共查询到20条相似文献,搜索用时 15 毫秒
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《Journal of molecular biology》2022,434(18):167776
The Sm protein Hfq chaperones small non-coding RNAs (sRNAs) in bacteria, facilitating sRNA regulation of target mRNAs. Hfq acts in part by remodeling the sRNA and mRNA structures, yet the basis for this remodeling activity is not understood. To understand how Hfq remodels RNA, we used single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes in OxyS sRNA upon Hfq binding. The results show that E. coli Hfq first compacts OxyS, bringing its 5′ and 3 ends together. Next, Hfq destabilizes an internal stem-loop in OxyS, allowing the RNA to adopt a more open conformation that is stabilized by a conserved arginine on the rim of Hfq. The frequency of transitions between compact and open conformations depend on interactions with Hfqs flexible C-terminal domain (CTD), being more rapid when the CTD is deleted, and slower when OxyS is bound to Caulobacter crescentus Hfq, which has a shorter and more stable CTD than E. coli Hfq. We propose that the CTDs gate transitions between OxyS conformations that are stabilized by interaction with one or more arginines. These results suggest a general model for how basic residues and intrinsically disordered regions of RNA chaperones act together to refold RNA. 相似文献
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Paola Turina 《Journal of bioenergetics and biomembranes》2000,32(4):373-381
F1F0-ATPase complexes undergo several changes in their tertiary and quaternary structureduring their functioning. As a possible way to detect some of these different conformationsduring their activity, an environment-sensitive fluorescence probe was bound to cysteineresidues, introduced by site-directed mutagenesis, in the subunit of the Escherichia colienzyme. Fluorescence changes and ATP hydrolysis rates were compared under variousconditions in F1 and in reconstituted F1F0. The results are discussed in terms of possible modes ofoperation of the ATP synthases. 相似文献
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Arluison V Derreumaux P Allemand F Folichon M Hajnsdorf E Régnier P 《Journal of molecular biology》2002,320(4):705-712
The Hfq polypeptide of Escherichia coli is a nucleic acid-binding protein involved in the expression of many proteins. Derivation of its three-dimensional structure is important for our understanding of its role in gene regulation at the molecular level. In this study, we combined computational and biophysical analysis to derive a possible structure for Hfq. As a first step towards determining the structure, we searched for possible sequence-structure compatibility, using secondary structure prediction and protein domain and fold-recognition methods available on the WEB. One fold, essentially beta sheet in character, the Sm motif of small nuclear ribonucleoproteins, even though it initially fell well below the confidence thresholds, was proposed and further validated by a series of biophysical and biochemical studies. The Hfq hexamer structure was modelled on the human Sm D3B structure using optimised sequence alignments and molecular mechanics methods. This structure accounts for the physico-chemical properties of Hfq and highlights amino acid residues that could interact with RNA. 相似文献
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The essential minichromosome maintenance (Mcm) proteins Mcm2 through Mcm7 likely comprise the replicative helicase in eukaryotes. In addition to Mcm2-7, other subcomplexes, including one comprising Mcm4, Mcm6, and Mcm7, unwind DNA. Using Mcm4/6/7 as a tool, we reveal a role for nucleotide binding by Saccharomyces cerevisiae Mcm2 in modulating DNA binding by Mcm complexes. Previous studies have shown that Mcm2 inhibits DNA unwinding by Mcm4/6/7. Here, we show that interaction of Mcm2 and Mcm4/6/7 is not sufficient for inhibition; rather, Mcm2 requires nucleotides for its regulatory role. An Mcm2 mutant that is defective for ATP hydrolysis (K549A), as well as ATP analogues, was used to show that ADP binding by Mcm2 is required to inhibit DNA binding and unwinding by Mcm4/6/7. This Mcm2-mediated regulation of Mcm4/6/7 is independent of Mcm3/5. Furthermore, the importance of ATP hydrolysis by Mcm2 to the regulation of the native complex was apparent from the altered DNA binding properties of Mcm2KA-7. Moreover, together with the finding that Mcm2K549A does not support yeast viability, these results indicate that the nucleotide-bound state of Mcm2 is critical in regulating the activities of Mcm4/6/7 and Mcm2-7 complexes. 相似文献
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Structural sites of RNA synthesis in Escherichia coli 总被引:6,自引:0,他引:6
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Beich-Frandsen M Vecerek B Konarev PV Sjöblom B Kloiber K Hämmerle H Rajkowitsch L Miles AJ Kontaxis G Wallace BA Svergun DI Konrat R Bläsi U Djinovic-Carugo K 《Nucleic acids research》2011,39(11):4900-4915
The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec). 相似文献
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Bojer MS Jakobsen H Struve C Krogfelt KA Løbner-Olesen A 《Microbes and infection / Institut Pasteur》2012,14(12):1034-1039
Escherichia coli is an important agent of Gram-negative bacterial infections worldwide, being one of the leading causes of diarrhoea and urinary tract infections. Strategies to understand pathogenesis and develop therapeutic compounds include the use of the nematode Caenorhabditis elegans as a model for virulence characterization and screening for novel antimicrobial entities. Several E. coli human pathotypes are also pathogenic towards C. elegans, and we show here that lack of the RNA chaperone Hfq significantly reduces pathogenicity of VTEC, EAEC, and UPEC in the nematode model. Thus, Hfq is intrinsically essential to pathogenic E. coli for survival and virulence exerted in the C. elegans host. 相似文献