Transcription factor-DNA Complexes Research Articles

Spin-column isolation of DNA–protein interactions from complex protein mixtures for AFM imaging

Applications of atomic force microscopy (AFM) to investigate structural–functional interactions between DNA and proteins, at the molecular level, should prove valuable for gaining a better understanding of gene expression. Specific genomic DNA–protein interactions occur within a sea of intracellular proteins. Successful AFM imaging requires isolating the specific DNA–protein complex free of background protein contamination. Using spin-column chromatography, we report the successful isolation and AFM imaging of transcription factor DNA complexes from DNA molecules incubated with crude cell lysates. This method should be applicable for the isolation and imaging of other specific DNA–protein complexes pertinent to functional genomic research.

A comparison of DNA-binding drugs as inhibitors of E2F1- and Sp1-DNA complexes and associated gene expression.

In this study, we examined how DNA-binding drugs prevented formation of transcription factor-DNA complexes and influenced gene transcription from the hamster dihydrofolate reductase promoter, which is regulated by E2F1 and Sp1. Gel mobility shift assay data showed that GC-binding drugs (e.g., mitoxantrone) inhibited the DNA binding of both E2F1 and Sp1. In contrast, AT-binding drugs (e.g., distamycin) interfered only with E2F1-DNA complex formation. In an in vitro transcription assay using HeLa nuclear extracts, inhibition of transcription was observed when mitoxantrone or distamycin was added either before or after assembly of the transcription complex on the DNA, although for the latter, higher drug concentrations were needed. Mitoxantrone, which was a stronger inhibitor of transcription factor-DNA complex, was more effective than distamycin at preventing transcript formation. Time course transcription in a cell-free assay with addition of various drug concentrations indicated that high drug concentrations of either mitoxantrone or distamycin completely blocked transcription, while low drug concentrations could delay the synthesis of transcripts.

転写因子におけるＤＮＡ‐蛋白質相互作用

The diversity of transcription factor-DNA complexes reviewed in this paper marks the extent of discovery since the first transcription factor-DNA co-crystal structures appeared less than ten years ago. The DNA-binding domains of transcription factors were classified into families, many of which have been identified by comparative amino acid sequence analysis. The three-dimensional views of their respective families revealed new designs as well as unanticipated similarities to older ones. Several hoped-for simplicities in their DNA-recognition mechanism have proved illusory, but other unexpected regularities have emerged.

Effect of DNA-binding Drugs on Early Growth Response Factor-1 and TATA Box-binding Protein Complex Formation with the Herpes Simplex Virus Latency Promoter

Adjacent binding sites for early growth response factor-1 (EGR1) and TATA box-binding protein (TBP) were identified on the herpes simplex virus latency promoter in previous work. The binding of EGR1 to the GC-rich region prevented TBP binding to the AT-rich region. With the simultaneous addition of both EGR1 and TBP, the intercalator nogalamycin prevented EGR1 complex formation, resulting in a dose-dependent increase of the TBP.DNA complex. The minor groove binder chromomycin A3 inhibited EGR1 complex formation but resulted in a smaller increase of the TBP complex. In contrast, an alkylating intercalator hedamycin strongly inhibited binding of both proteins. The ability of these GC-binding drugs to prevent EGR1.DNA complex formation was in the following order: hedamycin > nogalamycin > chromomycin A3, and the specificity was nogalamycin > chromomycin A3 > hedamycin. With transcription factor IIA (TFIIA) in the assay, TBP was able to bind the promoter whereas formation of the EGR1.DNA complex was reduced. An AT minor groove-binding drug, distamycin A, disrupted the TBP.TFIIA.DNA complex and restored the EGR1.DNA complex. We conclude that the binding motif and sequence preference of DNA-interactive drugs are manifested in their ability to inhibit the transcription factor-DNA complexes.

DNA recognition code of transcription factors.

Introduction Over 35 years have passed since the 'central dogma' of molecular biology (DNA makes RNA makes protein) was proposed (Crick, 1958). Despite its remarkable verification, it is being seen increasingly as limited, for if the whole flow of information in a cell were unidirectional, all cells with the same complement of genetic material would have identical function and morphology. The truth is manifestly otherwise. A group of proteins, transcription factors, selects the information used in cells by specifically binding to 'regulatory' DNA sequences. Among other effects, this causes the differentiation of cells. These factors act as the final messenger in a transduction pathway of signals which come from outside the cell. Thus, gene expression can be regulated by the environment. Recognition between a transcription factor and its target DNA is achieved through the physical interaction of the two molecules. Since the structures of both DNA and proteins are determined by their primary sequences, there must be a set of rules to describe DNA-protein interactions entirely on the basis of sequences. The fundamental question is whether these rules are simple and comprehensible, such that the DNA recognition code can be compared with the triplet code which summarizes the rules of how DNA and protein sequences are related in the central dogma. As we review in this paper, a simple code for DNA recognition by transcription factors does seem to exist. In fact, the recognition rules allow us (i) to predict DNA-protein interactions, (ii) to change the binding specificity of an existing transcription factor, and (iii) probably even to design in a rational way a new protein which binds to a particular DNA sequence. The code has been derived from crystal structures of transcription factor-DNA complexes (Table I) and the vast body of biochemical, genetic and statistical information about the binding specificity of transcription factors. Most of the transcription factors discussed here use an a-helix, which binds to the DNA major groove, for recognition. Those proteins which have a 'recognition helix' discussed here fall mainly into four families: probe helix (PH), helix-turnhelix (HTH), zinc finger (ZnF) and C4 Zn binding proteins (C4). There is, in addition, one transcription factor family described that uses a (J-sheet, the MetJ repressor-like (MR) family. [See Table I for members of these and other families. Note that (i) individual Zn fingers are further subdivided into A and B fingers, AF and BF (Suzuki et ai, 1994a), (ii) the PH family includes homeodomain and basic-zipper proteins (Suzuki, 1993) and (iii) the C4 family includes the hormone receptors and the GATA proteins (Suzuki and Chothia, 1994).]

A histone-binding protein, nucleoplasmin, stimulates transcription factor binding to nucleosomes and factor-induced nucleosome disassembly.

The binding of a GAL4-AH, USF or Sp1 to nucleosome cores was stimulated by the presence of the histone-binding protein, nucleoplasmin. Stimulation of factor binding by nucleoplasmin was specific for nucleosome reconstituted DNA and was not mimicked by non-specific histone sinks (i.e. polyglutamate or RNA). Upon GAL4-AH binding, nucleoplasmin specifically removed histones H2A and H2B from the nucleosome which enhanced the subsequent loss of the H3/H4 tetramers onto competing DNA. Thus, nucleoplasmin participated in the complete conversion of nucleosome cores to transcription factor-DNA complexes. These data indicate that proteins which bind histones can increase transcription factor binding to nucleosomal DNA and that transcription factor binding can initiate nucleosome disassembly. Similar activities of histone-binding proteins may participate in the displacement of nucleosomes at enhancers and promoters in vivo.

Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees.

The 3 angstrom resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with a 30-base pair DNA sequence shows that the DNA is bent by 90 degrees. This bend results almost entirely from two 40 degrees kinks that occur between TG/CA base pairs at positions 5 and 6 on each side of the dyad axis of the complex. DNA sequence discrimination by CAP derives both from sequence-dependent distortion of the DNA helix and from direct hydrogen-bonding interactions between three protein side chains and the exposed edges of three base pairs in the major groove of the DNA. The structure of this transcription factor--DNA complex provides insights into possible mechanisms of transcription activation.

Effects of temperature and single-stranded DNA on the interaction of an RNA polymerase III transcription factor with a tRNA gene.

It has previously been shown that a yeast RNA polymerase III transcription factor binds stably to tRNA genes. The principal protein-DNA contacts have now been mapped, by dimethylsulfate footprinting, to the vicinity of the A and B block promoter regions of the S. cerevisiae tRNALeu3 gene. Single-stranded DNA preferentially interferes with the binding of the transcription factor to one of the promoter regions. The effect of temperature on the formation of the transcription factor-DNA complex has been examined: the complex has the kind of "opening" property that has been previously associated with prokaryotic RNA polymerase-promoter complexes, with stability increasing as the temperature is raised.