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1.
PLoS One ; 19(3): e0301486, 2024.
Article in English | MEDLINE | ID: mdl-38530841

ABSTRACT

[This corrects the article DOI: 10.1371/journal.pone.0035263.].

4.
RNA ; 19(8): 1089-104, 2013 08.
Article in English | MEDLINE | ID: mdl-23804244

ABSTRACT

OxyS and RprA are two small noncoding RNAs (sRNAs) that modulate the expression of rpoS, encoding an alternative sigma factor that activates transcription of multiple Escherichia coli stress-response genes. While RprA activates rpoS for translation, OxyS down-regulates the transcript. Crucially, the RNA binding protein Hfq is required for both sRNAs to function, although the specific role played by Hfq remains unclear. We have investigated RprA and OxyS interactions with Hfq using biochemical and biophysical approaches. In particular, we have obtained the molecular envelopes of the Hfq-sRNA complexes using small-angle scattering methods, which reveal key molecular details. These data indicate that Hfq does not substantially change shape upon complex formation, whereas the sRNAs do. We link the impact of Hfq binding, and the sRNA structural changes induced, to transcript stability with respect to RNase E degradation. In light of these findings, we discuss the role of Hfq in the opposing regulatory functions played by RprA and OxyS in rpoS regulation.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Host Factor 1 Protein/metabolism , RNA, Bacterial/metabolism , RNA, Small Untranslated/metabolism , Repressor Proteins/metabolism , Sigma Factor/metabolism , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Biophysical Phenomena , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Host Factor 1 Protein/chemistry , Host Factor 1 Protein/genetics , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Structure, Quaternary , RNA Stability , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Small Untranslated/chemistry , RNA, Small Untranslated/genetics , Repressor Proteins/genetics , Scattering, Small Angle , Sigma Factor/genetics
5.
Protein Expr Purif ; 87(2): 136-40, 2013 02.
Article in English | MEDLINE | ID: mdl-23201446

ABSTRACT

Type I restriction-modification (R-M) systems are comprised of two multi-subunit enzymes with complementary functions: the methyltransferase (~160 kDa), responsible for methylation of DNA, and the restriction endonuclease (~400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits, including HsdM. Characterisation of either enzyme first requires the expression and purification of its constituent subunits, before reconstitution of the multisubunit complex. Previously, purification of the HsdM protein had proved problematic, due to the length of time required for the purification and its susceptibility to degradation. A new protocol was therefore developed to decrease the length of time required to purify the HsdM protein and thus prevent degradation. Finally, we show that the HsdM subunit exhibits a concentration dependent monomer-dimer equilibrium.


Subject(s)
Bacterial Proteins/isolation & purification , DNA Restriction-Modification Enzymes/isolation & purification , Deoxyribonucleases, Type I Site-Specific/chemistry , Methyltransferases/isolation & purification , Recombinant Proteins/isolation & purification , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA Restriction-Modification Enzymes/biosynthesis , DNA Restriction-Modification Enzymes/chemistry , DNA Restriction-Modification Enzymes/genetics , Escherichia coli , Methyltransferases/biosynthesis , Methyltransferases/chemistry , Methyltransferases/genetics , Protamines/chemistry , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sodium Chloride/chemistry , Ultracentrifugation
6.
Biophys J ; 103(3): 541-549, 2012 08 08.
Article in English | MEDLINE | ID: mdl-22947870

ABSTRACT

Calmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ~70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca(2+) or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca(2+)-signaling during viral processing.


Subject(s)
Calmodulin/metabolism , HIV Antigens/chemistry , HIV Antigens/metabolism , HIV-1 , Protein Refolding , gag Gene Products, Human Immunodeficiency Virus/chemistry , gag Gene Products, Human Immunodeficiency Virus/metabolism , Calmodulin/chemistry , Models, Molecular , Neutron Diffraction , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Scattering, Small Angle
7.
Nucleic Acids Res ; 40(17): 8698-710, 2012 09 01.
Article in English | MEDLINE | ID: mdl-22730296

ABSTRACT

In Vibrio cholerae, the RNA binding protein and chaperone Hfq (VcHfq) facilitates the pairing of the quorum regulatory RNA (Qrr) small regulatory RNAs (sRNAs) to the 5' untranslated regions of the mRNAs for a number of global regulators that modulate the expression of virulence genes. This Qrr-mediated sRNA circuit is an attractive antimicrobial target, but characterization at the molecular level is required for this to be realized. Here, we investigate the interactions between VcHfq and the Qrr sRNAs using a variety of biochemical and biophysical techniques. We show that the ring-shaped VcHfq hexamer binds the Qrrs with 1:1 stoichiometry through its proximal face, and the molecular envelope of the VcHfq-Qrr complex is experimentally determined from small angle scattering data to present the first structural glimpse of a Hfq-sRNA complex. This structure reveals that the VcHfq protein does not change shape on complex formation but the RNA does, suggesting that a chaperone role for VcHfq is a critical part of the VcHfq-Qrr interaction. Overall, these studies enhance our understanding of VcHfq-Qrr interactions.


Subject(s)
Host Factor 1 Protein/chemistry , RNA, Small Untranslated/chemistry , Vibrio cholerae , Binding Sites , Host Factor 1 Protein/metabolism , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , RNA, Small Untranslated/metabolism , Scattering, Small Angle
8.
Genes Dev ; 26(1): 92-104, 2012 01 01.
Article in English | MEDLINE | ID: mdl-22215814

ABSTRACT

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.


Subject(s)
DNA Restriction Enzymes/chemistry , DNA Restriction Enzymes/ultrastructure , Models, Molecular , Deoxyribonucleases, Type I Site-Specific/chemistry , Deoxyribonucleases, Type I Site-Specific/ultrastructure , Microscopy, Electron , Negative Staining , Protein Structure, Tertiary
9.
J Mol Biol ; 414(5): 735-48, 2011 12 16.
Article in English | MEDLINE | ID: mdl-22041450

ABSTRACT

New insights into the modular organization and flexibility of the N-terminal half of human cardiac myosin binding protein C (cMyBP-C) and information on the association state of the full-length protein have been deduced from a combined small-angle X-ray scattering (SAXS) and NMR study. SAXS data show that the first five immunoglobulin domains of cMyBP-C, which include those implicated in interactions with both myosin and actin, remain monodisperse and monomeric in solution and have a highly extended yet distinctively 'bent' modular arrangement that is similar to the giant elastic muscle protein titin. Analyses of the NMR and SAXS data indicate that a proline/alanine-rich linker connecting the cardiac-specific N-terminal C0 domain to the C1 domain provides significant structural flexibility at the N-terminus of the human isoform, while the modular arrangement of domains C1-C2-C3-C4 is relatively fixed. Domain fragments from the C-terminal half of the protein have a propensity to self-associate in vitro, while full-length bacterially expressed cMyBP-C forms flexible extended dimers at micromolar protein concentrations. In summary, our studies reveal that human cMyBP-C combines a distinctive modular architecture with regions of flexibility and that the N-terminal half of the protein is sufficiently extended to span the range of interfilament distances sampled within the dynamic environment of heart muscle. These structural features of cMyBP-C could facilitate its putative role as a molecular switch between actin and myosin and may contribute to modulating the transverse pliancy of the C-zone of the A-band across muscle sarcomeres.


Subject(s)
Carrier Proteins/chemistry , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Scattering, Small Angle
10.
J Mol Biol ; 409(2): 177-88, 2011 06 03.
Article in English | MEDLINE | ID: mdl-21440553

ABSTRACT

Controller proteins play a key role in the temporal regulation of gene expression in bacterial restriction-modification (R-M) systems and are important mediators of horizontal gene transfer. They form the basis of a highly cooperative, concentration-dependent genetic switch involved in both activation and repression of R-M genes. Here we present biophysical, biochemical, and high-resolution structural analysis of a novel class of controller proteins, exemplified by C.Csp231I. In contrast to all previously solved C-protein structures, each protein subunit has two extra helices at the C-terminus, which play a large part in maintaining the dimer interface. The DNA binding site of the protein is also novel, having largely AAAA tracts between the palindromic recognition half-sites, suggesting tight bending of the DNA. The protein structure shows an unusual positively charged surface that could form the basis for wrapping the DNA completely around the C-protein dimer.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Citrobacter/metabolism , DNA, Bacterial/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Promoter Regions, Genetic , Protein Conformation , Protein Multimerization , Sequence Homology, Amino Acid
11.
J Mol Biol ; 398(3): 391-9, 2010 05 07.
Article in English | MEDLINE | ID: mdl-20302878

ABSTRACT

The Type I R-M system EcoR124I is encoded by three genes. HsdM is responsible for modification (DNA methylation), HsdS for DNA sequence specificity and HsdR for restriction endonuclease activity. The trimeric methyltransferase (M(2)S) recognises the asymmetric sequence (GAAN(6)RTCG). An engineered R-M system, denoted EcoR124I(NT), has two copies of the N-terminal domain of the HsdS subunit of EcoR124I, instead of a single S subunit with two domains, and recognises the symmetrical sequence GAAN(7)TTC. We investigate the methyltransferase activity of EcoR124I(NT), characterise the enzyme and its subunits by analytical ultracentrifugation and obtain low-resolution structural models from small-angle neutron scattering experiments using contrast variation and selective deuteration of subunits.


Subject(s)
DNA Restriction-Modification Enzymes/metabolism , Escherichia coli Proteins/metabolism , Methyltransferases/metabolism , Recombinant Proteins/metabolism , DNA Restriction-Modification Enzymes/chemistry , DNA Restriction-Modification Enzymes/genetics , DNA, Bacterial/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Scattering, Small Angle , Ultracentrifugation
12.
Nucleic Acids Res ; 37(10): 3354-66, 2009 06.
Article in English | MEDLINE | ID: mdl-19336410

ABSTRACT

The convergently transcribed restriction (R) and methylase (M) genes of the Restriction-Modification system Esp1396I are tightly regulated by a controller (C) protein that forms part of the CR operon. We have mapped the transcriptional start sites from each promoter and examined the regulatory role of C.Esp1396I in vivo and in vitro. C-protein binding at the CR and M promoters was analyzed by DNA footprinting and a range of biophysical techniques. The distal and proximal C-protein binding sites at the CR promoter are responsible for activation and repression, respectively. In contrast, a C-protein dimer binds to a single site at the M-promoter to repress the gene, with an affinity much greater than for the CR promoter. Thus, during establishment of the system in a naïve host, the activity of the M promoter is turned off early, preventing excessive synthesis of methylase. Mutational analysis of promoter binding sites reveals that the tetranucleotide inverted repeats long believed to be important for C-protein binding to DNA are less significant than previously thought. Instead, symmetry-related elements outside of these repeats appear to be critical for the interaction and are discussed in terms of the recent crystal structure of C.Esp139I bound to the CR promoter.


Subject(s)
Bacterial Proteins/metabolism , DNA Restriction-Modification Enzymes/genetics , Gene Expression Regulation, Bacterial , Transcription Factors/metabolism , Transcription, Genetic , Base Sequence , Binding Sites , DNA Modification Methylases/genetics , DNA Restriction Enzymes/genetics , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Transcription Initiation Site
13.
J Mol Biol ; 376(2): 438-452, 2008 02 15.
Article in English | MEDLINE | ID: mdl-18164032

ABSTRACT

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.


Subject(s)
Biophysics , Deoxyribonucleases, Type I Site-Specific/chemistry , Protein Subunits/chemistry , Amino Acid Sequence , Base Sequence , Biophysical Phenomena , Codon, Terminator , DNA/metabolism , Holliday Junction Resolvases/chemistry , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Molecular Weight , Neutron Diffraction , Plasmids , Protein Binding , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/genetics , Protein Subunits/isolation & purification , Protein Subunits/metabolism , Pyrococcus furiosus/enzymology , Scattering, Small Angle , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sulfolobus solfataricus/enzymology , Templates, Genetic
14.
J Mol Biol ; 369(1): 177-85, 2007 05 25.
Article in English | MEDLINE | ID: mdl-17418232

ABSTRACT

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M(2)S(2) and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of (1)H:(2)H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (R(g)) and maximum dimensions (D(max)) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of R(g)=35 A and D(max)=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.


Subject(s)
Aeromonas hydrophila/enzymology , DNA Modification Methylases/chemistry , Protein Subunits/chemistry , Scattering, Small Angle , Amino Acid Sequence , Crystallography, X-Ray , Hydrogenation , Kinetics , Models, Molecular , Molecular Sequence Data , Neutron Diffraction , Sequence Alignment
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