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1.
Structure of prokaryotic SECIS mRNA hairpin and its interaction with elongation factor SelB 总被引:9,自引:0,他引:9
In prokaryotes, the recoding of a UGA stop codon as a selenocysteine codon requires a special elongation factor (EF) SelB and a stem-loop structure within the mRNA called a selenocysteine insertion sequence (SECIS). Here, we used NMR spectroscopy to determine the solution structure of the SECIS mRNA hairpin and characterized its interaction with the mRNA-binding domain of SelB. Our structural and biochemical data identified the conserved structural features important for binding to EF SelB within different SECIS RNA sequences. In the free SECIS mRNA structure, conserved nucleotides are strongly exposed for recognition by SelB. Binding of the C-terminal domain of SelB stabilizes the RNA secondary structure. In the protein-RNA complex, a Watson-Crick loop base-pair leaves a GpU sequence accessible for SelB recognition. This GpU sequence at the tip of the capping tetraloop and a bulge uracil five Watson-Crick base-pairs apart from the GpU are essential for interaction with SelB. 相似文献
2.
The kinetics of the interaction of GTP and GDP with SelB, the specific translation factor for the incorporation of selenocysteine into proteins, have been investigated using the stopped-flow method. Useful signals were obtained using intrinsic (i.e. tryptophan) fluorescence, the fluorescence of methylanthraniloyl derivatives of nucleotides, or fluorescence resonance energy transfer from tryptophan to the methylanthraniloyl group. The affinities of SelB for GTP (K(d) = 0.74 micrometer) and GDP (K(d) = 13.4 micrometer) were considerably lower than those of other translation factors. Of functional significance is the fact that the rate constant for GDP release from its complex with SelB (15 s(-)(1)) is many orders of magnitude larger than for elongation factor Tu, explaining why a GDP/GTP exchange factor is not required for the action of SelB. In contrast, the rate of release of GTP is 2 orders of magnitude slower and not significantly faster than for elongation factor Tu. Using a fluorescently labeled 17-nucleotide RNA minihelix that represents a binding site for the protein and that is part of the fdhF selenocysteine insertion sequence element positioned immediately downstream of the UGA triplet coding for selenocysteine incorporation, the kinetics of the interaction were studied. The high affinity of the interaction (K(d) approximately 1 nm) appeared to be increased even further when selenocysteyl-tRNA(Sec) was bound to SelB, but to be independent of the presence or nature of the guanosine nucleotide at the active site. These results suggest that the affinity of SelB for its RNA binding site is maximized when charged tRNA is bound and decreases to allow dissociation and reading of codons downstream of the selenocysteine codon after selenocysteine peptide bond formation. 相似文献
3.
Genetic probing of the interaction between the translation factor SelB and its mRNA binding element in Escherichia coli 总被引:1,自引:0,他引:1
Decoding of the UGA codon in mRNAs for selenoproteins as selenocysteine requires interaction of the translation factor SelB
with an mRNA structure, the SECIS element. A genetic analysis of this interaction was performed by selecting for intergenic
suppressor mutations in selB which counteracted the detrimental effect of defined mutations in the SECIS element. Both allele-nonspecific and allele-specific
mutations, as judged by readthrough of the UGA into the LacZ-encoding segment of fdhF ′-′lacZ fusions and by incorporation of selenium, were isolated. selB genes from ten suppressor mutants were sequenced and the corresponding mutations were localized to five positions within
the protein. Four of the suppressors had amino acid exchanges within a 23-amino acid stretch in domain 4b of SelB, which probably
represent sites of contact between the protein and the mRNA. A fifth mutation was localized in domain 4a of SelB; it promoted
allele-nonspecific readthrough. Since a truncated SelB species lacking domain 4b did not show complex formation with the SECIS
element, we speculate that the latter mutation affects the interaction between the tRNA-binding and the mRNA-binding domains.
None of the SelB variants was able to promote UGA readthrough when major structural changes that altered the length of the
helical part or enlarged the apical loop were introduced into the SECIS element. The results obtained also show that novel
pairs of SelB/SECIS derivatives can be generated which may be useful for the targeted insertion of selenocysteine into proteins.
Received: 29 June 1999 / Accepted: 10 July 1999 相似文献
4.
The bulged nucleotide in the Escherichia coli minimal selenocysteine insertion sequence participates in interaction with SelB: a genetic approach
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The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In Escherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the fdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing selB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selB suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a "single suppressible unit," suggesting that there is some flexibility to the nature of the interaction between this element and SelB. 相似文献
5.
Liu J Zhang J Yang Y Huang H Shen W Hu Q Wang X Wu J Shi Y 《Protein science : a publication of the Protein Society》2008,17(6):1003-1014
Leukemia-associated Rho guanine nucleotide exchange factor (LARG) is a RhoA-specific guanine nucleotide exchange factor (GEF) that can activate RhoA. The PDZ (PSD-95/Disc-large/ZO-1 homology) domain of LARG interacts with membrane receptors, which can relay extracellular signals to RhoA signal transduction pathways. Until now there is no structural and dynamic information about these interactions. Here we report the NMR structures of the LARG PDZ in the apo form and in complex with the plexin-B1 C-terminal octapeptide. Unobservable resonances of the residues in betaB/betaC and betaE/alphaB loops in apo state were observed in the complex state. A distinct region of the binding groove in the LARG PDZ was found to undergo conformational change compared with other PDZs. Analysis of the (15)N relaxation data using reduced spectral density mapping shows that the apo LARG PDZ (especially its ligand-binding groove) is flexible and exhibits internal motions on both picosecond to nanosecond and microsecond to millisecond timescales. Mutagenesis and thermodynamic studies indicate that the conformation of the betaB/betaC and betaE/alphaB loops affects the PDZ-peptide interaction. It is suggested that the conformational flexibility could facilitate the change of structures upon ligand binding. 相似文献
6.
Janin J 《Protein science : a publication of the Protein Society》2005,14(2):278-283
The Critical Assessment of PRedicted Interactions (CAPRI) experiment was designed in 2000 to test protein docking algorithms in blind predictions of the structure of protein-protein complexes. In four years, 17 complexes offered by crystallographers as targets prior to publication, have been subjected to structure prediction by docking their two components. Models of these complexes were submitted by predictor groups and assessed by comparing their geometry to the X-ray structure and by evaluating the quality of the prediction of the regions of interaction and of the pair wise residue contacts. Prediction was successful on 12 of the 17 targets, most of the failures being due to large conformation changes that the algorithms could not cope with. Progress in the prediction quality observed in four years indicates that the experiment is a powerful incentive to develop new procedures that allow for flexibility during docking and incorporate nonstructural information. We therefore call upon structural biologists who study protein-protein complexes to provide targets for further rounds of CAPRI predictions. 相似文献
7.
8.
Iakhiaeva E Bhuiyan SH Yin J Zwieb C 《Protein science : a publication of the Protein Society》2006,15(6):1290-1302
The signal recognition particle (SRP) plays an important role in the delivery of secretory proteins to cellular membranes. Mammalian SRP is composed of six polypeptides among which SRP68 and SRP72 form a heterodimer that has been notoriously difficult to investigate. Human SRP68 was purified from overexpressing Escherichia coli cells and was found to bind to recombinant SRP72 as well as in vitro-transcribed human SRP RNA. Polypeptide fragments covering essentially the entire SRP68 molecule were generated recombinantly or by proteolytic digestion. The RNA binding domain of SRP68 included residues from positions 52 to 252. Ninety-four amino acids near the C terminus of SRP68 mediated the binding to SRP72. The SRP68-SRP72 interaction remained stable at elevated salt concentrations and engaged approximately 150 amino acids from the N-terminal region of SRP72. This portion of SRP72 was located within a predicted tandem array of four tetratricopeptide (TPR)-like motifs suggested to form a superhelical structure with a groove to accommodate the C-terminal region of SRP68. 相似文献
9.
Longenecker KL Stamper GF Hajduk PJ Fry EH Jakob CG Harlan JE Edalji R Bartley DM Walter KA Solomon LR Holzman TF Gu YG Lerner CG Beutel BA Stoll VS 《Protein science : a publication of the Protein Society》2005,14(12):3039-3047
In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target. 相似文献
10.
Qing Ge Heini Ilves Anne Dallas Pavan Kumar Joshua Shorenstein Sergei A. Kazakov Brian H. Johnston 《RNA (New York, N.Y.)》2010,16(1):106-117
Small hairpin RNAs (shRNAs) are widely used in RNAi studies and typically consist of a stem of 19–29 base pairs (bp), a loop of at least 4 nucleotides (nt), and a dinucleotide overhang at the 3′ end. Compared with shRNAs with 21–29 bp stems, we have found that shRNAs with 19-bp or shorter stems (sshRNAs) possess some unique structure–activity features that depend on whether the antisense strand is positioned 5′ or 3′ to the loop (L- or R-type sshRNAs, respectively). L sshRNAs can have IC50s in the very low picomolar range, and sshRNAs with nominal loop sizes of 1 or 4 nt were at least as active as those with longer loops. L sshRNAs remained highly potent even when the 3′ end of the antisense strand was directly linked with the 5′ end of the sense strand. In this case, the sense strand can be shorter than the antisense strand, and the loop can be formed entirely by the 3′ end of the antisense strand. Monomer sshRNAs are not processed by recombinant Dicers in vitro. Although they can form dimers that are sometimes Dicer substrates, their RNAi activity is not dependent on the formation of such structures. Our findings have implications for the mechanism of action of sshRNAs, and the ability to design highly potent shRNAs with minimal length is encouraging for the prospects of the therapeutic use of direct-delivered shRNAs. 相似文献
11.
Quadt-Akabayov SR Chill JH Levy R Kessler N Anglister J 《Protein science : a publication of the Protein Society》2006,15(11):2656-2668
Type I interferons (IFNs) are a family of homologous helical cytokines that exhibit pleiotropic effects on a wide variety of cell types, including antiviral activity and antibacterial, antiprozoal, immunomodulatory, and cell growth regulatory functions. Consequently, IFNs are the human proteins most widely used in the treatment of several kinds of cancer, hepatitis C, and multiple sclerosis. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. The structure of the extracellular domain of IFNAR2 (R2-EC) was solved recently. Here we study the complex and the binding interface of IFNalpha2 with R2-EC using multidimensional NMR techniques. NMR shows that IFNalpha2 does not undergo significant structural changes upon binding to its receptor, suggesting a lock-and-key mechanism for binding. Cross saturation experiments were used to determine the receptor binding site upon IFNalpha2. The NMR data and previously published mutagenesis data were used to derive a docking model of the complex with an RMSD of 1 Angstrom, and its well-defined orientation between IFNalpha2 and R2-EC and the structural quality greatly improve upon previously suggested models. The relative ligand-receptor orientation is believed to be important for interferon signaling and possibly one of the parameters that distinguish the different IFN I subtypes. This structural information provides important insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN-like molecules. 相似文献
12.
Structural basis of the collagen-binding mode of discoidin domain receptor 2 总被引:3,自引:0,他引:3
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Discoidin domain receptor (DDR) is a cell-surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2-DS domain), and identified the binding site to fibrillar collagen by transferred cross-saturation experiments. The DDR2-DS domain structure adopts a distorted jellyroll fold, consisting of eight beta-strands. The collagen-binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen-binding site suggests that the DDR2-DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen-binding mode of the DDR2-DS domain. 相似文献
13.
14.
Columbus L Lipfert J Klock H Millett I Doniach S Lesley SA 《Protein science : a publication of the Protein Society》2006,15(5):961-975
Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins. 相似文献
15.
Identification of transient hub proteins and the possible structural basis for their multiple interactions 总被引:1,自引:0,他引:1
Higurashi M Ishida T Kinoshita K 《Protein science : a publication of the Protein Society》2008,17(1):72-78
Proteins that can interact with multiple partners play central roles in the network of protein-protein interactions. They are called hub proteins, and recently it was suggested that an abundance of intrinsically disordered regions on their surfaces facilitates their binding to multiple partners. However, in those studies, the hub proteins were identified as proteins with multiple partners, regardless of whether the interactions were transient or permanent. As a result, a certain number of hub proteins are subunits of stable multi-subunit proteins, such as supramolecules. It is well known that stable complexes and transient complexes have different structural features, and thus the statistics based on the current definition of hub proteins will hide the true nature of hub proteins. Therefore, in this paper, we first describe a new approach to identify proteins with multiple partners dynamically, using the Protein Data Bank, and then we performed statistical analyses of the structural features of these proteins. We refer to the proteins as transient hub proteins or sociable proteins, to clarify the difference with hub proteins. As a result, we found that the main difference between sociable and nonsociable proteins is not the abundance of disordered regions, in contrast to the previous studies, but rather the structural flexibility of the entire protein. We also found greater predominance of charged and polar residues in sociable proteins than previously reported. 相似文献
16.
17.
Roman Martin Philipp Hackert Maike Ruprecht Stefan Simm Lukas Brüning Oliver Mirus Katherine E. Sloan Grzegorz Kudla Enrico Schleiff Markus T. Bohnsack 《RNA (New York, N.Y.)》2014,20(8):1173-1182
Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis. 相似文献
18.
Xu QS Roberts RJ Guo HC 《Protein science : a publication of the Protein Society》2005,14(10):2590-2600
The crystal structure of the Type IIP restriction endonuclease MspI bound to DNA containing its cognate recognition sequence has been determined in both monoclinic and orthorhombic space groups. Significantly, these two independent crystal forms present an identical structure of a novel monomer-DNA complex, suggesting a functional role for this novel enzyme-DNA complex. In both crystals, MspI interacts with the CCGG DNA recognition sequence as a monomer, using an asymmetric mode of recognition by two different structural motifs in a single polypeptide. In the crystallographic asymmetric unit, the two DNA molecules in the two MspI-DNA complexes appear to stack with each other forming an end-to-end pseudo-continuous 19-mer duplex. They are primarily B-form and no major bends or kinks are observed. For DNA recognition, most of the specific contacts between the enzyme and the DNA are preserved in the orthorhombic structure compared with the monoclinic structure. A cation is observed near the catalytic center in the monoclinic structure at a position homologous to the 74/45 metal site of EcoRV, and the orthorhombic structure also shows signs of this same cation. However, the coordination ligands of the metal are somewhat different from those of the 74/45 metal site of EcoRV. Combined with structural information from other solved structures of Type II restriction enzymes, the possible relationship between the structures of the enzymes and their cleavage behaviors is discussed. 相似文献
19.
Solution structure of choline binding protein A, the major adhesin of Streptococcus pneumoniae
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Luo R Mann B Lewis WS Rowe A Heath R Stewart ML Hamburger AE Sivakolundu S Lacy ER Bjorkman PJ Tuomanen E Kriwacki RW 《The EMBO journal》2005,24(1):34-43
Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR. 相似文献
20.
Many viral mRNAs contain a 5′-UTR RNA element called internal ribosome-entry site (IRES), which bypasses the requirement of some canonical initiation factors allowing cap-independent translation. The IRES of hepatitis-C virus drives translation by directly recruiting 40S ribosomal subunits and binds to eIF3 which plays a critical role in both cap-dependent and cap-independent translation. However, the molecular basis for eIF3 activity in either case remains enigmatic. Here we report that subunit b of the eIF3 complex directly binds to HCV IRES domain III via its N-terminal-RRM. Because eIF3b was previously shown to be involved in eIF3j binding, biological implications are discussed. 相似文献