Dynamics of nucleosome invasion by DNA binding proteins

Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.     

Fractionation of chylomicrons by heparin-sepharose chromatography. Characterization of two heparin-binding proteins

Rat lymph chylomicrons were separated into two fractions using heparin-Sepharose chromatography: a major fraction which elutes from the column with the void volume at 0.05 M NaCl, and a smaller fraction which binds to the column at 0.05 M NaCl and elutes at 0.3 M NaCl. These two fractions differ in mean particle size, and lipid and protein compositions. Both fractions share apolipoproteins B, A-IV, E, A-I, and C, but the fraction which binds to heparin-Sepharose contains two additional proteins: protein I (Mr = 6.0 X 10(4)), and protein II (Mr = 8.0 X 10(4)). Both proteins are also present in the lipoprotein-free fraction of rat serum. Proteins I and II bind to heparin-Sepharose, and are highly amphiphilic: they bind with high affinity to phospholipid surfaces and form stable monolayers at the air-water interface. The molecular weight, amino acid composition, heparin binding, and amphiphilicity of protein I resemble that of beta 2-glycoprotein I; in addition, protein I from rat lymph chylomicrons cross-reacts with rabbit antiserum to human beta 2-glycoprotein I, suggesting that these two proteins are homologous. Protein II appears to be a previously undescribed protein. The possible functions of these two proteins are discussed.     

Structural proteins of two salmonid rhabdoviruses.

Purified infectious hematopoietic necrosis (IHN) virus and the virus of haemorrhagic septicaemia (VHS) (Egtved virus) each contain five structural proteins which were designated L, G, N, M-1, and M-2. The IHN viral polypeptides have molecular weights estimated to be 157,000, 72,000, 40,000, 25,000 and 20,000, respectively, whereas those of VHS viral polypeptides are estimated to be 157,000 74,000, 41,000, 21,500, and 19,000, respectively. The carbohydrate composition of the glycoprotein (G) was confirmed by demonstrating selective incorporation of [3H]glucosamine into the designated G protein of both viruses. Phosphoproteins were identified by incorporation of [32P]orthophosphate into the N and M-1 proteins of IHN virus and into the N protein of VHS virus. The glycoprotein of each virus was selectively solubilized by treatment with Triton X-100 in low salt buffer, whereas the M-1, and M-2 proteins along with the G protein were solubilized by Ttition X-100 in 0.43 M NaCl. The protein composition of the salmonid rhabdoviruses resembles that of the rabies virus group more closely than the vesicular stomatitis virus group.     

Synthesis of two lactogenic proteins by the mouse placenta in vitro.

The culture medium of mouse placental tissues was analyzed on acrylamide gel electrophoresis to localize lactogenic substances. Placental explants from 12- or 14-day-pregnant BALB/cHe mice were organ-cultured for 6 days in leucine-free Way-mouth's medium supplemented with 3H-leucine (10 mu/Ci/ml) and insulin (0.12I.U./ml). The medium was collected every other day and subjected to acrylamide gel electrophoresis. The electrophoretic pattern of radioactive leucine incorporated into proteins was examined on stained 7% gels. Five protein bands were associated with high radioactivity. The location of lactogenic activity on acrylamide gel was when investigated by the technique of organ culture of mouse mammary tissues. Placental explants from 12- to 14-day-pregnant BALB/cHe mice were organ-cultured in Waymouth's medium supplemented with insulin for 2 days. After electrophoresis, proteins were eluted by keeping the segment of acrylamide gel in phosphate buffer, dialyzed and dissolved in tissue culture medium 199 supplemented with insulin and cortisol. Mammary tissues from 8-day-pregnant KA2 mice were cultured for 3 days in the medium containing each eluate. Mammary glands always responded to eluted proteins from two positions of 7% gel, as judged in histological sections. The data suggest the presence of two lactogenic substances in the mouse placenta.     

Dynamics of mammalian NER proteins

Despite detailed knowledge on the genetic network and biochemical properties of most of the nucleotide excision repair (NER) proteins, cell biological analysis has only recently made it possible to investigate the temporal and spatial organization of NER. In contrast to several other DNA damage response mechanisms that occur in specific subnuclear structures, NER is not confined to nuclear foci, which has severely hampered the analysis of its arrangement in time and space. In this review the recently developed tools to study the dynamic molecular transactions between the NER factors and the chromatin template are summarized. First, different procedures to inflict DNA damage in a part of the cell nucleus are discussed. In addition, technologies to measure protein dynamics of NER factors tagged with the green fluorescent protein (GFP) will be reviewed. Most of the dynamic parameters of GFP-tagged NER factors are deduced from different variants of 'fluorescence recovery after photobleaching' (FRAP) experiments and FRAP analysis procedures will be briefly evaluated. The combination of local damage induction, genetic tagging of repair factors with GFP and microscopy innovations have provided the basis for the determination of NER kinetics within living mammalian cells. These new cell biological approaches have disclosed a highly dynamic arrangement of NER factors that assemble in an orderly fashion on damaged DNA. The spatio-temporal analysis tools developed for the study of NER and the kinetic model derived from these studies can serve as a paradigm for the understanding of other chromatin-associated processes.     

Dynamics of lipid transfer by phosphatidylinositol transfer proteins in cells

Of many lipid transfer proteins identified, all have been implicated in essential cellular processes, but the activity of none has been demonstrated in intact cells. Among these, phosphatidylinositol transfer proteins (PITP) are of particular interest as they can bind to and transfer phosphatidylinositol (PtdIns)--the precursor of important signalling molecules, phosphoinositides--and because they have essential functions in neuronal development (PITPalpha) and cytokinesis (PITPbeta). Structural analysis indicates that, in the cytosol, PITPs are in a 'closed' conformation completely shielding the lipid within them. But during lipid exchange at the membrane, they must transiently 'open'. To study PITP dynamics in intact cells, we chemically targeted their C95 residue that, although non-essential for lipid transfer, is buried within the phospholipid-binding cavity, and so, its chemical modification prevents PtdIns binding because of steric hindrance. This treatment resulted in entrapment of open conformation PITPs at the membrane and inactivation of the cytosolic pool of PITPs within few minutes. PITP isoforms were differentially inactivated with the dynamics of PITPbeta faster than PITPalpha. We identify two tryptophan residues essential for membrane docking of PITPs.     

Specificity of translational regulation by two DNA-binding proteins

The gene V protein of the filamentous bacteriophages f1, fd and M13, and the gene 32 protein of bacteriophage T4 share the property of binding strongly and co-operatively to single-stranded nucleic acids, especially DNA. Moreover, both are capable of repressing the translation of specific mRNAs (gene 32 protein its own, and gene V protein that of the filamentous phage gene II), both in vivo and in vitro. If the mechanism of repression by either of these proteins were based solely on its ability to bind single strands co-operatively, then the other would be expected to mimic or interfere with its effect in vitro. We have found no such mimicry or interference, even at protein concentrations high enough to have substantial non-specific effects on translation. This suggests that the sites of repression on the mRNAs must offer something other than simple “unstructuredness” for binding and repression to occur.     

Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene.

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.     

Analysis of Escherichia coli ribosomal proteins by an improved two dimensional gel electrophoresis. I. Detection of four new proteins

Kaltschmidt and Wittmann's two dimensional gel electrophoresis was improved in the following points. Preruns using radical scavengers were carried out to eliminate free radicals remaining in gels. Gelation of sample solutions was not performed to avoid immobilization of proteins in the sample gels. Instead, for preparing sample gels, prior to the first dimension (1-D) electrophoresis, another electrophoresis was performed to charge proteins into gel pieces polymerized previously. Proteins migrated together with charged reductants to avoid formation of artificial disulfide bridges during migration. The second dimension (2-D) electrophoresis was carried out at a more acidic pH, 3.6, to get better separation of very small and basic proteins. With these modifications, quantitative yield and reproducibility became better, and many faint spots disappeared not only at the high molecular weight side but also in the region containing primary spots of ribosomal proteins. The proportionality of the migration distance to the logarithm of molecular weight was also increased. On the improved 2-D electrophoretogram of Escherichia coli ribosomal proteins, four new spots, called protein A, B, C, and D, were found in the basic region. Proteins A, B, and C belong to 50S subunits but D to 30S. Their molecular weights were determined electrophoretically as 6,400, 4,900, 8,200, and 5,900, respectively. Their copy numbers in crude ribosomes were estimated to be 0.6, 0.4, 0.3, and 0.1, respectively, by using 14C-labeling.     

Analysis of Escherichia coli ribosomal proteins by an improved two dimensional gel electrophoresis. II. Characterization of four new proteins

Four new proteins, A, B, C, and D, found in Escherichia coli ribosomes by an improved two dimensional gel electrophoresis were characterized by oxidation, reduction, and carboxymethylation of cysteine residues, and CsCl fractionation. The cysteine contents of proteins A, B, C, and D were determined to be 1 +/- 0, 3 +/- 1, 5 +/- 1, and 0 +/- 0 by carboxymethylation with iodoacetic acid. The components of protein complexes, which formed numerously under non-reducing conditions, were analyzed. Including protein A, B, and C, every ribosomal protein (r-protein) having cysteine residue(s) except unconfirmed S1 was proved to form such complexes with various combinations. The cysteine residue in protein A, in particular, was highly reactive to make intermolecular S-S bridges so that spot A almost disappeared on the second dimension gel under the non-reducing conditions. Proteins B and C shifted their spots by reduction towards upper left side as do all known r-proteins having plural cysteine residues except S1. This suggests that proteins B and C change their conformation by intramolecular S-S bridges. The CsCl density gradient centrifugation of high salt washed 70S ribosomes showed that protein A belonged to the insoluble split proteins, proteins B and C to the core particles, and protein D and a small population of B to the soluble split proteins. The electrophoretic behaviors, CsCl fractionation and stoichiometry of the four new proteins suggested strongly that they were intrinsic ribosomal constituents different from known ribosomal proteins or factors.     

Yeast ribosomal proteins. VIII. Isolation of two proteins and sequence characterization of twenty-four proteins from cytoplasmic ribosomes

Summary Two proteins, YL41 and YL43, were isolated from 80S ribosomes of Saccharomyces cerevisiae by filtration through a Sephacryl S-200 column and by chromatography on a column of carboxymethylcellulose. Their amino acid compositions are presented. Twenty-four proteins including these two proteins were subjected to sequence analyses by automated Edman degradation. Amino-terminal amino acid sequences were determined for 17 proteins, YS3, YS9, YS23, YS24, YS29, YL6, YL8, YL11, YL15, YL17, YL23, YL28, YL33, YL37, YL39, YL41, and YL43. YL41, which has a 72.7% lysine and arginine content, was found to be particular to eukaryotic ribosomes. The aminotermini of another seven proteins, YS2, YS5, YS8, YS12, YS13, YS20, and YS27, were suggested to be blocked.Comparison of the amino-terminal sequences with all other ribosomal protein sequences so far available indicates that YS9 shows sequence homology to rat liver ribosomal protein S8 (Wittmann-Liebold et al. 1979).     

Evidence for zinc binding by two structural proteins of Plodia interpunctella granulosis virus.

Workers in our laboratory previously reported the possibility of cation involvement in the in vitro dissociation of the Plodia interpunctella granulosis virus nucleocapsids (K. A. Tweeten, L. A. Bulla, Jr., and R. A. Consigli, J. Virol. 33:866-876, 1980; M. E. Wilson and R. A. Consigli, Virology 143:516-525, 1985). The current study found zinc associated with both granulosis virus nucleocapsids and granulin by atomic absorption analysis. A blotting assay with 65Zn2+ specifically identified the radioactive cation as binding to two viral structural proteins, granulin and VP12. These findings indicate that zinc may have a critical role in maintaining virus stability.     

Recognition of a DNA operator by a dimer composed of two different homeodomain proteins.

The yeast homeodomain proteins a1 and alpha 2 interact to form a heterodimer that binds DNA with high specificity. The DNA recognition element consists of two similar half sites, arranged with dyad symmetry and separated by a fixed number of base pairs. We demonstrate that in the a1 alpha 2-DNA complex, one of these half-sites is bound by a1 while the other is bound by alpha 2. These assignments allow a comparison of the chemical and nuclease protection patterns produced by both proteins when bound together to the hsg operator. Contrary to simple expectations, we propose that the a1 and alpha 2 homeodomains are arranged on the DNA in tandem, despite the fact that the recognition sequence is dyad symmetric.     

Comparison of fractionation of groundnut proteins by two different methods

The salt-soluble proteins of groundnut meal were fractionated by precipitation with (NH₄)₂SO₄ by increasing the (NH₄)₂SO₄ saturation in steps of 10%. The sharp separation into arachin and conarachin claimed by earlier workers was not achieved, as protein was precipitated at each stage from 20 to 100% saturation with (NH₄)₂SO₄. The fractions so obtained were examined by disc electrophoresis on polyacrylamide gel and the amino acid compositions were determined by ion-exchange chromatography. Differences in both electrophoretic pattern and amino acid composition were found. The protein precipitated by CaCl₂ solution was similar in yield, nitrogen content, electrophoretic pattern, and amino acid composition to the fraction precipitating at 10–20% (NH₄)₂SO₄ saturation. The main differences in amino acid composition of the various fractions precipitated by (NH₄)₂SO₄ were found in the amino acids cystine, methionine, and lysine, which increased with increase in (NH₄)₂SO₄ saturation. The electrophoretic pattern and amino acid composition of “conarachin” varied according to the method of preparation.     

Dynamics in transient complexes of redox proteins

Recent studies have provided experimental information about the initial stage of protein complex formation, the encounter complex. This stage is particularly important in the weak and transient complexes formed between electron transfer proteins and their partners. These studies are discussed and the role of the encounter complex is interpreted in terms of the specific requirements that the biological function puts on these complexes.