule FRET analysis and in vitro biochemical assays. Our study provides a structural insight on how Bdp1 is assembled into TFIIIB complexes, reveals structural and functional similarities between Bdp1 and Pol II factors TFIIA and TFIIF, and unravels essential interactions with DNA and with the upstream factor SNAPc. Furthermore, our data support the idea of a concerted mechanism involving TFIIIB and RNA polymerase III subunits for the closed to open pre-initiation complex transition.Transcription initiation by RNA polymerase III requires TFIIIB, a complex formed by Brf1/Brf2, TBP and Bdp1. Here, the authors describe the crystal structure of a Brf2-TBP-Bdp1 complex bound to a DNA promoter and characterize the role of Bdp1 in TFIIIB assembly and pre-initiation complex formation.


Assuntos
RNA Polimerase III/metabolismo , Fator de Transcrição TFIIIB/metabolismo , Iniciação da Transcrição Genética , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIIB/química , Fator de Transcrição TFIIIB/genética
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