A model for the complex between the helix destabilizing protein GP32 of bacteriophage T4 and single-stranded DNA

A model for the structure of the complex between the helix-destabilizing protein of bacteriophage T4, GP32, and single-stranded DNA is proposed. In this model the bases are arranged in a helix, that is characterized by a relatively large distance between successive bases, a substantial base tilt, in combination with a small rotation per base. This helix is further organized into a tertiary structure, possibly a superhelix, of which the corresponding protein shell corresponds to the relatively rigid and rod-like structure that is observed in hydrodynamic experiments. It is proposed that similar structural features apply to other single-stranded DNA binding proteins in complex with polynucleotides.     

On the thermodynamics and kinetics of the cooperative binding of bacteriophage T4-coded gene 32 (helix destabilizing) protein to nucleic acid lattices.

In this paper we summarize a series of thermodynamic, and preliminary kinetic, studies on the molecular details and specificity of interaction of phage T4-coded gene 32-protein (GP32) with nucleic acid lattices. It is shown that the binding of GP32 to short (l = 2--8 residues) oligonucleotides is essentially independent of base composition and sugar-type, as well as of salt concentration. In contrast, cooperative (continuous) or isolated binding of GP32 to single-stranded polynucleotides is base and sugar composition-dependent (binding is tighter to DNA than to RNA) and highly dependent on salt concentrations. Binding constants (K), cooperativity parameters (w), and binding site sizes (n) are determined for binding to various nucleic acid lattices under a variety of environmental conditions. These results are used to show that GP32 can bind to nucleic acid lattices in two different conformations, and to characterize the molecular details of these binding species. Further insight into the molecular origins of binding cooperativity is obtained by determining these thermodynamic parameters also for the specifically proteolytically degraded GP32 fragments GP32 I (C-terminal peptide removed) and GP32 III (C- and N-terminal peptides removed). It is also shown that these GP32-nucleic acid binding measurements can be used to provide a quantitative molecular interpretation of the sequential (competitive) binding equilibria involved in the autogenous translational regulation of GP32 synthesis (Lemaire et al., 1978, J. Mol. Biol. 126:73, 1978), and to illustrate some general principles of the development of interactional specificity in cooperatively binding protein-nucleic acid complexes. Preliminary experiments have also been carried out on the kinetics of GP32 association to, and dissociation from, single-stranded nucleic acid lattices. In particular, fluorescence stopped-flow measurements of the dissociation of GP32 from such lattices as a function of lattice saturation (and protein cluster size) can be interpreted to suggest that the protein may translocate ("slide") on the lattice before dissociation, These studies permit an approach to possible rates and mechanisms of such translocation events.     

DNA binding site of the helix destabilizing protein gp32 from bacteriophage T4.

A helix destabilizing protein, the product of gene 32 (gp32) of bacteriophage T4, was subjected to limited proteolysis to produce three types of products with differing affinities for DNA. Previous work has suggested that the 18 amino acids at the N-terminus are required for tight binding to single-stranded DNA (Hosoda &; Moise, 1978). This paper reports the sequence of the N-terminal region and predicts the amino acid residues responsible for DNA binding.     

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: I. Characterization of the binding interactions

In this paper we examine molecular details of the interaction of bacteriophage T4-coded gene 32 protein with oligo- and polynucleotides. It is shown that the binding affinity (K_oligo) of oligonucleotides of length (l) from two to eight nucleotide residues for gene 32 protein is essentially independent of base composition or sugar type. This binding also shows little dependence on salt concentration and on oligonucleotide length; even the expected statistical length factor in K_oligo is not observed, suggesting that binding occurs at the end of the oligonucleotide lattice and that the oligonucleotide is not free to move across the binding site. Co-operative (contiguous) or isolated binding of gene 32 protein to polynucleotides is very different; here binding is highly salt dependent ($\text{? log Kω}\text{? log}\text{[NaCl]}\text{～- ?7}\text{)}$ and essentially stoichiometric at salt concentrations less than ~0.2 m (for poly(rA)). Binding becomes much weaker and the binding isotherms appear typically co-operative (sigmoid) in protein concentration at higher salt concentrations. We demonstrate, by fitting the co-operative binding isotherms to theoretical plots at various salt concentrations and also by measuring binding at very low protein binding density (ν), that the entire salt dependence of Kω is in the intrinsic binding constant (K); the co-operativity parameter (ω) is essentially independent of salt concentration. Furthermore, by determining titration curves in the presence of salts containing a series of different anions and cations, it is shown that the major part of the salt dependence of the gene 32 protein-polynucleotide interaction is due to anion (rather than to cation) displacement effects. Binding parameters of oligonucleotides of length sufficient to bind two or more gene 32 protein monomers show behavior intermediate between the oligonucleotide and the polynucleotide binding modes. These different binding modes probably reflect different conformations of the protein; the results are analyzed to produce a preliminary molecular model of the interactions of gene 32 protein with nucleic acids in its different binding modes.     

The conformation of the complex of the helix destabilizing protein GP32 of bacteriophage T4 and single stranded DNA

The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32). On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation. On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (less than or equal to -10 degrees) and either a small rotation per base or a small helix diameter. This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements. Our calculations show that also the model proposed by Alma (Ph.D. Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex. For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.     

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: II. Specificity of binding to DNA and RNA

In this paper we examine the specificity of the co-operative binding (in the polynucleotide mode) of bacteriophage T4-coded gene 32 protein to synthetic and natural single-stranded nucleic acids differing in base composition and sugar type. It is shown by competition experiments in a tight-binding (low salt) environment that there is a high degree of binding specificity under these (protein-limiting) conditions, with one type of nucleic acid lattice binding gene 32 protein to saturation before any binding to the competing lattice takes place; it is also shown that the same differential specificities apply at high salt concentrations. Procedures developed in the preceding paper (Kowalczykowski et al., 1980) are used to measure the net binding affinities (Kω) of gene 32 protein to a variety of polynucleotides, as well as to determine individual values of K and ω for some systems. For all polynucleotides, virtually the entire specificity and salt dependence of binding of Kω appears to be in K. In ~0.2 m-NaCl, the net binding affinities (Kω) range from ~10⁶ to ~10¹¹m^?1; in order of increasing affinities we find: poly(rC) < poly(rU) < poly(rA) < poly(dA) < poly(dC) < poly(dU) < poly(rI) < poly(dI) < poly-(dT). In general, Kω for a particular homopolyribonucleotide at constant salt concentration is 10¹ to 10⁴smaller than Kω for the corresponding homopoly-deoxyribopolynucleotide. Values of Kω for randomly copolymerized polynucleotides and for natural DNA fall at the compositionally weighted average of the values for the individual homopolynucleotides (except for poly(dT), which appears to bind somewhat tighter), indicating that the net affinity represents the sum of the binding free energy contributions of the individual nucleotides. It is shown that these results, on a competition basis under physiological salt conditions, can account quantitatively for the autogenous regulation of the synthesis of gene 32 protein at the translational level (Russel et al., 1976; Lemaire et al., 1978). In addition, these results suggest possible mechanisms by which gene 32 messenger RNA might be specifically recognized (by gene 32 protein) and functionally discriminated from the other mRNAs of phage T4.     

Structural analysis of T4 DNA helix destabilizing protein (gp32 I) crystal by electron microscopy

Low dose electron diffraction and imaging techniques have been applied to the study of the crystalline structure of gp32*I, a DNA helix destabilizing protein derived from bacteriophage T4 gene 32 protein. A quantitative analysis of intensities from electron diffraction patterns from tilted, multilayered gp32*I crystal has provided the unit cell thickness of the crystal. The three-dimensional phases indicate that the space group P2(1)2(1)2. By taking into account the unit cell volume and the solvent content in the crystal, it was deduced that there is one gp32*I molecule in each asymmetric unit. A projected density map of unstained, glucose-embedded gp32*I crystal was synthesized with amplitudes from electron diffraction intensities and phases from electron images with reflections out to 7.6 A. Because of the similarity in the scattering density between glucose and protein, this projected map cannot be interpreted with certainty. A low resolution three-dimensional reconstruction shows that the protein molecule is about 90 A long and about 20 A in diameter. Because the dimer is formed around a dyad axis, the protein molecules comprising it must be arranged head-to-head. This dimeric arrangement of the proteins in the unit cell may be implicated as one of the conformational states of this protein in solution.     

Interaction between bacteriophage T4 coded gene 32 protein and poly(rA)

The cooperative binding of T4 gene 32 protein with polynucleotides, of which the quantitative aspects in the literature have not satisfied the requirements of thermodynamics, is studied by adopting a modified formula of the lattice theory. A moderate value is found for the cooperativity parameter (q approximately 200 at 0.2 M NaCl), which is weakly dependent on salt concentration. The cation effect on the binding suggests that the shielding of negative charges of the protein or a loose cation bridge between the bound protein molecules plays a role in the cooperative binding process.     

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: III. Binding properties of two specific proteolytic digestion products of the protein (G32P1I and G32P1III)

Brief treatment of gene 32 protein with proteolytic enzymes produces two specific digestion products in good yield (Moise & Hosoda, 1976). One, representing the native protein with ~60 amino acid residues removed from the C-terminus, is G32P¹I. The other, for which ~20 amino acid residues have been removed from the N-terminus in addition to the 60 residues from the C-terminus, is G32P¹III. Both of these specific “core” fragments of gene 32 protein have been isolated and purified, and their binding properties to single-stranded oligo- and polynucleotides have been studied. We find that the binding properties of G32P¹I are relatively little changed from those characteristic of the native gene 32 protein: (1) the apparent binding constants to short (l = 2 to 8) oligonucleotides are independent of lattice length and essentially independent of base and sugar composition, but do show an increased salt dependence of binding relative to that of the native protein; (2) the intrinsic association constants (K) for polynucleotides binding in the co-operative mode show the same binding specificities as seen with the native protein, but with absolute values increased two to fourfold; (3) the polynucleotide binding co-operativity parameter (ω^?2 × 10³) and the binding site size (n ～-7 nucleotide residues) are the same as for the native protein; (4) essentially the entire salt dependence of the net affinity (Kω) remains in K. However, unlike native gene 32 protein, G32P¹I can melt native DNA to equilibrium (Hosoda et al., 1974; Greve et al., 1978); this suggests that the kinetic pathways for DNA melting by these two species must differ, since the changes in equilibrium binding parameters measured here are far too small to account for the differences in melting behavior. In contrast to G32P¹I, for G32P¹III we find that: (1) binding is non-cooperative (ω ～-1); (2) the binding site size (n) for the protein has decreased by one to two nucleotide residues relative to that characteristic of the native protein and G32P¹I; (3) binding to short (l = 2 to 8) oligonucleotides is length and salt concentration dependent; (4) while binding to polynucleotides continues to show approximately the same base composition dependence as the native protein, the absolute values of K are somewhat different and the salt concentration dependencies of K are less. Polynucleotide ultraviolet light and circular dichroism spectra obtained in the presence of G32P¹I and G32P¹III are indistinguishable from those measured with the native protein at similar binding densities, indicating that all three protein species distort the polynucleotide lattice to comparable extents.These results are combined with the equilibrium binding data for native gene 32 protein (Kowalczykowski et al., 1980a: Newport et al., 1980) to obtain further insight into the molecular details of the interactions of this protein with its nucleic acid binding substrates.     

Autoregulation of gene expression: Quantitative evaluation of the expression and function of the bacteriophage T4 gene 32 (single-stranded DNA binding) protein system

The free concentration of bacteriophage T4-coded gene 32 (single-stranded DNA binding) protein in the cell is autoregulated at the translational level during T4 infection of Escherichia coli. The control of the synthesis of this protein reflects the following progression of net (co-operative) binding affinities for the various potential nucleic acid binding targets present: single-stranded DNA > gene 32 mRNA > other T4 mRNAs ? double-stranded DNA. In this paper we show that the free concentration of gene 32 protein is maintained at 2 to 3 μm, and use the measured binding parameters for gene 32 protein, extrapolated to intracellular conditions, to provide a quantitative molecular interpretation of this system of control of gene expression. These results are then further utilized to define the specific autoregulatory binding sequence (translational operator site) on the gene 32 mRNA as a uniquely unstructured finite binding lattice terminated by elements of secondary structure not subject to melting by gene 32 protein at the autoregulated concentration, and to predict how this site must differ from those found on other T4 messenger RNAs. It is shown that these predictions are fully consistent with available T4 DNA sequence data. The control of free protein concentration as a method of genome regulation is discussed in terms of other systems to which these approaches may apply.     

Regulation of the synthesis of bacteriophage T4 gene 32 protein

The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment.Mutations in genes required for late gene expression (genes 33 and 53) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not dependent on DNA synthesis.These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by its own function. Thus, in the absence of P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently in the maturation of replicated DNA, the increased P32 production is caused by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41, 56, 58, 60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.     

A novel function for zinc(II) in a nucleic acid-binding protein. Contribution of zinc(II) toward the cooperativity of bacteriophage T4 gene 32 protein binding

The contribution of Zn(II) toward the binding of bacteriophage T4 gene 32 single-stranded nucleic acid-binding protein (gp32) has been examined by the use of two independent approaches. Studies carried out with successively longer oligonucleotides which have the general structure p(dT)n, where n is equal to 8, 16, 24, or 32 nucleotides, suggest that removal of Zn(II) decreases the cooperativity of binding by as much as 30-fold. Hence, whereas apo-gp32 and native gp32 have similar apparent affinities for the single-site lattice p(dT)8, native gp32 has an approximately 10-fold higher affinity compared to apo-gp32 for a two-site lattice, such as p(dT)16. In contrast to native gp32, where full cooperativity (in terms of the strength of a single gp32-gp32 interaction) is reached with only a two-site lattice, the cooperativity of apo-gp32 binding appears to increase approximately 4-fold upon going from a two- to a four-site lattice such as p(dT)32. The conclusion reached from these oligonucleotide studies agrees well with a series of titrations with polyribo(ethenoadenylic) acid, in 0.275-0.40 M NaCl. These latter studies indicate that the 6-38-fold higher affinity of native gp32 as compared to apo-gp32 for polyribo(ethenoadenylic) acid results primarily from the higher cooperativity of binding of native gp32. By stabilizing a specific subdomain within gp32 that is essential along with the NH2-terminal domain (residues 1-9), Zn(II) contributes from 20 to 50% of the free energy of cooperative gp32-gp32 interactions that occur along a polynucleotide lattice.     

Suppressors of gene 32 am mutants that specifically overproduce P32 (unwinding protein) in bacteriophage T4.

A gene 32 amber (am) mutant, amNG364, fails to grow on Escherichia coli Su3+ high temperatures, suggesting that the tyrosine residue inserted at the am codon by Su3+ leads to a temperature-sensitive gene 32 protein (P32). By plating amNG364 on E. coli Su3+ 45 degrees C, several pseudorevertants were found that proved to contain a suppressor (su) mutant in addition to the original am mutation. Crosses of two of these amNG364su strains to am+ phage indicated that the suppressors themselves are in or close to gene 32. Phage strains carrying either of the two su mutations, without amNG364, grew normally. When cells were infected by these su mutants and the proteins produced were examined by sodium dodecyl sulfate-gel electrophroesis, specific overproduction of P32 was found. Maximum overproduction compared to am+ phage was 6.6-fold for one su mutant and 2.4-fold for the other. Other proteins were produced in normal amounts and in normal time sequence. When amNG364su phage were allowed to infect E. coli S/6/5(Su-), the gene 32 am fragments produced were present at the same derepressed levels as in an infection by amNG364 without a suppressor. The suppressor mutations are interpreted as causing derepression of P32 by altering sites in this autogenously regulated protein involved in template recognition. Previously, specific derepression of gene 32 had only been shown using gene 32 conditional lethal mutants grown under restrictive conditions. We have shown that P32 can also be derepressed under permissive conditions, indicating that loss of P32 function is not necessary for specific derepression.     

Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure

In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.     

On the role of the single-stranded DNA binding protein of bacteriophage T4 in DNA metabolism. I. Isolation and genetic characterization of new mutations in gene 32 of bacteriophage T4

Summary The product of gene 32 of bacteriophage T4 is a single-stranded DNA binding protein involved in T4 DNA replication, recombination and repair. Functionally differentiated regions of the gene 32 protein have been described by protein chemistry. As a preliminary step in a genetic dissection of these functional domains, we have isolated a large number of missense mutants of gene 32. Mutant isolation was facilitated by directed mutagenesis and a mutant bacterial host which is unusually restrictive for missense mutations in gene 32. We have isolated over 100 mutants and identified 22 mutational sites. A physical map of these sites has been constructed and has shown that mutations are clustered within gene 32. The possible functional significance of this clustering is considered.     

Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA

The gene 4 protein of bacteriophage T7 is a multifunctional enzyme that catalyzes (i) the hydrolysis of nucleoside 5'-triphosphates, (ii) the synthesis of tetraribonucleotide primers at specific recognition sequences on a DNA template, and (iii) the unwinding of duplex DNA. All three activities depend on binding of gene 4 protein to single-stranded DNA followed by unidirectional 5' to 3' translocation of the protein (Tabor, S., and Richardson, C. C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 205-209). Binding of gene 4 protein to single-stranded DNA, assayed by retention of DNA-protein complexes on nitrocellulose filters, is random with regard to DNA sequence. Although gene 4 protein does not bind to duplex DNAs, the presence of a 240-nucleotide-long single-stranded tail on a 7200-base pair duplex DNA molecule is sufficient for gene 4 protein to cause retention of the DNA on a filter. The binding reaction requires, in addition to MgCl2, the presence of a nucleoside 5'-triphosphate, but binding is not dependent on hydrolysis; nucleoside 5'-diphosphate will substitute for nucleoside 5'-triphosphate. Of the eight common nucleoside triphosphates, dTTP promotes optimal binding. The half-life of the gene 4 protein-DNA complex depends on both the secondary structure of the DNA and on whether or not the nucleoside 5'-triphosphate cofactor can be hydrolyzed. Using the nonhydrolyzable nucleoside 5'-triphosphate analog, beta,gamma-methylene dTTP, the half-life of the gene 4 protein-DNA complex is greater than 80 min. In the presence of the hydrolyzable nucleoside 5'-triphosphate, dTTP, the half-life of the gene 4 protein-DNA complex using circular M13 DNA is at least 4 times longer than that observed using linear M13 DNA.     

Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.     

Mutator mutations in bacteriophage T4 gene 32 (DNA unwinding protein).

Bacteriophage T4 gene 32 encodes a DNA unwinding protein required for DNA replication, repair, and recombination. Gene 32 temperature-sensitive mutations enhance virtually all base pair substitution mutation rates.