Nature Structural & Molecular Biology

Efficiency and specificity in microRNA biogenesis.

Publication Date: 2012 May 13 PMID: 22580560
Authors: Barad, O. - Mann, M. - Chapnik, E. - Shenoy, A. - Blelloch, R. - Barkai, N. - Hornstein, E.
Journal: Nat Struct Mol Biol

Primary microRNA cleavage by the Drosha-Dgcr8 'Microprocessor' complex is critical for microRNA biogenesis. Yet, the Microprocessor may also cleave other nuclear RNAs in a nonspecific manner. We studied Microprocessor function using mathematical modeling and experiments in mouse and human tissues. We found that the autoregulatory feedback on Microprocessor expression is instrumental for balancing the efficiency and specificity of its activity by effectively tuning Microprocessor levels to those of its pri-miRNA substrate.

post to: CiteULike

A locally closed conformation of a bacterial pentameric proton-gated ion channel.

Publication Date: 2012 May 13 PMID: 22580559
Authors: Prevost, M. S. - Sauguet, L. - Nury, H. - Van Renterghem, C. - Huon, C. - Poitevin, F. - Baaden, M. - Delarue, M. - Corringer, P. J.
Journal: Nat Struct Mol Biol

Pentameric ligand-gated ion channels mediate signal transduction through conformational transitions between closed-pore and open-pore states. To stabilize a closed conformation of GLIC, a bacterial proton-gated homolog from Gloeobacter violaceus whose open structure is known, we separately generated either four cross-links or two single mutations. We found all six mutants to be in the same 'locally closed' conformation using X-ray crystallography, sharing most of the features of the open form but showing a locally closed pore as a result of a concerted bending of all of its M2 helices. The mutants adopt several variant conformations of the M2-M3 loop, and in all cases an interacting lipid that is observed in the open form disappears. A single cross-linked mutant is functional, according to electrophysiology, and the locally closed structure of this mutant indicates that it has an increased flexibility. Further cross-linking, accessibility and molecular dynamics data suggest that the locally closed form is a functionally relevant conformation that occurs during allosteric gating transitions.

post to: CiteULike

A rule of seven in Watson-Crick base-pairing of mismatched sequences.

Publication Date: 2012 May 13 PMID: 22580558
Authors: Cisse, I. I. - Kim, H. - Ha, T.
Journal: Nat Struct Mol Biol

Sequence recognition through base-pairing is essential for DNA repair and gene regulation, but the basic rules governing this process remain elusive. In particular, the kinetics of annealing between two imperfectly matched strands is not well characterized, despite its potential importance in nucleic acid-based biotechnologies and gene silencing. Here we use single-molecule fluorescence to visualize the multiple annealing and melting reactions of two untethered strands inside a porous vesicle, allowing us to precisely quantify the annealing and melting rates. The data as a function of mismatch position suggest that seven contiguous base pairs are needed for rapid annealing of DNA and RNA. This phenomenological rule of seven may underlie the requirement for seven nucleotides of complementarity to seed gene silencing by small noncoding RNA and may help guide performance improvement in DNA- and RNA-based bio- and nanotechnologies, in which off-target effects can be detrimental.

post to: CiteULike

A glutamate switch controls voltage-sensitive phosphatase function.

Publication Date: 2012 May 6 PMID: 22562138
Authors: Liu, L. - Kohout, S. C. - Xu, Q. - Muller, S. - Kimberlin, C. R. - Isacoff, E. Y. - Minor, D. L. Jr
Journal: Nat Struct Mol Biol

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) couples a voltage-sensing domain (VSD) to a lipid phosphatase that is similar to the tumor suppressor PTEN. How the VSD controls enzyme function has been unclear. Here, we present high-resolution crystal structures of the Ci-VSP enzymatic domain that reveal conformational changes in a crucial loop, termed the 'gating loop', that controls access to the active site by a mechanism in which residue Glu411 directly competes with substrate. Structure-based mutations that restrict gating loop conformation impair catalytic function and demonstrate that Glu411 also contributes to substrate selectivity. Structure-guided mutations further define an interaction between the gating loop and linker that connects the phosphatase to the VSD for voltage control of enzyme activity. Together, the data suggest that functional coupling between the gating loop and the linker forms the heart of the regulatory mechanism that controls voltage-dependent enzyme activation.

post to: CiteULike

Structural basis for cisplatin DNA damage tolerance by human polymerase eta during cancer chemotherapy.

Publication Date: 2012 May 6 PMID: 22562137
Authors: Ummat, A. - Rechkoblit, O. - Jain, R. - Roy Choudhury, J. - Johnson, R. E. - Silverstein, T. D. - Buku, A. - Lone, S. - Prakash, L. - Prakash, S. - Aggarwal, A. K.
Journal: Nat Struct Mol Biol

A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase eta (Pol-eta) is a crucial polymerase that allows cancer cells to cope with the cisplatin-DNA adducts that are formed during chemotherapy. We present here a structure of human Pol-eta inserting deoxycytidine triphosphate (dCTP) opposite a cisplatin intrastrand cross-link (PtGpG). We show that the specificity of human Pol-eta for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG-dCTP base pair and to accommodate the lesion without steric hindrance. This specificity is augmented by the residues Gln38 and Ser62, which interact with PtGpG, and Arg61, which interacts with the incoming dCTP. Collectively, the structure provides a basis for understanding how Pol-eta in human cells can tolerate the DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy.

post to: CiteULike

Real-time assembly landscape of bacterial 30S translation initiation complex.

Publication Date: 2012 May 6 PMID: 22562136
Authors: Milon, P. - Maracci, C. - Filonava, L. - Gualerzi, C. O. - Rodnina, M. V.
Journal: Nat Struct Mol Biol

Initiation factors guide the ribosome in the selection of mRNA and translational reading frame. We determined the kinetically favored assembly pathway of the 30S preinitiation complex (30S PIC), an early intermediate in 30S initiation complex formation in Escherichia coli. IF3 and IF2 are the first factors to arrive, forming an unstable 30S-IF2-IF3 complex. Subsequently, IF1 joins and locks the factors in a kinetically stable 30S PIC to which fMet-tRNA(fMet) is recruited. Binding of mRNA is independent of initiation factors and can take place at any time during 30S PIC assembly, depending on the cellular concentration of the mRNA and the structural determinants at the ribosome-binding site. The kinetic analysis shows both specific and cumulative effects of initiation factors as well as kinetic checkpoints of mRNA selection at the entry into translation.

post to: CiteULike

Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine.

Publication Date: 2012 May 6 PMID: 22562135
Authors: Glynn, S. E. - Nager, A. R. - Baker, T. A. - Sauer, R. T.
Journal: Nat Struct Mol Biol

In the Escherichia coli ClpXP protease, a hexameric ClpX ring couples ATP binding and hydrolysis to mechanical protein unfolding and translocation into the ClpP degradation chamber. Rigid-body packing between the small AAA+ domain of each ClpX subunit and the large AAA+ domain of its neighbor stabilizes the hexamer. By connecting the parts of each rigid-body unit with disulfide bonds or linkers, we created covalently closed rings that retained robust activity. A single-residue insertion in the hinge that connects the large and small AAA+ domains and forms part of the nucleotide-binding site uncoupled ATP hydrolysis from productive unfolding. We propose that ATP hydrolysis drives changes in the conformation of one hinge and its flanking domains and that the changes are propagated around the AAA+ ring through the topologically constrained set of rigid-body units and hinges to produce coupled ring motions that power substrate unfolding.

post to: CiteULike

Mad1 attachment.

Publication Date: 2012 PMID: 22551711
Authors: Montoya, M.
Journal: Nat Struct Mol Biol

post to: CiteULike

Un-arrested development.

Publication Date: 2012 PMID: 22551710
Authors: Finkelstein, J. M.
Journal: Nat Struct Mol Biol

post to: CiteULike

Understanding decoding.

Publication Date: 2012 PMID: 22551709
Authors: Heinrichs, A.
Journal: Nat Struct Mol Biol

post to: CiteULike

Small RNA-mediated DNA repair.

Publication Date: 2012 PMID: 22551708
Authors: Mason, S.
Journal: Nat Struct Mol Biol

post to: CiteULike

Dynein dynamics.

Publication Date: 2012 PMID: 22551707
Authors: Hook, P. - Vallee, R.
Journal: Nat Struct Mol Biol

Dyneins are the largest of the cytoskeletal motor proteins, and their mechanochemical behavior is complex. Recent high-resolution crystallographic structures have revealed new surprises regarding motor domain organization and new insights into how force and movement are achieved.

post to: CiteULike

Enforcing silencing: dynamic HP1 complexes in Neurospora.

Publication Date: 2012 PMID: 22551706
Authors: Wallrath, L. L. - Elgin, S. C.
Journal: Nat Struct Mol Biol

Analysis of the Neurospora crassa chromodomain protein CDP-2, a component of a newly characterized HP1-containing complex, reveals a second gene-silencing mechanism and provides insights into the dynamic nature of chromatin domains that possess shared components.

post to: CiteULike

The cryo-EM structure of the UPF-EJC complex shows UPF1 poised toward the RNA 3' end.

Publication Date: 2012 May PMID: 22522823
Authors: Melero, R. - Buchwald, G. - Castano, R. - Raabe, M. - Gil, D. - Lazaro, M. - Urlaub, H. - Conti, E. - Llorca, O.
Journal: Nat Struct Mol Biol

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that degrades aberrant mRNAs containing premature termination codons (PTCs). NMD is triggered upon the assembly of the UPF surveillance complex near a PTC. In humans, UPF assembly is prompted by the exon junction complex (EJC). We investigated the molecular architecture of the human UPF complex bound to the EJC by cryo-EM and using positional restraints from additional EM, MS and biochemical interaction data. The heptameric assembly is built around UPF2, a scaffold protein with a ring structure that closes around the CH domain of UPF1, keeping the helicase region in an accessible and unwinding-competent state. UPF2 also positions UPF3 to interact with the EJC. The geometry is such that this transient complex poises UPF1 to elicit helicase activity toward the 3' end of the mRNP.

post to: CiteULike

Rapid oligomer formation of human muscle acylphosphatase induced by heparan sulfate.

Publication Date: 2012 May PMID: 22522822
Authors: Motamedi-Shad, N. - Garfagnini, T. - Penco, A. - Relini, A. - Fogolari, F. - Corazza, A. - Esposito, G. - Bemporad, F. - Chiti, F.
Journal: Nat Struct Mol Biol

Many human diseases are caused by the conversion of proteins from their native state into amyloid fibrils that deposit in the extracellular space. Heparan sulfate, a component of the extracellular matrix, is universally associated with amyloid deposits and promotes fibril formation. The formation of cytotoxic prefibrillar oligomers is challenging to study because of its rapidity, transient appearance and the heterogeneity of species generated. The process is even more complex with agents such as heparan sulfate. Here we have used a stopped-flow device coupled to turbidometry detection to monitor the rapid conversion of human muscle acylphosphatase into oligomers with varying heparan sulfate and protein concentrations. We also analyzed mutants of the 15 basic amino acids of acylphosphatase, identifying the residues primarily involved in heparan sulfate-induced oligomerization of this protein and tracing the process with unprecedented molecular detail.

post to: CiteULike