Circular Permutation Analysis Research Articles

Site-directed photo-cross-linking of rRNA transcription initiation complexes.

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

Estrogen receptor-induced DNA bending: orientation of the bend and replacement of an estrogen response element with an intrinsic DNA bending sequence.

The estrogen receptor (ER) binds to DNA fragments containing estrogen response elements (EREs) and causes them to bend. To characterize this ER-induced DNA bend and determine if it is involved in transcription activation, three different lines of investigation were used. Using MCF-7 human breast cancer cell extracts and circular permutation analysis, it was determined that molybdate-stabilized, unoccupied cytosolic ER was unable to bind to ERE-containing DNA fragments when maintained at 4 C, but that thermal activation enabled the cytosolic receptor to bind and bend ERE-containing DNA fragments to the same extent as ER present in whole cell extracts. DNA phasing analysis was utilized to determine that ER binding induced DNA fragments containing EREs to bend toward the major groove of the DNA helix. The orientation of this bend was the same with thermally activated, unoccupied cytosolic ER and with unoccupied ER, 17 beta-estradiol-occupied ER, and 4-hydroxytamoxifen-occupied ER present in whole cell extracts. Using transient cotransfection assays, the ability of an intrinsically bent DNA sequence to replace an ERE was tested. When a single consensus ERE, which is induced to bend 56 degrees on ER binding, was replaced with a 54 degrees intrinsic DNA bending sequence, transcription was effectively activated. Similar levels of transcription were also observed when promoters contained either a 108 degrees intrinsic DNA bending sequence or two consensus EREs. However, the 54 degrees DNA bending sequence and a single ERE were unable to cooperatively activate transcription. Because the magnitude and orientation of ER-induced DNA bends are the same with the unoccupied and occupied receptor, DNA bending alone probably does not function as a transcriptional switch to turn on gene transcription. However, DNA bending may be required to provide the architecture needed for modulation of target genes.

The Role of DNA Bending in Flp-mediated Site-specific Recombination

The Flp recombinase of the 2 μm plasmid of Saccharomyces cerevisiaebinds to a recognition target site, induces DNA bending and catalyses DNA cleavage and strand exchange to bring about recombination. The minimal Flp recognition target site contains two Flp binding sequences flanking an 8 bp core region; binding of Flp results in the formation of two Flp:DNA complexes (complexes I and II). Binding of a Flp monomer to a single symmetry element generates a DNA bend of about 60 ° (a type I bend), whereas binding of two Flp monomers to the FRT site generates a DNA bend of>144 ° (a type II bend). We have used circular permutation analysis to locate the centre of the type I and type II DNA bends induced by Flp, and the Flp peptides P27 (27 kDa; amino acid residues 124 to 346) and P32 (32 kDa; amino acid residues 124 to 423). The location of the centre of the type I bend depends upon whether the substrate contains one or two Flp binding elements. When the substrate contains one symmetry element, the centre of the type I bend induced by Flp is located at the core-distal end of the belement. However, it is located at the core-proximal end of the belement when the substrate contains two Flp-binding elements. The P27 and P32 peptides, which lack the NH 2-terminal 13 kDa region of Flp, do not show this behavior. We deduce that the 13 kDa region of Flp is critical for the positioning of the type I bend centre on a minimal Flp recognition site. We propose a model in which a single molecule of Flp interactions with two symmetry elements to account for these results. The centre of the type II bend induced by Flp is in the middle of the core region. We used ligation-defective Flp proteins to determine the location of the type II bend centres in complexes where either the top or bottom strand was cleaved. The bend centres of such complexes depend upon which strand is cleaved. We propose a model which associates the position of Flp-induced type II bends with a defined order of strand exchanges in the recombination reaction.

Protein-induced bending or flexing at the 5′-end of the duck β A-globin promoter

The 5′-end of the duck β A-globin promoter contains a protein binding site BS-3/Spl, the A + T-rich part of which could be involved in DNA bending. Plasmids were constructed using the pBend2 plasmid containing BS-3/Spl. Circular permutation analysis of the fragments cut out from the plasmids using various restriction endonucleases, in the presence of a partially purified protein extract from embryonic duck erythrocytes, was performed. The results indicate that a rather complicated change in the fragment shape takes place upon protein binding, which is best explained as an induction of two points of bending or flexure within the fragment. Analogical points of flexure may exist at the protein-binding sites of the duck and chicken β A-globin promoters in spite of differing DNA sequences.

Characterization of the Elk-1 ETS DNA-binding Domain

The ETS domain family of transcription factors is comprised of several important proteins that are involved in controlling key cellular events such as proliferation, differentiation, and development. One such protein, Elk-1, regulates the activity of the c-fos promoter in response to extracellular stimuli. Elk-1 is representative of a subgroup of ETS domain proteins that utilize a bipartite recognition mechanism that is mediated by both protein-DNA and protein-protein interactions. In this study, we have overexpressed, purified, and characterized the ETS DNA-binding domain of Elk-1 (Elk-93). Elk-93 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and purified to homogeneity from both the soluble and insoluble fractions using a two-column protocol. A combination of CD, NMR, and fluorescence spectroscopy demonstrates that Elk-93 represents an independently folded domain of mixed alpha/beta structure in which the three conserved tryptophans appear to contribute to the hydrophobic core of the protein. Moreover, DNA binding studies demonstrate that Elk-93 binds DNA with both high affinity (Kd approximately 0.85 x 10(-10)M) and specificity. Circular permutation analysis indicates that DNA binding by Elk-93 does not induce significant bending of the DNA. Our results are discussed with respect to predictive models for the structure of the ETS DNA-binding domain.

DNA bending in the ternary nucleoprotein complex at the c-fos promoter.

Transcriptional induction of the c-fos proto-oncogene in response to serum growth factors is mediated in part by a ternary complex that forms on the serum response element (SRE) within its promoter. This complex consists of Elk-1, serum response factor (SRF) and the SRE. Elk-1 is phosphorylated by MAP kinase, which correlates with the induction of c-fos transcription. In this study we have investigated the protein-induced DNA bending which occurs during the formation and post-translational modification of the ternary complex that forms at the c-fos SRE. Circular permutation analysis demonstrates that the minimal DNA-binding domain of SRF, which contains the MADS box, is sufficient to induce flexibility into the centre of its binding site within the SRE. Phasing analysis indicates that at least part of this flexibility results in the production of a directional bend towards the minor groove. The isolated ETS domains from Elk-1 and SAP-1 induce neither DNA bending nor increased DNA flexibility. Formation of ternary complexes by binding of Elk-1 to the binary SRF:SRE complex results in a change in the flexibility of the SRE. Phosphorylation of Elk-1 by MAP kinase (p42/ERK2) induces further minor changes in this DNA flexibility. However, phasing analysis reveals that the recruitment of Elk-1 to form the ternary complex affects the SRF-induced directional DNA bend in the SRE. The potential roles of DNA bending at the c-fos SRE are discussed.

Selection of circularly permuted ribozymes from Bacillus subtilis RNAse P by substrate binding.

The effect of a single break in the phosphodiester backbone of Bacillus subtilis RNAse P RNA (P RNA) was examined using circular permutation analysis (CPA). This method reveals that many of the phosphodiester bonds in this catalytic RNA can be broken with little or no effect on substrate binding. Phosphate positions that show strong effects are located mostly in regions conserved among all RNAse P RNAs, or they are in regions known to interact directly with the pre-tRNA substrate. Two circularly permuted isomers of P RNA were constructed and analyzed in detail. The KM for both circularly permuted isomers is nearly identical to that of the wild-type P RNA. Since the KM of the P RNA is essentially the same as the binding constant to the substrate, this finding confirms the CPA results. The implications of backbone breakage are discussed with respect to folding and catalysis of the RNAse P RNA.

The C/EBP family of proteins distorts DNA upon binding but does not introduce a large directed bend.

The DNA-bending properties of several C/EBP family proteins, bound to two different sites, have been determined using circular permutation and phasing analyses. All the proteins examined by circular permutation analysis induced a significant distortion in the DNA (40-66 degrees), and this distortion was of the same magnitude at both C/EBP sites. However, phasing analysis revealed that the size of the directed bend induced in the DNA by C/EBP proteins was only 1-4 degrees. Both the magnitude and orientation of the directed bend were affected by the specific sequence of the DNA-binding site. All proteins induced a directed bend of similar magnitude and orientation (toward the minor groove) when bound to the C/EBP site derived from the IgH enhancer, but the induced bend was smaller when the protein was bound to the C/EBP site derived from the VH1 promoter. The study also included a heterodimer between Ig/EBP and ATF4. Similar to C/EBP protein homodimers, this heterodimer induces a small directed bend of 4 degrees oriented toward the minor groove when bound to the enhancer-derived C/EBP site.

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains.

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.

Bending of DNA in solution caused by a protein from Arabidopsis that binds to a TATA element.

The TFIID complex, which is a general transcription factor, helps to activate and regulate the transcription associated with eukaryotic promoters. A TATA element-binding protein (TBP), a central subunit of the TFIID complex, binds to TATA elements. Circular permutation analysis showed that purified recombinant TBP-1 from Arabidopsis (aTBP-1) introduced a bend in DNA around a TATA element in DNA fragments in solution. The bending was a slow process. A bend in DNA caused by a TBP-1 was seen under an electron microscope, as was an aTBP-DNA complex after immunochemical staining.

Using circular permutation analysis to redefine the R17 coat protein binding site.

The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem. Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone. This method revealed that breakage of all but one phosphodiester bond within a well-defined binding site substantially reduced the binding affinity. This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions. One circularly permuted isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA. CPA also locates the 5' and 3' boundaries of protein binding sites on the RNA. The 5' boundary of the R17 coat protein site as defined by CPA was two nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal truncation experiments. A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein. The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used.

DNA bending by retinoid X receptor-containing retinoid and thyroid hormone receptor complexes.

Retinoid X receptors (RXR) have been identified as common subunits in the regulation of multiple hormonal signaling pathways. Using circular permutation and phasing analysis of specific response elements, we present evidence that RXR-retinoic acid receptor and RXR-thyroid hormone receptor heterodimer or RXR-RXR homodimer complexes induce directed DNA bends when bound to their cognate response elements. The extent of DNA bending induced by the RXR alpha-containing complexes varied and depended on the structure of the DNA-binding sites and the RXR partners. The overall bending orientation for RXR-containing complexes is directed toward the major groove of the DNA helix at the center of hormone response elements. Our observation implicates DNA bending as a possible mechanism underlying transcriptional regulation of distinct retinoid and thyroid hormone responsive genes.

DNA bending by retinoid X receptor-containing retinoid and thyroid hormone receptor complexes.

Retinoid X receptors (RXR) have been identified as common subunits in the regulation of multiple hormonal signaling pathways. Using circular permutation and phasing analysis of specific response elements, we present evidence that RXR-retinoic acid receptor and RXR-thyroid hormone receptor heterodimer or RXR-RXR homodimer complexes induce directed DNA bends when bound to their cognate response elements. The extent of DNA bending induced by the RXR alpha-containing complexes varied and depended on the structure of the DNA-binding sites and the RXR partners. The overall bending orientation for RXR-containing complexes is directed toward the major groove of the DNA helix at the center of hormone response elements. Our observation implicates DNA bending as a possible mechanism underlying transcriptional regulation of distinct retinoid and thyroid hormone responsive genes.

Zebrafish pou[c]: a divergent POU family gene ubiquitously expressed during embryogenesis.

We report the isolation and characterization of cDNA for a novel zebrafish (Brachyodanio rerio) POU domain gene, pou[c], which is ubiquitously expressed during embryonic development. This gene encodes a 610 amino acids long protein with a 149 amino acid POU domain ending only 8 residues before the C terminus. The 453 amino acids long region N-terminal to the POU domain contains several features typical of transcriptional activation domains such as an acidic region with a putative amphipathic alpha-helix, a glutamine-rich region, and short threonine- and/or serine-rich regions. Comparison of the POU domain of pou[c] to other known POU sequences clearly show that pou[c] has the most divergent POU domain sequence reported to date. Thus, we suggest that pou[c] should be placed as the presently sole member of a new, sixth class of POU proteins. DNA-binding studies revealed that pou[c] is not an octamer-binding transcription factor like the Oct proteins described from mammals, chicken and Xenopus. Rather, pou[c] binds with high affinity to the TAATGARAT motif found in the promoters of the herpes simplex virus immediate early genes and to degenerate octamer-TAATGA motifs. Circular permutation analyses also show that pou[c] induces DNA bending upon sequence-specific binding.

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending.

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.

Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending.

We have used circular permutation analysis to determine whether binding of purified Xenopus laevis estrogen receptor DNA-binding domain (DBD) to a DNA fragment containing an estrogen response element (ERE) causes the DNA to bend. Gel mobility shift assays showed that DBD-DNA complexes formed with fragments containing more centrally located EREs migrated more slowly than complexes formed with fragments containing EREs near the ends of the DNA. DNA bending standards were used to determine that the degree of bending induced by binding of the DBD to an ERE was approximately 34 degrees. A 1.55-fold increase in the degree of bending was observed when two EREs were present in the DNA fragment. These in vitro studies suggest that interaction of nuclear receptors with their hormone response elements in vivo may result in an altered DNA conformation.

The promoter of the tgt/sec operon in Escherichia coli is preceded by an upstream activation sequence that contains a high affinity FIS binding site.

The tgt/sec operon in E. coli consists of five genes: queA, tgt, ORF12, secD, and secF. QueA and Tgt participate in the biosynthesis of the hypermodified t-RNA nucleoside Queuosine, whereas SecD and SecF are involved in protein secretion. Examination of the promoter region of the operon showed structural similarity to promoter regions of the rrn-operons. An upstream activation sequence (UAS) containing a potential binding site for the factor of inversion stimulation (FIS) was found. Gel retardation assays and DNaseI footprinting indicated, that FIS binds specifically and with high affinity to a site centred at position -58. Binding of FIS caused bending of the DNA, as deduced from circular permutation analysis. Various 5' deletion mutants of the promoter region were constructed and fused to a lacZ reporter gene to determine the influence of the UAS element on the promoter strength. An approximately two-fold activation of the promoter by the UAS element was observed.

Folding of Circularly Permuted Transfer RNAs

All of the ribose-phosphate linkages in yeast tRNA(Phe) that could be cleaved without affecting the folding of the molecule have been determined in a single experiment. Circular permutation analysis subjects circular tRNA molecules to limited alkaline hydrolysis in order to generate one random break per molecule. Correctly folded tRNAs were identified by lead cleavage at neutral pH, a well-characterized reaction that requires proper folding of tRNA(Phe). Surprisingly, most of the circularly permuted tRNA molecules folded correctly. This result suggests that the tRNA folding motif could occur internally within other RNA sequences, and a computer search of Genbank entries has identified many examples of such motifs.

Fos-Jun heterodimers and jun homodimers bend DNA in opposite orientations: Implications for transcription factor cooperativity

Association of Fos and Jun with the AP-1 site results in a conformational change in the basic amino acid regions that constitute the DNA-binding domain. We show that Fos and Jun induce a corresponding alteration in the conformation of the DNA helix. Circular permutation analysis indicated that both Fos-Jun heterodimers and Jun homodimers induce flexure at the AP-1 site. Phasing analysis demonstrated that Fos-Jun heterodimers and Jun homodimers induce DNA bends that are directed in opposite orientations. Fos-Jun heterodimers bend DNA toward the major groove, whereas Jun homodimers bend DNA toward the minor groove. Fos and Jun peptides encompassing the dimerization and DNA-binding domains bend DNA in the same orientations as the full-length proteins. However, additional regions of both proteins influence the magnitude of the DNA bend angle. Thus, despite the amino acid sequence similarity in the basic region Fos-Jun heterodimers and Jun homodimers form topologically distinct DNA-protein complexes.

Upstream sequence activation of Escherichia coli argT promoter in vivo and in vitro

Escherichia coli argT promoter in a galK fusion construct is shown by BAL 31 deletion to require its upstream region for high in vivo activity. The extent of activation conferred by the upstream sequence from -130 to -38 is 25-fold. A spontaneous mutant containing a T to G transversion at -37 (i.e., the T-37G promoter) shows a similar requirement; however, the upstream sequence producing a 10-fold effect spans only -130 to -60. The difference in upstream sequence boundaries between the wild-type and T-37G promoters suggests the possible existence of two activating elements. Gel mobility investigation points to the presence of bent DNA in the argT promoter, and the bent center was localized to the -90 to -95 region by circular permutation analysis. The role of the upstream activating sequence (UAS) in promoter function was probed by competitive transcription experiments in vitro. Results of this type of analysis indicate that the full UAS activates transcription through a combined effect on KB and k2. Of these, KB is significantly strengthened by the proximal element, and k2 is stimulated to a smaller extent by the distal element. The evidence from deletion analysis, gel mobility investigation, and competitive transcription together support a "two-element" model of UAS function for the argT promoter.