Topography of simian virus 40 A protein-DNA complexes: arrangement of protein bound to the origin of replication.

DNA binding regions I, II, and III at the origin of replication have different arrangements of A protein (T antigen) recognition pentanucleotides. The A protein also protects each region from DNase in distinctly different patterns. Footprint and fragment assays led to the following conclusions: (i) in some cases a single recognition pentanucleotide is sufficient to direct the binding and accurate alignment of A protein on DNA; (ii) the A protein binds within isolated region I or II in a sequential process leading to multiple overlapping areas of DNase protection within each region; and (iii) the 23-base pair span of recognition sequences in region II allows binding and protection of a longer length of DNA than the 23-base pair span in region I. We propose a model of protein binding that addresses the problem of variations in the arrangement of pentanucleotides in regions I and II and explains the observed DNase protection patterns. The central feature of the model requires each protomer of A protein to bind to a pentanucleotide in a unique direction. The resulting orientation of protein would protect more DNA at the 5' end of the 5'-GAGGC-3' recognition sequence than at the 3' end. The arrangement of multiple protomers at the origin of simian virus 40 replication is discussed.     

Characterization of two distinct positive cis-acting elements in the mouse alpha 1 (III) collagen promoter

We have identified two distinct sequence elements in the mouse alpha 1(III) collagen promoter which are protected from DNase I digestion by the binding of factors present in crude nuclear extracts of NIH 3T3 fibroblasts. Small substitution mutations were introduced into these promoter elements and shown by the gel retardation (gel mobility shift) and DNase I protection assays to decrease or eliminate factor binding to the mutated element but not to the remaining wild-type element, indicating that two distinct factors recognize these separate promoter regions. Region A appears to bind a factor related to the Jun/AP-1 protein, whereas the factor binding to region B remains as yet unidentified. Mutagenesis of either region decreased the activity of the alpha 1(III) collagen promoter in DNA transfection assays by about 3-fold for the A region (located between - 122 and - 106) and about 5-fold for the B region (located between -83 and -61). These results indicate that regions A and B in the mouse alpha 1(III) collagen promoter are positive cis-regulatory elements, independently binding two distinct trans-activating factors.     

Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor.

The promoter region of the pColV-K30-encoded operon specifying biosynthesis and transport of the siderophore aerobactin was subjected to deletion analysis to determine the smallest DNA sequence affording iron regulation of a iucA'-'lacZ gene fusion. A 78-base-pair (bp) region containing the main (P1) promoter retained the character of inducibility under iron starvation. A 250-bp fragment carrying this sequence was examined for protection against DNase I by the Fur protein, the product of a gene (fur) required for negative control of several iron-regulated functions. The DNase I footprints, in the presence of various divalent heavy-metal ions added as corepressors, revealed two contiguous binding sites with different lengths and affinities for Fur. Increased concentrations of the protein appeared to elicit formation of repressor oligomers which bind to the upstream and downstream regions of the P1 promoter in a metal-dependent fashion, but with a presently undefined stoichiometry. The primary site for Fur binding spans 31 bp and contains two overlapping symmetry dyads which share the sequence 5'-TCATT-3'. It also contains extensive homology with a 19-bp consensus sequence for iron-regulated genes as deduced from comparison with the fhuA and fepA putative promoter sequences.     

T7 RNA polymerase interacts with its promoter from one side of the DNA helix

The interactions of T7 RNA polymerase with its promoter DNA have been previously probed in footprinting experiments with either DNase I or (methidiumpropyl-EDTA)-Fe(II) to cleave unprotected DNA [Basu, S., & Maitra, U. (1986) J. Mol. Biol. 190, 425-437. Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614-3618]. Both of these reagents have drawbacks; DNase I is a bulky reagent and so provides low resolution, and (methidiumpropyl-EDTA)-Fe(II) intercalates into DNA and is therefore biased toward cleavage of double-stranded DNA. In this study, the interaction between the polymerase and the promoter has been probed with Fe(II)-EDTA. This reagent generates reactive hydroxyl radicals free in solution, which produces a more detailed picture of the polymerase-promoter complex. Two protected regions are observed on each of the two promoter DNA strands: from position -17 to position -13 and from position -7 to position -1 on the coding strand and from position -14 to position -9 and from position -3 to position +2 on the noncoding strand. From this pattern it is clear that if recognition occurs via double-stranded B-form DNA, then the protected regions lie on one face of the DNA helix, and therefore the enzyme must interact predominantly from one side of the DNA helix. Digestion of the DNA in a polymerase-promoter complex with a single-strand-specific endonuclease shows that a small region of the noncoding strand near position -5 is susceptible to cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Terminase host factor: a histone-like E. coli protein which can bind to the cos region of bacteriophage lambda DNA.

Terminase Host Factor (THF), an E. coli protein capable of fulfilling the host factor requirement for in vitro bacteriophage lambda terminase activity, displays properties characteristic of the prokaryotic type II DNA-binding or "histone-like" proteins. It is a 22 K basic, heat- and acid-stable protein which binds non-specifically to various DNAs. Conditions can be established, however, where THF binds preferentially to the cohesive end site (cos) of lambda DNA forming several distinct complexes as visualized by band retardation in polyacrylamide gels. DNase I footprinting reveals that THF can protect several regions of the top strand on the right side (+) of cos but does not bind as well to the left side (-). The binding regions are separated either by unprotected or by DNase I- hypersensitive bases. Under the conditions used in these experiments, DNA which does not contain cos lambda sequences does not show this pattern of protection. Several repeated motifs in the cos lambda nucleotide sequence may represent a consensus sequence for THF interaction. THF may be similar to other "histone-like" proteins which display both non-specific and selective DNA-binding capacities.     

High-resolution analysis of a histone H1 binding site in a rat albumin gene

Interaction of rat liver histone H1 fraction with the 5'-end of the rat serum albumin gene was localized within a 346 base pair (bp) restriction fragment. Sequence analysis of the fragment showed the fragment was 72 mol % adenosine-thymidine, which is significantly greater than the mole percent adenosine-thymidine composition of the rat genome. Gel retardation assays of the histone H1-DNA interaction indicate the complex formed behaves as previously characterized H1-DNA and shows a high-affinity H1 binding site within the enriched albumin restriction site. Deoxyribonuclease I (DNase I) protection assays on the H1 binding site define three protected regions only on the template strand of the DNA fragment. The three sites lie 55 and 110 bp apart (165 bp between the first and third binding site) with a consensus binding sequence of 5'-GA-ATA-CTGGCTT-C-TT-CTA-G-3'. The sequences between the protected DNA regions are highly enriched in adenosine-thymidine bases (79.3 and 86 mol % adenosine-thymidine, respectively). The functional significance is not understood.