Filter Binding Technique Research Articles

Modification of DNA damage in transcriptionally active vs. bulk chromatin

Our previous experiments have demonstrated that regions of nuclear chromatin, containing transcriptionally active DNA sequences and associated with the nuclear matrix, are hypersensitive to the production of both single-strand breaks and DNA-protein cross-links upon γ-irradiation of exponentially growing mammalian cells. In this study, we have irradiated Chinese hamster V79 cells in buffered saline with or without DMSO to scavenge hydroxyl radicals and in buffered salines of various tonicities to expand or condense chromatin. The yield of DNA-protein cross-links was assayed by a nitrocellulose filter binding technique and the DNA recovered from the cross-links hybridized to 251-poly(A+)RNA to determine the relative frequency of transcriptionally active sequences in the cross-links compared to the bulk DNA. In all cases, the data show that active DNA is affected to a greater extent than bulk, primarily inactive DNA. The more extensive alteration of the level of ionizing radiation-induced damage in active DNA by the diffusible agents tested suggests that other agents, such as chemical sensitizers and protectors, which need to diffuse to the nuclear DNA, may also be acting primarily on active, matrix-associated DNA.

In vitro characterization of hybrid promoters and altered tryptophan operon promoters.

This study examines the in vitro interaction of hybrid and altered Escherichia coli promoters and other promoters with purified E. coli RNA polymerase. Three parameters of polymerase activity were examined: the time for open complex formation; the temperature of transitions; and the time required for productive initiation. The results indicate the rate of in vitro binding as measured by the filter binding technique does not completely correlate with the in vivo activities among these diverse promoters. Transition temperatures ranged from 13 to 27 degrees C with the lowest transition temperatures associated with the relatively weak in vivo beta-lactamase and anti-tet promoters. The productive initiation studies showed a dependence on labeled nucleoside triphosphate concentration when that nucleotide was present early and frequently in the transcript. Promoters containing the -10 region of the lac promoter had slow productive initiation rates while trp -10 promoter derivatives were generally very fast. In the promoters studied here, a trend was noted between the binding rate and transition temperature studies in that the promoters with the lower transition temperatures tended to bind more rapidly.

Cooperative non-specific DNA binding of the N-terminal core of the cyclic AMP receptor protein of Escherichia coli and its modulation by cyclic AMP

The non-specific DNA binding of CRP and its N-terminal core, αCRP, to a 298 base pair DNA fragment, in the presence and absence of cAMP, has been studied using the nitrocellulose filter binding technique and analysed quantitatively using the theory of Clore et al. [J. Mol. Biol. (1982) 155, 447–466]. It is shown that both CRP and αCRP bind cooperatively to DNA. At an ionic strength of 100 mM and pH 7.5, the intrinsic equilibrium association constant for the binding of αCRP to DNA is ∼ 10-times smaller than that for CRP, but the cooperativity parameter is ∼ 17-times larger for αCRP than CRP. cAMP exerts its effect soley on the intrinsic equilibrium constant and does not alter the cooperativity. In the case of αCRP, cAMP reduces the intrinsic equilibrium association constant by a factor of 3, in contrast to the case of CRP where cAMP increases it by a factor of 3. The possible location of the DNA binding site present in the N-terminal core of CRP is discussed in the light of crystallographic data on the cAMP·CRP complex [McKay (1982) J. Biol. Chem. 257, 9518–9524].

Properties of a DNA-binding protein in a culture medium of thymus cells.

A DNA-binding protein, which migrated as one major protein band, with a molecular weight of 14,000, on sodium dodecylsulfate polyacrylamide gel, was purified from a culture medium of mouse thymus cells. The interaction of the isolated protein with DNA in vitro was assayed by a nitrocellulose filter binding technique. Equilibrium competition experiments demonstrated that the DNA-binding protein had the ability to differentiate among sequences of polynucleotides, indicating that the DNA-binding protein-DNA interaction was at least partially specific. This protein increased the helix melting temperature of DNA and inhibited the incorporation of [3H]dTMP into DNA by the DNA polymerase of calf thymus in vitro.

Specific protein-DNA interaction at four sites flanking the chicken lysozyme gene.

Cloned DNA containing 22.2 kb of the chicken lysozyme gene region was screened with use of a nitrocellulose filter binding technique for specific recognition by nuclear DNA binding proteins from chicken oviduct cells. The analysis showed specific retention of four restriction fragments (BS1-BS4), which map approximately 6.1 and 3.9 kb upstream from the transcription start, and 2.8 and 6.2 kb downstream from the poly(A)-addition point of the lysozyme gene. The four DNA fragments mutually served as efficient competitors, indicating that only one class of proteins is involved in the recognition of all four sites. An apparent binding constant of KD = 6 X 10(-12) M was estimated for one of the binding fragments (BS1). Fine mapping of this fragment resolved two closely spaced contact areas at least 43 bp apart.

Interactions of Qβ replicase with Qβ RNA

The interactions of Qβ replicase with Qβ RNA were investigated by treating replicase-Qβ RNA complexes under various conditions with ribonuclease T 1, and by characterizing enzyme-bound RNA fragments recovered by a filter binding technique. Evidence for replicase binding at two internal regions of Qβ RNA was obtained. One region (at about 1250 to 1350 nucleotides from the 5′ end) overlaps with the initiation site for coat protein synthesis; this interaction is thought to be inessential for template activity but rather to be involved in the regulation of protein synthesis. Binding to this site (called the S-site) requires moderate concentrations of salt but no magnesium ions. The other region (at about 2550 to 2870 nucleotides from the 5′ end) is probably essential for template activity; binding to this site (called the M-site) is dependent on the presence of magnesium ions. The nucleotide sequences of the RNA fragments from the two sites were determined and found to have no common features. Under the conditions tested, replicase binding at the 3′ end of Qβ RNA could not be demonstrated, except when initiation of RNA synthesis was allowed to occur in the presence of GTP and host factor. If instead of intact Qβ RNA, a complete RNAase T 1 digest of Qβ RNA was allowed to bind to replicase, oligonucleotides from the S-site and the M-site, and oligonucleotides from a region close to the 3′ end, were found to have the highest affinity to the enzyme. The RNA fragments recovered in highest yield, M-2 and S-3 from the M and S-site, respectively, were isolated on a preparative scale and their enzyme binding properties were studied. In competition assays with random RNA fragments of the same size, selective binding was observed both for the M and the S-site fragment. Partial competition for replicase binding was found if M-2 and S-3 were presented simultaneously to the enzyme. Either fragment, if preincubated with replicase, caused a specific inhibition of initiation of Qβ RNA-directed RNA synthesis, without inhibiting the poly(rC)-directed reaction. The results are discussed in terms of a model of replicase-Qβ RNA recognition. Template specificity is attributed to binding of internal RNA regions to replicase, resulting in a specific spatial orientation of the RNA by which the inherently weak, but essential, interaction at the 3′ end is allowed to occur and to lead to the initiation of RNA synthesis.

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.

ATP-induced changes in the binding of RNA synthesis termination protein rho to RNA

The interaction between rho protein, an RNA synthesis termination factor from Escherichia coli , and synthetic RNA polymers has been studied using a membrane filter binding technique and by the inhibition of enzymes that digest RNA. The binding studies show that rho forms a very stable, salt-resistant complex with poly(C). The high affinity of the interaction correlates with the high activity of poly(C) as an effector of rho-ATPase. Poor rho-ATPase effectors, such as poly(U) and poly(A) also bind to rho but with much lower affinities. However, good binding is not a sufficient condition for activation of rho-ATPase; poly(dC) binds to rho with a higher affinity than poly(U) yet it does not activate rho-ATPase. At low ratios of rho to poly(C), one poly(C) molecule is bound for each molecule of hexameric rho. At higher ratios, a single poly(C) can bind several rho molecules independently. The sites for rho on poly(C) become staturated at a level of one rho molecule per 100 nucleotides, which is about the level expected for close packing of rho on a single-stranded RNA. From the size of poly(C) protected by rho against ribonuclease A digestion, it is concluded that the binding site on rho is large enough to contain 60 residues of poly(C). Poly(C) bound to rho is subject to a bimolecular displacement reaction by free poly(C). The rate of that reaction is decreased by ATP and β-γ imido ATP and increased by ADP. The tight binding of rho to poly(C) or poly(U,C) inhibits the action of polynucleotide phosphorylase, bovine brain exonuclease and Q β replicase on the RNA. The addition of ATP increases the effectiveness of rho in inhibiting these enzymes by a factor of two. ATP also decreases the maximum amount of rho that can be bound to poly(C) by a factor of two. However, ATP does not change the binding stoichiometry when sites on poly(C) are not limiting nor the amount or size of poly(C) protected from ribonuclease A digestion. A model to explain these effects plus the effects of nucleotides on the poly(C) displacement reaction is proposed in which ATP causes poly(C) to become wrapped around rho. We suggest that this occurs through multiple interactions at secondary RNA binding sites on rho.

E coli RNA polymerase-rRNA promoter interaction and the effect of ppGpp.

The interaction between RNA polymerase and the E coli r (ibosomal) RNA promoters of the rrnX and rrnE operon was studied with the filter-binding technique. Quantitative differences were observed between the rrnX and rrnE promoters: stable rrnX promoter complexes are formed faster, and are less sensitive towards heparin and salt than stable rrnE promoter complexes. The effect of ppGpp, the specific inhibitor of rRNA synthesis, on rrn promoter complex formation was studied. In the presence of ppGpp complexes are formed which cannot be trapped in a transcription complex by addition of the start nucleotides, and are therefore considered to be non-productive. A tentative mode for the action of ppGpp is proposed.

Biotinyl 5'-adenylate: corepressor role in the regulation of the biotin genes of Escherichia coli K-12.

A DNA filter-binding technique was used to study the interaction of the biotin repressor and operator site. From a biotin saturation curve, the concentration for half-maximal binding (K0.5) was calculated to be 1 microM. However, in a similar study with the in vitro coupled transcription-translation system in which biotin served as the corepressor, the K0.5 for repression was 7.1 nM. This marked difference of over 2 orders of magnitude was attributed to the activation of biotin by the partially purified repressor preparation in the in vitro system. The activated product formed from biotin, ATP, and repressor preparation was identified as biotinyl 5'-adenylate by paper chromatography and hydroxamic acid formation. Synthetic biotinyl 5'-adenylate was as effective as biotin in the in vitro system (K0.5, 10 nM) and much more effective than biotin in the DNA-binding assay (K0.5 1.1 nM versus 1 microM). These studies indicate that biotinyl 5'-adenylate has a more direct role in the regulation of the biotin genes than does biotin per se.

Isolation and characterization of mutations in the cIII gene of bacteriophage λ which increase the efficiency of lysogenization of Escherichia coli K-12

Mutations of phage λ are described which permit lysogenization of singly infected cells under conditions where λ + lysogenizes only by multiple infection. Deletion mapping places them between the end points of bio252 and bio10, most probably within the cIII gene. In addition to their effects on lysogenization, these mutations ( cIIIs1 and cIIIs2) cause a lengthening of the latent period in one-step growth experiments with lytic infections. This growth defect is relieved by a mutation in the cII gene, but not by a mutation in the Y region. A one-step growth experiment in which cells were mixedly infected by cIIIs cI − and cIII+ cI − showed a latent period intermediate between that of infections by each strain alone. A similar result was obtained in a mixed infection with cIIIs cI − and cIII − cI −. I conclude that the intermediate latent period obtained in mixed infections depends upon the ratio of cIIIs cI − to cIII + cI − or cIII − cI − chromosomes. cIIIs mutants accumulate more repressor than cIII + by 30 min after infection, as assayed by the DNA filter binding technique. These properties are consistent with the idea of a “super cIII” protein, which may be more stable or more active than the wild-type protein.

Specific interaction between mouse liver non-histone chromosomal proteins and mouse DNA demonstrated by a sequential DNA-protein binding procedure

The binding of mouse liver chromosomal proteins to DNA has been investigated using the nitrocellulose filter binding technique. Careful purification of the DNA involving nuclease S 1 digestion and prefiltration through a nitrocellulose filter is used to reduce background binding in the absence of protein to less than 1%. Procedures involving direct binding of protein to labeled DNA, competition of binding of labeled DNA by unlabeled DNA, and dissociation of DNA · protein complexes with time do not indicate significant preference for binding to mouse DNA relative to Escherichia coli DNA. This specificity is demonstrated much more clearly by a novel type of procedure, which we call a sequential binding procedure. In this procedure non-specific binding proteins are sequestered by incubation with an excess of unlabeled E. coli DNA prior to addition of labeled DNA. Under these conditions, labeled mouse DNA is bound to filters to a 3- to 4-fold greater extent than labeled E. coli DNA.

Interaction of contractile proteins with DNA.

The interaction of contractile proteins (myosin, actin, tropomyosin and troponin) with DNA was studied in vitro using a nitrocellulose filter binding technique. The data indicate a high affinity of myosin and troponin for DNA, a lesser interaction between DNA and tropomyosin and the absence of binding of actin to DNA. When binding to DNA was detected, the interaction was higher with single-stranded DNA than with RNA or double-stranded DNA, although in some conditions myosin binds equally as well to native as to denatured eukaryotic DNA. Myosin binds better to eukaryotic than to phage native DNA.

RNA polymerase binding sites in λp lac 5 DNA

The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of lambdaplac5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p(R)', the INT promoter (p(I)), several sites in the b2 region, the mis promoter, the oop promoter (or p(O)), and p(rm). Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the cIII-t(L) region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites. Additional polymerase binding sites have been detected by examining which HindII and HindIII sites were not cleaved when digestion was performed after RNA polymerase had been bound to the DNA. This technique revealed polymerase binding at p(L), at p(R), at a site between R and cos, and at a site at the junction of the gamma and cIII-t(L) fragments. A comparison of the location of polymerase binding fragments with the partial denaturation map of the lambda genome indicates that RNA polymerase binding sites are located within A-T rich regions. It is suggested that RNA polymerase binding is a function both of specific sequences (where recognition occurs) and of the base composition of the surrounding regions (which affects the stability of the helix at the specific site).

The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme.

The extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.

Messenger RNA binding of a ribosome-associated protein kinase and a ribosomal phosphoprotein(s) in mouse plasmacytoma

A specific ribosome-associated protein kinase (protein kinase II) and phosphoprotein(s) from the ribosomal KCl wash fraction termed “PPx” have been isolated from plasmacytoma, and tested for their ability to bind to poly(A) and to different plasmacytoma polynucleotides. The nitrocellulose filter binding technique was used to measure RNA-protein interaction. Protein kinase II and PPx preferentially bound mRNA compared to poly(A). They did not bind ribosomal RNA, soluble RNA or DNA. The optimal conditions (temperature, time, protein concentration, ionic strength) for mRNA-protein interaction were determined. Ribosomal protein kinase (protein kinase II) phosphorylated PPx proteins which bound to mRNA represented at least two bands as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis ( M r = 90 000 and 80 000 ). The high affinity of protein kinase II and PPx for mRNA suggests that they may function in regulating protein synthesis.

Binding of Escherichia coli RNA polymerase to T7 DNA. Displacement of holoenzyme from promoter complexes by heparin.

Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.

Synthesis of polyadenylic acid RNA during zoospore differentiation and germination in Blastocladiella emersonii

Blastocladiella emersonii zoospores and germlings were assayed for the presence of poly(A) RNA by the poly(U) glass-fiber filter binding technique and oligo(dT)-cellulose chromatography. Results from assays of sucrose density gradient fractions show that poly(A) RNA is synthesized de novo during germination of zoospores. In addition, a portion of the performed mRNA in the zoospore has a poly(A) fragment attached to the 3′-terminus. Zoospore poly(A) fragments migrate on polyacrylamide gels slightly faster than the 4S RNA and have an AMP:adenosine ratio of 49:1. During germination some of the performed mRNA is adenylated. Experiments with actinomycin D demonstrated that transcription is not required for polyadenylation of zoospore mRNA. These findings indicate that lack of a poly(A) fragment is not an explanation for the inability of the zoospore messages to participate in protein synthesis.

The binding of polynucleotides to the DNA polymerase of avian myeloblastosis virus

The interaction between avian myeloblastosis virus DNA polymerase and synthetic nucleic acids was studied by an adaptation of the membrane filter binding technique. Bacillus subtilis DNA was used as a substrate for the binding reaction and was retained on the filters in the presence of the viral polymerase. The polymerase activity was demonstrated to be retained on the filter in either the presence or absence of the bacterial DNA. Characterization of the polymerase-DNA interaction demonstrated a marked similarity to previous data regarding the binding of Escherichia coli DNA-dependent RNA polymerase to nucleic acids when studied using related techniques. In contrast, the association between methylated bovine serum albumin and the B. subtilis DNA was found to differ significantly in both reaction stoichiometry and stability. Synthetic polynucleotides were shown to inhibit the binding of the bacterial DNA to the viral DNA polymerase and poly 2′-fluoro-2′-deoxyuridylic acid was found to be the most potent inhibitor of this reaction. Results from the binding-inhibition studies correlated well with studies concerning the inhibition of enzyme activity and it is concluded that the inhibitory polynucleotides act by interfering with binding of nucleic acid template to the viral enzyme.

Stability of different ribosomal complexes with initiator transfer RNA and synthetic messenger RNA

Stability of the mRNA-initiator tRNA-ribosomal complex has been studied by nitrocellulose filter binding and thermal dissociation techniques. There is no significant difference between the stability of the 70 s ribosomal complex involving fMet-tRNA f§ and its coding triplet and the stability of other synthetic mRNA-aminoacyl-tRNA systems including the ApUpG-Met-tRNA f complex. However, the 30 s subunit complex with fMet-tRNA f and ApUpG is significantly more stable than Met-tRNA f-ApUpG and other aminoacyl-tRNA-mRNA complexes. Further, the 30 s subunit complex with acetylphenylalanyl-tRNA and poly U is unstable. A stable 70 s ribosomal complex with Met-tRNA m is formed with poly (A,U,G) but not with the triplet ApUpG.