Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros











Base de dados
Intervalo de ano de publicação
1.
Proc Natl Acad Sci U S A ; 120(28): e2303849120, 2023 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-37406096

RESUMO

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)-consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA-and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.


Assuntos
Proteínas de Escherichia coli , RNA Bacteriano , RNA Bacteriano/metabolismo , Cinética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Transcrição Gênica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
2.
Proc Natl Acad Sci U S A ; 119(5)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35078937

RESUMO

Hsp70 and Hsp90 chaperones provide protein quality control to the cytoplasm, endoplasmic reticulum (ER), and mitochondria. Hsp90 activity is often enhanced by cochaperones that drive conformational changes needed for ATP-dependent closure and capture of client proteins. Hsp90 activity is also enhanced when working with Hsp70, but, in this case, the underlying mechanistic explanation is poorly understood. Here we examine the ER-specific Hsp70/Hsp90 paralogs (BiP/Grp94) and discover that BiP itself acts as a cochaperone that accelerates Grp94 closure. The BiP nucleotide binding domain, which interacts with the Grp94 middle domain, is responsible for Grp94 closure acceleration. A client protein initiates a coordinated progression of steps for the BiP/Grp94 system, in which client binding to BiP causes a conformational change that enables BiP to bind to Grp94 and accelerate its ATP-dependent closure. Single-molecule fluorescence resonance energy transfer measurements show that BiP accelerates Grp94 closure by stabilizing a high-energy conformational intermediate that otherwise acts as an energetic barrier to closure. These findings provide an explanation for enhanced activity of BiP and Grp94 when working as a pair, and demonstrate the importance of a high-energy conformational state in controlling the timing of the Grp94 conformational cycle. Given the high conservation of the Hsp70/Hsp90 system, other Hsp70s may also serve dual roles as both chaperones and closure-accelerating cochaperones to their Hsp90 counterparts.


Assuntos
Chaperona BiP do Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Camundongos , Dobramento de Proteína
3.
J Mol Biol ; 431(17): 3312-3323, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31202885

RESUMO

The Hsp90 family of chaperones requires ATP-driven cycling to perform their function. The presence of two bound ATP molecules is known to favor a closed conformation of the Hsp90 dimer. However, the structural and mechanistic consequences of subsequent ATP hydrolysis are poorly understood. Using single-molecule FRET, we discover novel dynamic behavior in the closed state of Grp94, the Hsp90 family member resident in the endoplasmic reticulum. Under ATP turnover conditions, Grp94 populates two distinct closed states, a relatively static ATP/ATP closed state that adopts one conformation, and a dynamic ATP/ADP closed state that can adopt two conformations. We constructed a Grp94 heterodimer with one arm that is catalytically dead, to extend the lifetime of the ATP/ADP state by preventing hydrolysis of the second ATP. This construct shows prolonged periods of cycling between two closed conformations. Our results enable a quantitative description of how ATP hydrolysis influences Grp94, where sequential ATP hydrolysis steps allow Grp94 to transition between closed states with different dynamic and structural properties. This stepwise transitioning of Grp94's dynamic properties may provide a mechanism to propagate structural changes to a bound client protein.


Assuntos
Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas
4.
Elife ; 72018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29932423

RESUMO

Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.


Assuntos
Éxons , Íntrons , Precursores de RNA/genética , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Trifosfato de Adenosina/metabolismo , Biologia Computacional/métodos , Células HEK293 , Humanos , Precursores de RNA/química , Precursores de RNA/metabolismo , Sítios de Splice de RNA , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Spliceossomos/ultraestrutura
5.
Nat Struct Mol Biol ; 24(3): 309-315, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28191892

RESUMO

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.


Assuntos
Replicação do DNA , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/metabolismo , Origem de Replicação , Trifosfato de Adenosina/metabolismo , DNA Fúngico/metabolismo , Hidrólise , Modelos Biológicos , Conformação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo
6.
Elife ; 52016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27244240

RESUMO

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation.


Assuntos
Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Spliceossomos/genética , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Simulação de Dinâmica Molecular , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Spliceossomos/metabolismo
7.
Science ; 331(6022): 1289-95, 2011 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-21393538

RESUMO

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.


Assuntos
Precursores de RNA/metabolismo , Splicing de RNA , RNA Fúngico/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Spliceossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes , Íntrons , Cinética , Microscopia de Fluorescência , Ligação Proteica , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA