Carla W. Gray

Adsorption complex of filamentous fd virus

Abstract We have directly visualized what appears to be the adsorption apparatus of a filamentous virus. Electron microscopy reveals at least three knobs, located specifically at one end of the virion, which is more tapered than is the other end. The knobs are attached to the virion tip by connecting stems that are too thin to be seen in most preparations. The protease subtilisin digests away the adsorption protein from the virus, as shown by electrophoretic analysis, and it removes the knobs seen by electron microscopy. Resistance of virion DNAs to endonucleases, even after removal of the terminal knobs with subtilisin, indicates that the thin stems do not consist of DNA. The size of the knobs indicates that each is principally a monomer of the viral adsorption protein; each stem is presumed to be a connecting protein bridge.

Three-dimensional structure of complexes of single-stranded DNA-binding proteins with DNA. IKe and fd gene 5 proteins form left-handed helices with single-stranded DNA.

Specimen-tilting in an electron microscope was used to determine the three-dimensional architecture of the helical complexes formed with DNA by the closely related single-stranded DNA binding proteins of fd and IKe filamentous viruses. The fd gene 5 protein is the only member of the DNA-helix-destabilizing class of proteins whose structure has been determined crystallographically, and yet a parameter essential to molecular modeling of the co-operative interaction of this protein with DNA, the helix handedness, has not been available prior to this work. We find that complexes formed by titrating fd viral DNA with either the fd or IKe gene 5 protein have a left-handed helical sense. Complexes isolated from Escherichia coli infected by fd virus are also found to be left-handed helical; hence, the left-handed fd helices are not an artefact of reconstitution in vitro. Because the proteins and nucleic acid of the complexes are composed of asymmetric units which cannot be fitted equivalently to right-handed and left-handed helices, these results rule out a previous computer graphics atomic model for the helical fd complexes: a right-handed helix had been assumed for the model. Our work provides a defined three-dimensional structural framework within which to model the protein-DNA and protein-protein interactions of two structurally related proteins that bind contiguously and co-operatively on single-stranded DNAs.

A nucleoprotein complex in bacteria infected with Pf1 filamentous virus: Identification and electron microscopic analysis

Abstract We report the discovery, partial purification, and high-resolution electron microscopic characterization of an intracellular complex from Pseudomonas aeruginosa bacteria infected by Pf1 filamentous virus. The Pf1 complex resembles the virion precursor complex of DNA and viral gene 5 protein formed by fd virus of Escherichia coli , but the two complexes differ in structure. Image reconstruction indicates that both complexes are single-start morphological helices; specimen tilting shows the Pf1 helix to be right-handed. Although the Pf1 and fd complexes contain a similar number of nucleotides per axial unit length, the mean distance between helical turns is 61 A for Pf1 but 91 A for fd under the conditions used for our measurements; two turns of the fd nucleoprotein helix contain about as many nucleotides as do three turns of the Pf1 helix. The Pf1 complex is much shorter than are Pf1 virions, in contrast to the similar lengths of the fd virion and complex. The fd complex is extremely flexible, but the Pf1 complex is more highly regular in structure. Most significant, calculations based on our data indicate that the DNA in the Pf1 complex is probably located at a smaller radius than in the bulk of the protein. If the DNA and morphological helices coincide, the DNA in the Pf1 complex must be well inside of (axial to) the outeer protein surfaces of the complex, rather than being wrapped around the protein subunits as proposed by others for fd complex.

Structure of the fd DNA--gene 5 protein complex in solution. A neutron small-angle scattering study.

Neutron small-angle scattering has been used to investigate the fd DNA-gene 5 protein complex in solution. Results are as follows. 1. (1) The mass per unit length is found to be 1380 or 1610 daltons/A, depending upon whether one gene 5 protein molecule is assumed to bind to four or five nucleotides, respectively. These values correspond to axial subunit repeats of 7.9 or 7.0 A and to total contour lengths in solution of 1.27 or 0.90 μm. For a helix of pitch 90 A there are between about 11 and 13 proteins per turn. 2. (2) The cross-sectional radius of gyration at infinite contrast of the complex is 34.5 ± 1 A. 3. (3) The structure must be quite open and solvated as indicated by the dry volume per subunit, the mass per unit length, and the radius of gyration. The volume occupied per subunit in a uniform cylinder having the measured radius of gyration and mass per unit length is about four times greater than the measured subunit dry volume in the complex. 4. (4) From the change with contrast of both the measured radius of gyration and the position of a subsidiary maximum we conclude that the DNA could not be on the outer periphery of the helical structure. This is supported by a calculation of the maximum radius of the DNA backbone. With the possible exception of the positioning of the DNA, our results for the complex in solution are in good agreement with a model proposed by McPherson et al. (1979b) for the complex structure. 5. (5) The complex formed by reconstitution in vitro is not substantially different in its solution structure from the in vivo complex isolated from infected cells.

CD of single-stranded, double-stranded, and G-quartet nucleic acids in complexes with a single-stranded DNA-binding protein

We review CD studies of a single-stranded DNA binding protein, g5p, of the Ff group of bacterial viruses. The CD spectrum of the g5p is dominated by a positive tyrosine La band at 229 nm, to which all five of the protein tyrosines contribute. The La band becomes much less positive upon binding of g5p to nucleic acids. CD spectra of mutant proteins identified a single tyrosine, Y34, that is largely responsible for this CD perturbation. At >250 nm, CD perturbations of nucleic acids can be monitored during g5p binding, and CD titrations have identified two distinct modes of binding of the g5p at physiological ionic strength (0.2 M NaCl). SELEX selection of sequences bound preferentially by g5p yielded a G-rich sequence that is closely related to telomere sequences and has CD properties of a G-tetraplex. CD spectroscopy showed that the presumed G-quadruplex form is maintained within saturated g5p x DNA complexes.

THE BINDING AFFINITY OF FF GENE 5 PROTEIN DEPENDS ON THE NEAREST-NEIGHBOR COMPOSITION OF THE SSDNA SUBSTRATE

The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Komega (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Komega values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA48, dC48, d(AAC)16, d(ACC)16, d(AACC)12, and d(AAACC)9A3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein.

The reduction of Raf-1 protein by phosphorothioate ODNs and siRNAs targeted to the same two mRNA sequences.

Two sets of 20-mer phosphorothioate-modified oligodeoxynucleotide DNAs (sODN) and 21-mer or 22-mer small interfering RNAs (siRNAs), targeted to the same coding sites in raf-1 mRNA, were compared for their abilities to reduce the amount of endogenously expressed Raf-1 protein in T24 cells. The amount of Raf-1 protein was monitored by careful quantitation of Western blots. We found that the siRNAs were somewhat less effective than the S-ODNs in reducing the Raf-1 protein level 20 hours after a 4-hour transfection. The siRNA duplexes were characterized by circular dichroism (CD) spectra, and melting temperatures (Tm) were obtained for the siRNA duplexes and DNA x RNA hybrids formed by the S-ODNs. The S-ODNs differed in their effectiveness, the S-ODN that formed the more stable hybrid being the more effective in reducing the Raf-1 protein level, but the two siRNAs were equally effective despite a difference in Tm of about 20 degrees C. Finally, the siRNAs and S-ODNs had a comparable nonspecific effect on a nontargeted (Bcl-2) protein. Our data add to others in the literature that show it can be difficult to select siRNAs that are more effective than antisense ODNs in downregulating endogenously expressed proteins.

Ultrafast Fluorescence Decay Profiles Reveal Differential Unstacking of 2-Aminopurine from Neighboring Bases in Single-Stranded DNA-Binding Protein Subsites

Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.

Complex of fd gene 5 protein and double-stranded RNA☆

We report the formation of complexes of the single-stranded DNA binding protein encoded by gene 5 of fd virus, with natural double-stranded RNAs. In the first direct visualization of a complex of the fd gene 5 protein with a double-stranded nucleic acid, we show by electron microscopy that the double-stranded RNA complex has a structure which is distinct from that of complexes with single-stranded DNA and is consistent with uniform coating of the exterior of the double-stranded RNA helix by the protein. Circular dichroism spectral data demonstrate that the RNA double helix in the complex is undisrupted, and that perturbation of the 228-nm circular dichroism assigned to protein tyrosines can occur in the absence of intercalation of nucleotide bases with protein aromatic residues. Our findings emphasize the potential importance of interaction with the sugar-phosphate polynucleotide backbone in binding of the fd gene 5 protein to nucleic acids.

Antisense DNA parameters derived from next-nearest-neighbor analysis of experimental data

BackgroundThe enumeration of tetrameric and other sequence motifs that are positively or negatively correlated with in vivo antisense DNA effects has been a useful addition to the arsenal of information needed to predict effective targets for antisense DNA control of gene expression. Such retrospective information derived from in vivo cellular experiments characterizes aspects of the sequence dependence of antisense inhibition that are not predicted by nearest-neighbor (NN) thermodynamic parameters derived from in vitro experiments. However, quantitation of the antisense contributions of motifs is problematic, since individual motifs are not isolated from the effects of neighboring nucleotides, and motifs may be overlapping. These problems are circumvented by a next-nearest-neighbor (NNN) analysis of antisense DNA effects in which the overlapping nature of nearest-neighbors is taken into account.ResultsNext-nearest-neighbor triplet combinations of nucleotides are the simplest that include overlapping sequence effects and therefore can encompass interactions beyond those of nearest neighbors. We used singular value decomposition (SVD) to fit experimental data from our laboratory in which phosphorothioate-modified antisense DNAs (S-DNAs) 20 nucleotides long were used to inhibit cellular protein expression in 112 experiments involving four gene targets and two cell lines. Data were fitted using a NNN model, neglecting end effects, to derive NNN inhibition parameters that could be combined to give parameters for a set of 49 sequences that represents the inhibitory effects of all possible overlapping triplet interactions in the cellular targets of these antisense S-DNAs. We also show that parameters to describe subsets of the data, such as the mRNAs being targeted and the cell lines used, can be included in such a derivation. While NNN triplet parameters provided an adequate model to fit our data, NN doublet parameters did not.ConclusionsThe methodology presented illustrates how NNN antisense inhibitory information can be derived from in vivo cellular experiments. Subsequent calculations of the antisense inhibitory parameters for any mRNA target sequence automatically take into account the effects of all possible overlapping combinations of nearest-neighbors in the sequence. This procedure is more robust than the tallying of tetrameric motifs that have positive or negative antisense effects. The specific parameters derived in this work are limited in their applicability by the relatively small database of experiments that was used in their derivation.