A comparison of the phage T4 gene 32 protein and Escherichia coli RNA polymerase binding sites on hamster papovavirus DNA

Phage T4 gene 32 protein and Escherichia coli RNA polymerase were bound to hamster papovavirus DNA. The binding regions were identified by electron microscopy employing a protein-free spreading technique. After gene 32 protein treatment four denaturation regions could be mapped, at 0.04–0.12, 0.30–0.36, 0.50–0.60 and 0.75–0.90 DNA map units, respectively, using the unique BamHI cleavage site as zero point. Eight RNA polymerase binding sites can be found which are localized at positions 0.05; 0.11; 0.18; 0.31; 0.57; 0.66; 0.76 and 0.82. A comparison of the RNA polymerase binding sites with the gene 32 protein denaturation pattern reveals a correspondence of six of eight polymerase binding sites with (A + T)-rich regions within the hamster papovavirus genome.     

DNA of the Streptomyces phage SH10: Binding sites for Escherichia coli RNA polymerase and denaturation map

Summary Escherichia coli RNA polymerase bound to Streptomyces phage SH10 DNA was visualized by electron microscopy. Six specific binding sites were observed at map units 53, 85, 93, 97, 98, and 99 on the physical map of the 48 kb long genome. Electron microscopy of partially denatured SH10 DNA revealed a characteristic melting pattern of A+T-rich regions around map units 1, 3, 48, 52, and 99. A comparison of the denaturation map with the RNA polymerase binding sites indicates that three binding sites are located in the most A+T-rich regions, two in other early melting regions and one in a segment of higher DNA helix stability.     

Electron microscopic mapping of wheat germ RNA polymerase II binding sites on cloned CaMV DNA.

The binding sites of wheat germ RNA polymerase II were mapped on the cloned CaMV genome by observation of enzyme-linear DNA complexes by electron microscopy. Twelve sites are observed. Three of them are relatively stable in the presence of heparin and are found at positions 8-9, 21-23, and 41-44 map units on the physical map of the genome. These positions correspond to AT-rich regions of the viral genome which contain potential promoter sites. These results are discussed with reference to current information on the structure and expression of the CaMV genome.     

Mung bean nuclease cleavage of a dA + dT-rich sequence or an inverted repeat sequence in supercoiled PM2 DNA depends on ionic environment

We have determined the nucleotide sequences around two alternative sites cleaved in supercoiled PM2 DNA by single-strand-specific mung bean nuclease in different ionic environments. In 10 mM Tris-HC1 (pH 7.0, 37 degrees C), the major site is a dA+dT-rich sequence which maps with a known early denaturation region at 0.75 map units. About 30 cleavages occurred in a 135 bp region. Cleavages were largely excluded at (dA)n . (dT)n (n = 3-7) sequences. Cleavage patterns of this type have not been previously observed in dA+dT-rich sequences. With the addition of 0.1 M NaC1 the major alternative site occurred in a hyphenated inverted repeat sequence 500 bp away (0.70 map units) and did not map to an early denaturation region. One major and 4 minor cleavages occurred in the region between the repeats, suggesting that a hairpin containing at most a 12 bp stem and 10 base loop is recognized. The basis for nuclease recognition of the dA+dT-rich sequence is not clear. The differences in the sequences and cleavage patterns at the alternative sites indicate that their secondary structures differ.     

Deoxyribonucleic acid sequence organization of a yeast plasmid.

Two-micrometer deoxyribonucleic acid (DNA), a circular plasmid of Saccharomyces cerevisiae, contains two nontandem repeated sequences which are inverted with respect to one another. These repeated sequences together account for 21% of the molecule length. Restriction endonuclease analysis and electron microscopy demonstrated the existence of two forms of 2-mum DNA differing in the orientation of the interstitial segments bounded by the inverted repeated sequences. The two forms of 2-mum DNA could result from an intramolecular reciprocal recombination between inverted repeat elements. A map containing the restriction endonuclease sites and the units of the inverted repeat has been constructed.     

Transcription of polyoma virus DNA in vitro. III. Localization of calf thymus RNA polymerase II binding sites.

The binding sites of calf thymus RNA polymerase II on polyoma DNA were monitored by electron microscopy. Six discrete binding sites were located at positions 0.06, 0.25, 0.57, 0.66, 0.85 and 0.98 on the physical map of polyoma DNA. Although most of these sites are located in easily denaturable regions of the DNA, the strongest binding sites do not overlap with the major A + T-rich regions. In addition, the same binding sites were observed on superhelical or linear polyoma DNA. These results suggest that the eucaryotic RNA polymerase II can recognize specific sequences on double-stranded DNA and not only easily denaturable regions. At least five of these sites correspond to the binding and initiation sites mapped previously for the Escherichia coli RNA polymerase (Lescure et al., 1976).Stable initiation complexes can be formed with both E. coli and calf thymus RNA polymerases in the presence of a single dinucleotide (GpU) and a specific ribotriphosphate (CTP). Under these conditions, the binding of both enzymes to the sites in positions 0.06 and 0.57 is stimulated whereas the binding in positions 0.65 and 0.84 is partially suppressed. Both eucaryotic and procaryotic RNA polymerases may recognize similar sequences of the viral DNA in vitro.     

Study of the binding of RNA polymerase by a recombinant plasmid using electron microscopy

RNA-polymerase of E. coli was bound in vitro under physiological conditions to a recombinant plasmid pBR322 carrying two identical segments of bacteriophage T4 DNA, each containing a complete gene coding for T4 DNA ligase. After fixation of the complex with formaldehyde it was analysed by electron microscopy. A map of binding sites of the enzyme to DNA was obtained after a statistical assessment of micrographs. The inserted repeat revealed itself in the map as two regions of identical binding patterns, thus proving the adequacy of the preparation procedure. In pBR322 the strong binding sites correlate with the position of promoters. Also, apart from this, there are other strong binding sites within the T4 sequences which correlate strongly with the regions of abnormally high AT content. That means that under physiological conditions the RNA polymerase forms strong binding complexes with any AT rich DNA regions, as well as with real promoters.     

Rescue of a herpes simplex virus type 1 neurovirulence function with a cloned DNA fragment.

A herpes simplex virus type 1 (HSV-1) genetic function that is required for viral replication in the murine central nervous system was unambiguously localized. Thus, cosmid clones of either HSV-1 HindIII fragment C (0.64 to 0.87 map units) or fragment B (0.64 to 0.83 plus 0.91 to 1.0 map units) were employed to restore neurovirulence to an intertypic recombinant (RE6) that is specifically deficient in this property. The neurovirulent recombinants were generated in cell culture by cotransfecting the clone fragments and unit-length RE6 DNA and then selected in mouse brains. Either fragment efficiently conferred neurovirulence to RE6, demonstrating that no short region unique sequences are required. Analyses of the genomic structures of the neurovirulent recombinants showed that, in every case, HSV-1 information from 0.71 to 0.83 map units was incorporated into the RE6 genome. Cleavage of HindIII fragment C with EcoRI eliminated its capacity to rescue RE6. Virulence could be restored by the addition of HSV-1 BamHI fragment L (0.71 to 0.74 map units) that spans an EcoRI site at 0.72 map units. The precise location of this HSV-1 neurovirulence function is discussed.     

A DNA-binding protein specific for the early replicated region of the chromosome obtained from Escherichia coli membrane fractions

The binding sites of calf thymus RNA polymerase (B) II, wheat germ RNA polymerase B and of the Escherichia coli RNA polymerase were mapped on the simian virus 40 genome by observation of enzyme-linear DNA complexes by electron microscopy. Three to four major sites and several minor sites are observed for each enzyme; common binding sites for the three enzymes are found in positions 0.17, 0.53 and 0.90 of the viral physical map. Initiation complexes with these enzymes can be stabilized with specific ribodinucleotides and a single ribonucleoside triphosphate. Whereas ApA and ATP greatly enhances the binding of the E. coli enzyme at position 0.17, they stabilize the binding of the eukaryotic enzyme at many sites, some of them located in close proximity of the origin of replication.     

Binding sites of calmodulin and actin on the brain spectrin, calspectin

We used rotary-shadowing electron microscopy to map the calmodulin-and actin-binding sites on the brain spectrin, calspectin (or fodrin). Calspectin dimers appeared as rods 110 nm long and joined in a head-to- head manner to form tetramers 220 nm long. We determined calmodulin- binding sites by a ferritin-labeling method combined with biotin-avidin complex formation. Ferritin particles were found to attach to the head parts of calspectin dimers at a position 10-20 nm from the top of the head. The number of the calmodulin-binding sites seemed to be only one for each dimer and two for each tetramer. In contrast, the actin- binding sites were localized at the tail ends of the calspectin molecules. The tetramers attached to muscle F-actin with their tail ends and often cross-linked adjacent filaments. The results are discussed in view of the analogy to the erythrocyte spectrin.     

Location of ribosome binding sites on the right end of lambda DNA.

The locations of ribosome binding sites on the right end of bacteriophage lambda DNA were determined by measurement of the positions of ribosomes bound to single-stranded DNA visualized by electron microscopy. A total of ten ribosome binding sites were found between map co-ordinates 0.90 and 1.0. Four of these ribosome binding sites probably correspond to the polypeptide initiation sites for genes Q (0.910), S (0.928), R (0.936) and Rz (0.945). Six other ribosome binding sites were found which presumably indicate the presence of new lambda genes. Four of these ribosome binding sites (0.958, 0.967, 0.975, 0.995) are located to the right of Rz, which is the most rightward known lambda gene. A ribosome binding site is located at 0.923, between genes Q and S, in or near the 6 S RNA sequence. Another is located left of gene Q at 0.900.     

Three-dimensional structure of the respiratory chain supercomplex I1III2IV1 from bovine heart mitochondria

The respiratory chain complexes can arrange into multienzyme assemblies, so-called supercomplexes. We present the first 3D map of a respiratory chain supercomplex. It was determined by random conical tilt electron microscopy analysis of a bovine supercomplex consisting of complex I, dimeric complex III, and complex IV (I1III2IV1). Within this 3D map the positions and orientations of all the individual complexes in the supercomplex were determined unambiguously. Furthermore, the ubiquinone and cytochrome c binding sites of each complex in the supercomplex could be located. The mobile electron carrier binding site of each complex was found to be in proximity to the binding site of the succeeding complex in the respiratory chain. This provides structural evidence for direct substrate channeling in the supercomplex assembly with short diffusion distances for the mobile electron carriers.     

Torsional stress induces left-handed helical stretches in DNA of natural base sequence: circular dichroism and antibody binding

Above a threshold of torsional stress, the c.d. spectrum of covalently closed circular DNA of natural base sequence acquires a Z-like contribution and antibodies raised against Z-DNA are bound. Mapping of the antibody binding sites by electron microscopy reveals sites which correlate with stretches enriched in alternating purine-pyrimidine sequences and GC base pairs.     

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density).     

A large inverted repeat sequence overlaps two acceptor splice sites in adenovirus

The distribution of nucleotide sequences resembling functional sites for mRNA splicing was examined by computer-directed searches in order to determine what factors may influence splice site selection in nuclear precursors. In particular, the distribution of large potentially stable hairpin structures or regions of extensive dyad symmetry was studied in adenovirus sequences. One region, spanning 106 nucleotides, was found at 66.4 map units, overlapping back-to-back acceptor sites for two mRNA molecules, those coding for the 100K protein and the 72K DNA binding protein, which are transcribed from opposite strands. This region displays exceptional dyad symmetry and is potentially capable of forming a single, highly stable hairpin when transcribed. It seems likely that the secondary structure as well as the primary structure of RNA plays a role in determining the correct splicing of these mRNA molecules.     

Carbohydrate binding sites in a pancreatic alpha-amylase-substrate complex, derived from X-ray structure analysis at 2.1 A resolution.

The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) that was soaked with the substrate maltopentaose showed electron density corresponding to two independent carbohydrate recognition sites on the surface of the molecule. Both binding sites are distinct from the active site described in detail in our previous high-resolution study of a complex between PPA and a carbohydrate inhibitor (Qian M, Buisson G, Duée E, Haser H, Payan F, 1994, Biochemistry 33:6284-6294). One of the binding sites previously identified in a 5-A-resolution electron density map, lies at a distance of 20 A from the active site cleft and can accommodate two glucose units. The second affinity site for sugar units is located close to the calcium binding site. The crystal structure of the maltopentaose complex was refined at 2.1 A resolution, to an R-factor of 17.5%, with an RMS deviation in bond distances of 0.007 A. The model includes all 496 residues of the enzyme, 1 calcium ion, 1 chloride ion, 425 water molecules, and 3 bound sugar rings. The binding sites are characterized and described in detail. The present complex structure provides the evidence of an increased stability of the structure upon interaction with the substrate and allows identification of an N-terminal pyrrolidonecarboxylic acid in PPA.     

Natural occurrence of left-handed (Z) regions in PM2 DNA

Bacteriophage PM2 DNA, a ccc genome of high apparent superhelical density, contains left-handed (Z) regions as detected by competitive radioimmunoassay, agarose gel electrophoresis of DNA: antibody complexes and immunoelectron microscopy. The latter technique, in conjunction with partial blockage of restriction endonuclease sites by bound antibody, was used to map the left-handed regions along the DNA molecule. A cluster of four to five antibody molecules (approximately 25% of bound antibody) was located within map units 0.05-0.18 of the single Hpa II restriction site. Sequence analysis of part of this region showed the presence of several areas of high alternating purine-pyrimidine content. A strong correlation is observed between alternating pyrimidine-purine tracts of significant length and antibody binding sites.