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Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation (2015)

Taylor, J. A., Pastrana, C. L., Butterer, A., Pernstich, C., Gwynn, E. J., Sobott, F., Moreno-Herrero, F., Dillingham, M. S.

Oxford University Press

In: Nucleic Acids Research

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Publikationsdatum: 2015-01-24

Beschreibung: The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans . The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed.

Print ISSN: 0305-1048

Digitale ISSN: 1362-4962

Thema: Biologie

Publiziert von Oxford University Press

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A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy (2015)

Garcia-Sacristan, A., Moreno, M., Ariza-Mateos, A., Lopez-Camacho, E., Jaudenes, R. M., Vazquez, L., Gomez, J., Martin-Gago, J. A., Briones, C.

Oxford University Press

In: Nucleic Acids Research

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Publikationsdatum: 2015-01-10

Beschreibung: The 5' untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II–IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg 2+ -induced switch between two alternative conformations: from ‘open’, elongated morphologies at 0–2 mM Mg 2+ concentration to a ‘closed’, comma-shaped conformation at 4–6 mM Mg 2+ . This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4–6 mM Mg 2+ in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I–II)+(V–VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.

Print ISSN: 0305-1048

Digitale ISSN: 1362-4962

Thema: Biologie

Publiziert von Oxford University Press

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SEVA 2.0: an update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities (2015)

Martinez-Garcia, E., Aparicio, T., Goni-Moreno, A., Fraile, S., de Lorenzo, V.

Oxford University Press

In: Nucleic Acids Research

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Publikationsdatum: 2015-01-16

Beschreibung: The Standard European Vector Architecture 2.0 database (SEVA-DB 2.0, http://seva.cnb.csic.es ) is an improved and expanded version of the platform released in 2013 (doi: 10.1093/nar/gks1119) aimed at assisting the choice of optimal genetic tools for de-constructing and re-constructing complex prokaryotic phenotypes. By adopting simple compositional rules, the SEVA standard facilitates combinations of functional DNA segments that ease both the analysis and the engineering of diverse Gram-negative bacteria for fundamental or biotechnological purposes. The large number of users of the SEVA-DB during its first two years of existence has resulted in a valuable feedback that we have exploited for fixing DNA sequence errors, improving the nomenclature of the SEVA plasmids, expanding the vector collection, adding new features to the web interface and encouraging contributions of materials from the community of users. The SEVA platform is also adopting the Synthetic Biology Open Language (SBOL) for electronic-like description of the constructs available in the collection and their interfacing with genetic devices developed by other Synthetic Biology communities. We advocate the SEVA format as one interim asset for the ongoing transition of genetic design of microorganisms from being a trial-and-error endeavor to become an authentic engineering discipline.

Print ISSN: 0305-1048

Digitale ISSN: 1362-4962

Thema: Biologie

Publiziert von Oxford University Press

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In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs (2015)

Martinez de Alba, A. E., Moreno, A. B., Gabriel, M., Mallory, A. C., Christ, A., Bounon, R., Balzergue, S., Aubourg, S., Gautheret, D., Crespi, M. D., Vaucheret, H., Maizel, A.

Oxford University Press

In: Nucleic Acids Research

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Publikationsdatum: 2015-03-14

Beschreibung: Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5' cap structure and their subsequent 5' to 3' degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs.

Print ISSN: 0305-1048

Digitale ISSN: 1362-4962

Thema: Biologie

Publiziert von Oxford University Press

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