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PubMed2000

Characterization of a major histocompatibility complex class II X-box-binding protein enhancing tat-induced transcription directed by the human immunodeficiency virus type 1 long terminal repeat.

The X-box element present within the promoter region of genes belonging to the major histocompatibility complex (MHC) plays a pivotal role in the expression of class II molecules, since it contains the binding sites for several well-characterized transcription factors. We have analyzed a randomly selected compilation of viral genomes for the presence of elements homologous to the X box of the HLA-DRA gene. We found that human immunodeficiency virus type 1 (HIV-1) shows the highest frequency of X-like box elements per 1,000 bases of genome. Within the HIV-1 genome, we found an X-like motif in the TAR region of the HIV-1 long terminal repeat (LTR), a regulative region playing a pivotal role in Tat-induced HIV-1 transcription. The use of a decoy approach for nuclear proteins binding to this element, namely, XMAS (X-like motif activator sequence), performed by transfection of multiple copies of this sequence into cells carrying an integrated LTR-chloramphenicol acetyltransferase construct, suggests that this element binds to nuclear proteins that enhance Tat-induced transcription. In this report we have characterized two proteins, one binding to the XMAS motif and the other to the flanking regions of XMAS. Mobility shift assays performed on crude nuclear extracts or enriched fractions suggest that similar proteins bind to XMAS from HIV-1 and the X box of the HLA-DRA gene. Furthermore, a UV cross-linking assay suggests that one protein of 47 kDa, termed FAX (factor associated with XMAS)-1, binds to the XMAS of HIV-1. The other protein of 56 kDa was termed FAX-2. In a decoy ex vivo experiment, it was found that sequences recognizing both proteins are required to inhibit Tat-induced HIV-1 LTR-driven transcription. Taken together, the data reported in this paper suggest that XMAS and nearby sequences modulate Tat-induced HIV-1 transcription by binding to the X-box-binding proteins FAX-1 and FAX-2. The sequence homology between XMAS and X box is reflected in binding of a common protein, FAX-1, and similar functional roles in gene expression. To our knowledge, this is the first report showing that transcription factors binding to the X box of the MHC class II genes enhance the transcription of HIV-1.

Base Sequence↗

PubMed2003

Effect of Box-Cox transformation on power of Haseman-Elston and maximum-likelihood variance components tests to detect quantitative trait Loci.

Non-normality of the phenotypic distribution can affect power to detect quantitative trait loci in sib pair studies. Previously, we observed that Winsorizing the sib pair phenotypes increased the power of quantitative trait locus (QTL) detection for both Haseman-Elston (HE) least-squares tests [Hum Hered 2002;53:59-67] and maximum likelihood-based variance components (MLVC) analysis [Behav Genet (in press)]. Winsorizing the phenotypes led to a slight increase in type 1 error in H-E tests and a slight decrease in type I error for MLVC analysis. Herein, we considered transforming the sib pair phenotypes using the Box-Cox family of transformations. Data were simulated for normal and non-normal (skewed and kurtic) distributions. Phenotypic values were replaced by Box-Cox transformed values. Twenty thousand replications were performed for three H-E tests of linkage and the likelihood ratio test (LRT), the Wald test and other robust versions based on the MLVC method. We calculated the relative nominal inflation rate as the ratio of observed empirical type 1 error divided by the set alpha level (5, 1 and 0.1% alpha levels). MLVC tests applied to non-normal data had inflated type I errors (rate ratio greater than 1.0), which were controlled best by Box-Cox transformation and to a lesser degree by Winsorizing. For example, for non-transformed, skewed phenotypes (derived from a chi2 distribution with 2 degrees of freedom), the rates of empirical type 1 error with respect to set alpha level=0.01 were 0.80, 4.35 and 7.33 for the original H-E test, LRT and Wald test, respectively. For the same alpha level=0.01, these rates were 1.12, 3.095 and 4.088 after Winsorizing and 0.723, 1.195 and 1.905 after Box-Cox transformation. Winsorizing reduced inflated error rates for the leptokurtic distribution (derived from a Laplace distribution with mean 0 and variance 8). Further, power (adjusted for empirical type 1 error) at the 0.01 alpha level ranged from 4.7 to 17.3% across all tests using the non-transformed, skewed phenotypes, from 7.5 to 20.1% after Winsorizing and from 12.6 to 33.2% after Box-Cox transformation. Likewise, power (adjusted for empirical type 1 error) using leptokurtic phenotypes at the 0.01 alpha level ranged from 4.4 to 12.5% across all tests with no transformation, from 7 to 19.2% after Winsorizing and from 4.5 to 13.8% after Box-Cox transformation. Thus the Box-Cox transformation apparently provided the best type 1 error control and maximal power among the procedures we considered for analyzing a non-normal, skewed distribution (chi2) while Winzorizing worked best for the non-normal, kurtic distribution (Laplace). We repeated the same simulations using a larger sample size (200 sib pairs) and found similar results.

Chromosome Mapping↗

PubMed2000

The period E-box is sufficient to drive circadian oscillation of transcription in vivo.

The minimum element from the Drosophila period promoter capable of driving in vivo cycling mRNA is the 69 bp circadian regulatory sequence (CRS). In cell culture, an 18 bp E-box element from the period promoter is regulated by five genes that are involved in the regulation of circadian expression in flies. This E-box is a target for transcriptional activation by bHLH-PAS proteins dCLOCK (dCLK) and CYCLE (CYC), this activation is inhibited by PERIOD (PER) and TIMELESS (TIM) together, and inhibition of dCLK/CYC by PER and TIM is blocked by CRYPTOCHROME (CRY) in the presence of light. Here, the same 18 bp E-box region generated rhythmic expression of luciferase in flies under both light-dark cycling and constant conditions. Flies heterozygous for the Clke(jrk) mutation maintained rhythmic expression from the E-box although at a lower level than wild type. Homozygous mutant Clk(jrk) animals had drastically lowered and arrhythmic expression. In a per01 background, expression from the E-box was high and not rhythmic. Transcription mediated by the per E-box was restricted to the same spatial pattern as the CRS. The per E-box DNA element and cognate binding proteins can confer per-like temporal and spatial expression. This demonstrates in vivo that the known circadian genes that form the core of the circadian oscillator in Drosophila integrate their activities at a single DNA element.

Animals↗

PubMed2006

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome.

BACKGROUND: The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs. RESULTS: Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The first-generation consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR. CONCLUSION: Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system.

4-Butyrolactone↗

PubMed2001

Ligands specify coactivator nuclear receptor (NR) box affinity for estrogen receptor subtypes.

Nuclear receptors (NRs) require coactivators to efficiently activate transcription of their target genes. Many coactivators including the p160 proteins utilize a short NR box motif to recognize the ligand-binding domain of the NR when it is activated by ligand. To investigate the ability of various ligands to specify the affinity of NR boxes for a ligand-bound NR, we compared the capacity of p160 NR boxes to be recruited to estrogen receptor (ERalpha) and ERbeta in the presence of 17beta-estradiol, diethylstilbestrol, and genestein. A time-resolved fluorescence-based binding assay was used to determine the dissociation constants for the 10 NR boxes derived from the three p160 coactivators for both ER subtypes in the presence of the each of the agonists. While the affinity of some NR boxes for ER was independent of the agonist, we identified several NR boxes that had significantly different affinities for ER depending on which agonist was bound to the receptor. Therefore, an agonist may specify the affinity of an NR for various NR boxes and thus regulate the coactivator selectivity of the receptor.

Acetyltransferases↗

PubMed1994

The effects of P-box substitutions in thyroid hormone receptor on DNA binding specificity.

Three "P-box" amino acids within the DNA recognition alpha-helix of members of the steroid hormone and thyroid hormone families of nuclear receptors are known to determine the identity of two of the six base pairs within the half-sites of cognate DNA elements. We introduced P-box substitutions derived from different members of the thyroid hormone/estrogen receptor (T3R/ER) family into the beta-isoform of human thyroid hormone receptor (hT3R beta) and tested the DNA binding and transactivation activities of these mutants using thyroid hormone response elements (TREs) with half-sites composed of different sequences and arranged in different orientations. Different P-box sequences derived from the T3R/ER family resulted in distinct DNA binding specificities determined by the fourth base pair of the half-site. Thyroid hormone receptor mutants containing EGA, EAA, EGS substitutions for the wild type EGG P-box bound with wild type affinity to consensus AGGTCA half-sites, regardless of orientation. TREs composed of AGGACA half-sites bound hT3R beta s with an EGG or EAA P-box sequence, but not those with EGA or EGS P-box sequence. A reversal of this specificity was observed on a direct repeat TRE with AGGGCA half-sites. Additionally, an ESG P-box substitution in hT3R beta prevented the receptor from binding to a direct repeat as a homodimer, but this mutant could bind as a heterodimer with retinoid X receptor or to the everted repeat TRE from the chicken lysozyme promoter.

Amino Acid Sequence↗

PubMed2000

A developmentally regulated GAGA box-binding factor and Sp1 are required for transcription of the hsp70.1 gene at the onset of mouse zygotic genome activation.

We have investigated the onset of zygotic genome transcription in early two-cell mouse embryos by analyzing the regulation of hsp70.1, one of the first genes expressed after fertilization. The transcriptional activation of both an episomic hsp70 promoter and the endogenous hsp70.1 gene requires the contiguity of the GC box proximal to the TATA box with a GAGA box and involves GC box- and GAGA box-binding factors. In vivo transcription factor titrations with double-stranded oligodeoxyribonucleotides and antibodies pinpoint these factors as Sp1 and a novel murine GAGA box-binding factor, which is structurally related to the Drosophila GAGA factor and acts as transcriptional coactivator/potentiator of Sp1. Mouse unfertilized eggs and one-cell and two-cell embryos display a GAGA box-binding activity of maternal origin that disappears at the four-cell stage and is also abundant in the gonads, but is barely detectable in other adult tissues. In light of the well-established nucleosome-disruption role of the Drosophila GAGA factor, these findings suggest a novel mechanism of enhancer-independent gene derepression in early mouse embryos.

Animals↗

PubMed2003

Polo boxes form a single functional domain that mediates interactions with multiple proteins in fission yeast polo kinase.

Polo kinases play multiple roles in cell cycle regulation in eukaryotic cells. In addition to the kinase domain, conservation at the primary sequence level is also found in the non-catalytic region mainly in three blocks, namely the polo boxes. Although several studies have implicated the polo boxes in protein localisation, no systematic study to elucidate the roles of individual polo boxes has been carried out. Here we show, by using fission yeast, that the polo boxes form a single functional unit that is essential for both cellular function and cell-cycle-regulated localisation to the spindle pole bodies. Various polo box mutations abolish the mitotic arrest seen upon overexpression of plo1 but do not prevent the untimely septation seen under these conditions, showing that the functions of Plo1 may be separated. Plo1 interacts with multiple proteins including cell cycle regulators in a polo-box-dependent manner. Isolation of mutants that differentially disrupt these interactions revealed a role for the polo boxes in mediating protein-protein interactions.

Cell Cycle↗

PubMed1999

DNA binding of Myc/Max/Mad network complexes to oligonucleotides containing two E box elements: c-Myc/Max heterodimers do not bind DNA cooperatively.

Myc proteins function in heterodimeric complexes with Max proteins as transcriptional regulators at least in part by binding to E box sequences with a 5'-CACGTG core. Since such E boxes are found frequently in the human genome and since other proteins besides Myc/Max can bind to similar or identical sequences it is unclear how the specificity of E box-mediated gene transcription is determined. Recent findings were interpreted to suggest that Myc/Max, but not Max/Max or USF complexes, bind cooperatively to DNA sequences that contain two E box elements. This provides a potential mechanism for selective E box-mediated gene transcription. To extend this finding we analyzed DNA binding of c-Myc/Max complexes using a transient COS-7 expression system. In both competition and titration experiments no cooperative binding of c-Myc/Max heterodimers to probes with two E boxes was observed. Furthermore, c-Myc-specific transcription of reporter gene constructs did not reveal cooperativity. Thus ourfindings argue against cooperative DNA binding of Myc proteins as a selection mechanism for E box-dependent transcription.

Animals↗

PubMed2004

Aromatherapy in the Mediterranean fruit fly (Diptera: Tephritidae): sterile males exposed to ginger root oil in prerelease storage boxes display increased mating competitiveness in field-cage trials.

Previous research showed that exposure to ginger root, Zingiber officinale Roscoe, oil increased the mating success of mass-reared, sterile males of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). This work, however, involved the exposure of small groups of males (n = 25) in small containers (volume 400 ml). Several sterile male release programs use plastic adult rearing containers (so-called PARC boxes; hereafter termed storage boxes; 0.48 by 0.60 by 0.33 m) to hold mature pupae and newly emerged adults before release (approximately = 36,000 flies per box). The objective of the current study was to determine whether the application of ginger root oil to individual storage boxes increases the mating competitiveness of sterile C. capitata males. Irradiated pupae were placed in storage boxes 2 d before adult emergence, and in the initial experiment (adult exposure) ginger root oil was applied 5 d later (i.e., 3 d after peak adult emergence) for 24 h at doses of 0.0625, 0.25, 0.5, 1.0, and 2.0 ml. In a second experiment (pupal-adult exposure), ginger root oil was applied to storage boxes immediately after pupal placement and left for 6 d (i.e., 4 d after peak adult emergence) at doses of 0.25 and 1.0 ml. Using field cages, we conducted mating trials in which ginger root oil-exposed (treated) or nonexposed (control) sterile males competed against wild-like males for copulations with wild-like females. After adult exposure, treated males had significantly higher mating success than control males for all doses of ginger root oil, except 2.0 ml. After pupal-adult exposure, treated males had a significantly higher mating success than control males for the 1.0-ml but not the 0.25-ml dose of ginger root oil. The results suggest that ginger root oil can be used in conjunction with prerelease, storage boxes to increase the effectiveness of sterile insect release programs.

Animals↗

PubMed1999

Role of -35 sequence and its cooperativity with vir-box for the expression of virE gene.

To elucidate the role of the -35 sequence and its cooperativity with vir box in the expression of the virE gene, various mutants were constructed by either site-directed mutation or deletional mutation of the virE promoter. The expression level of pHBAV, a mutant where its putative -35 sequences (CCGAGT) have been substituted with the consensus -35 sequences of the Escherichia coli promoter (TTGACA), was increased by 386%. pECHV, containing the conserved -35 sequence but lacking the vir box and the 5'-half of the imperfect dyad symmetry region (DSR) showed an increase of 286% in its promoter activity. pESHV, containing the conserved -35 sequence but lacking the complete 5'-upstream region from the mid-region of imperfect DSR, exhibited 244% of the native virE promoter activity. pHBCA, containing the conserved -35 sequence but destroying the vir box, was constructed by substitution of A, C, T at the positions -62, -63, and -65 on the vir-box to T, A, C, respectively. These mutations increased promoter activities by 319%. On the other hand, when the vir box was mutated from imperfect DSR to almost perfect DSR with T to A and G to T substitutions at -60 and -61 positions of the virE promoter containing the conserved -35 sequence (pHBNA), a higher activity of 671% was observed. These results demonstrate that when the putative -35 sequence of virE promoter is replaced with the consensus -35 sequence, the virE gene can be expressed independently without the binding of VirG protein to the vir-box and/or the induction of acetosyringone. Moreover, the presence of an almost perfect dyad symmetry of the vir-box can increase the expression of virE synergistically with the consensus -35 sequence.

Agrobacterium tumefaciens↗

PubMed1992

[The effect of 2 finishing methods on the micromorphology of the proximal box margin. An in-vivo study].

Minipreparations of class II cavities often are not employed in daily practice mainly because adequate instruments for finishing the axial box margin and the proximo-cervical curvature were not available. The purpose of this in vivo study was to assess the morphology of the proximal box margin after finishing with two different methods. 81 class II minicavities for amalgam restorations were prepared by 4 dental practitioners. One method for finishing the axial box margin and the proximo-cervical curved border was the use of a modified EVA-System with the total amplitude reduced to 0.4 mm and a highly flexible file (Cavishape, grain 25 microns). The other method was the use of an axial margin trimmer. The micromorphology of the two methods was investigated by means of scanning electron microscopy and a score system as described earlier (Lussi et al. 1987). The margins of the axial box as well as of the proximo-cervical curvature were significantly better finished with the EVA-System and the highly flexible file compared to the axial margin trimmer (p < 0.001). The finishing time for the box margin was not significantly different using the EVA instrument or the axial margin trimmer. These findings confirm other in vitro investigations. This in vivo study shows that the modified EVA instrument with the highly flexible file (Cavishape, grain 25 microns) is clearly superior to the axial margin trimmer in finishing the box margin. This device allowed a significant better finishing of the axial box margin and the proximo-cervical curvature of minicavities in daily practice.

Chi-Square Distribution↗

PubMed1993

The Schizosaccharomyces pombe mating-type gene mat-Mc encodes a sequence-specific DNA-binding high mobility group box protein.

The Schizosaccharomyces pombe gene mat-Mc plays a determinative role in the sexual differentiation of the fission yeast. The mat-Mc protein has been suggested to belong to a novel family of so-called high mobility group (HMG) box proteins, characterized by homology to high mobility group-1 and -2 proteins. Several HMG box proteins, including the mammalian sex-determining gene product SRY and the lymphoid transcription factors TCF-1 and LEF-1, have been shown to bind to DNA in a sequence-specific fashion. To analyze possible DNA-binding properties of mat-Mc, we have cloned and expressed its putative HMG box in Escherichia coli. Gel retardation analysis revealed that the mat-Mc HMG box recognizes the AACAAAG heptamer in a sequence-specific fashion. Combined T-->C and A-->I substitutions on both strands of the AACAAAG heptamer, which change the surface of the major groove while leaving the minor groove intact, did not interfere with sequence-specific binding of mat-Mc. Methylation interference analysis confirmed that the mat-Mc HMG box contacts adenine residues in the minor groove. By using a circular permutation assay, the mat-Mc HMG box was observed to bend DNA. These results indicate that mat-Mc is indeed a member of the HMG box family with DNA-binding characteristics assigned earlier to other members of this novel transcription factor family.

Amino Acid Sequence↗

PubMed1993

A second box-end scoring artifact in the Farnsworth-Munsell 100-hue test.

PURPOSE: To investigate and describe a hitherto unreported scoring artifact in the Farnsworth-Munsell 100-hue Test, arising from the grouping of the caps into four boxes, which causes caps near the ends of a box to score less than caps near the center of a box. This artifact is in addition to a previously reported one, which causes caps near the end of a box to score more than caps in the center of the box. METHODS: Two different statistical simulations were used to generate synthetic cap sequences, which were scored in the normal way. RESULTS: For error scores less than about 500, the new artifact, which depresses scores at the ends of boxes, was found to dominate the pattern of scores. CONCLUSION: The existing published correction for the box-end scoring artifact is inappropriate for scores less than about 500, and therefore should be applied cautiously.

Artifacts↗

PubMed1996

Multiple interactions amongst floral homeotic MADS box proteins.

Most known floral homeotic genes belong to the MADS box family and their products act in combination to specify floral organ identity by an unknown mechanism. We have used a yeast two-hybrid system to investigate the network of interactions between the Antirrhinum organ identity gene products. Selective heterodimerization is observed between MADS box factors. Exclusive interactions are detected between two factors, DEFICIENS (DEF) and GLOBOSA (GLO), previously known to heterodimerize and control development of petals and stamens. In contrast, a third factor, PLENA (PLE), which is required for reproductive organ development, can interact with the products of MADS box genes expressed at early, intermediate and late stages. We also demonstrate that heterodimerization of DEF and GLO requires the K box, a domain not found in non-plant MADS box factors, indicating that the plant MADS box factors may have different criteria for interaction. The association of PLENA and the temporally intermediate MADS box factors suggests that part of their function in mediating between the meristem and organ identity genes is accomplished through direct interaction. These data reveal an unexpectedly complex network of interactions between the factors controlling flower development and have implications for the determination of organ identity.

Amino Acid Sequence↗

PubMed1997

Formation of a regulatory factor X/X2 box-binding protein/nuclear factor-Y multiprotein complex on the conserved regulatory regions of HLA class II genes.

Coordinate regulation of MHC class II genes occurs in a tissue-specific and cytokine-inducible manner. While the upstream regulatory sequences are conserved among all MHC class II genes, multiple base pair changes are found, even within the essential X box region. Analysis of all class II X boxes reveals differential binding between two transcription factors known to interact with the X box region, regulatory factor X and X2 box-binding protein (RFX and X2BP) of the HLA-DRA gene. These data presented a paradox with regard to the coordinate regulation of the class II genes if the factors though to regulate the HLA-DRA gene do not bind to the homologous sequence of all class II genes. Previous results suggested that cooperative interactions between the DNA binding proteins may be the key to understanding this paradox. Here RFX/X2BP/DNA complexes were formed on all class II isotypes regardless of the ability of the X box region to bind either factor individually. To further determine the role of the interactions between the X and Y factors, multiprotein/DNA complexes containing RFX, X2BP, NF-Y, and X-Y box DNA of the DRA and DRB genes were formed. This quaternary complex was extremely stable to competitor DNA, with a half-life > 4 h. These results suggest that the conserved X and Y boxes of class II genes function similarly and define a single multiprotein regulatory complex for class II expression in B cells.

Base Composition↗

PubMed2002

Isolation and characterization of a novel F-box protein Pof10 in fission yeast.

The SCF complex is a type of ubiquitin-protein ligase (E3) that consists of invariable components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Using a yeast two-hybrid system, we isolated six proteins that interact with Schizosaccharomyces pombe Skp1. Among them, Pof10 is a novel F-box protein consisting of 662 amino acids, harboring the F-box domain required for the binding to Skp1 and followed by four WD40 repeats. Overexpression of Pof10 in fission yeast resulted in loss of viability with marked morphological changes that are similar to those in pop1 mutant yeast. Coexpression of Skp1 with Pof10 prevented the lethality, suggesting that the lethality from Pof10 overexpression results from the sequestration of Skp1 from other F-box proteins including Pop1. Whereas most F-box proteins show rapid turnover, Pof10 has a remarkably long half-life in vivo and has been shown to be localized predominantly in cytoplasm. These results suggest that the stable F-box protein Pof10 might target abundant cytoplasmic proteins for degradation in fission yeast.

Amino Acid Sequence↗

PubMed1999

The RNA polymerase III-recruiting factor TFIIIB induces a DNA bend between the TATA box and the transcriptional start site.

TFIIIB, the RNA polymerase III-recruiting factor of Saccharomyces cerevisiae, may be assembled upstream of the transcriptional start site, either through the interaction of its constituent TATA-binding protein (TBP) with a strong TATA-box, or by means of the multisubunit assembly factor, TFIIIC. Missing nucleoside interference analysis of TFIIIC-dependent TFIIIB-DNA complex formation revealed enhanced complex formation at 0 degreesC when the DNA is missing nucleosides in two broad 7-10 bp regions centered around base-pairs -17 and -3 relative to the transcriptional start site; no effect of missing nucleosides was evident at 20 degreesC. The implication of these results for required DNA flexure in TFIIIC-mediated TFIIIB-DNA complex formation was pursued in a TFIIIC-independent context, using DNA with a suboptimal 6 bp TATA box (TATAAA). A unique missing nucleoside at the downstream end of the TATA box, corresponding to the position of one of two TBP-mediated DNA kinks, significantly enhances TBP-DNA complex formation. In contrast, TFIIIB displays a broad preference for missing nucleosides within an approximately 15 bp region immediately downstream of the TATA box. Consecutive mismatches (4-nt loops), either at the sites of TBP-mediated DNA kinking at both ends of the TATA box or within the identified region where missing nucleosides promote TFIIIB-DNA complex formation, also result in enhanced and specific TFIIIB assembly; 4-nt loops further downstream do not lead to preferential placement of TFIIIB. We conclude that TFIIIB induces an additional DNA deformation between the TATA box and the start site of transcription that is likely to be more extended than the sharp kinks generated by TBP.

Base Sequence↗