Binding Sites For Regulatory Proteins Research Articles

C-myc Intron Element-binding Proteins Are Required for 1,25-Dihydroxyvitamin D3 Regulation of c-myc during HL-60 Cell Differentiation and the Involvement of HOXB4

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) suppresses c-myc expression during differentiation of HL-60 cells along the monocytic pathway by blocking transcriptional elongation at the first exon/intron border of the c-myc gene. In the present study, the physiological relevance of three putative regulatory protein binding sites found within a 280-base pair region in intron 1 of the c-myc gene was explored. HL-60 promyelocytic leukemia cells were transiently transfected with three different c-myc promoter constructs cloned upstream of a chloramphenicol acetyltransferase (CAT) reporter gene. With the wild-type c-myc promoter construct (pMPCAT), which contains MIE1, MIE2, and MIE3 binding sites, 1,25-(OH)2D3 was able to decrease CAT activity by 45.4 +/- 7.9% (mean +/- S.E., n = 8). The ability of 1, 25-(OH)2D3 to inhibit CAT activity was significantly decreased to 18. 5 +/- 4.3% (59.3% reversal, p < 0.02) when examined with a MIE1 deletion construct (pMPCAT-MIE1). Moreover, 1,25-(OH)2D3 was completely ineffective at suppressing CAT activity in cells transfected with pMPCAT-287, a construct without MIE1, MIE2, and MIE3 binding sites (-6.5 +/- 10.9%, p < 0.002). MIE1- and MIE2-binding proteins induced by 1,25-(OH)2D3 had similar gel shift mobilities, while MIE3-binding proteins migrated differently. Furthermore, chelerythrine chloride, a selective protein kinase C (PKC) inhibitor, and a PKCbeta antisense oligonucleotide completely blocked the binding of nuclear proteins induced by 1,25-(OH)2D3 to MIE1, MIE2, and MIE3. A 1,25-(OH)2D3-inducible MIE1-binding protein was identified to be HOXB4. HOXB4 levels were significantly increased in response to 1,25-(OH)2D3. Taken together, these results indicate that HOXB4 is one of the nuclear phosphoproteins involved in c-myc transcription elongation block during HL-60 cell differentiation by 1,25-(OH)2D3.

Using sequence logos and information analysis of Lrp DNA binding sites to investigate discrepancies between natural selection and SELEX.

In vitro experiments that characterize DNA-protein interactions by artificial selection, such as SELEX,are often performed with the assumption that the experimental conditions are equivalent to natural ones. To test whether SELEX gives natural results, we compared sequence logos composed from naturally occurring leucine-responsive regulatory protein (Lrp) binding sites with those composed from SELEX-generated binding sites. The sequence logos were significantly different, indicating that the binding conditions are disparate. A likely explanation is that the SELEX experiment selected for a dimeric or trimeric Lrp complex bound to DNA. In contrast, natural sites appear to be bound by a monomer. This discrepancy suggests that in vitro selections do not necessarily give binding site sets comparable with the natural binding sites.

Upstream Elements Involved in Vivo in Activation of the Brain-specific Rat Aldolase C Gene: ROLE OF BINDING SITES FOR POU AND WINGED HELIX PROTEINS

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a 115-base pair (bp) promoter fragment was able to ensure the brain-specific expression of the chloramphenicol acetyltransferase (CAT) reporter gene in transgenic mice, but only at a low level (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). Here we show that in vivo activation of this promoter at a high level requires cooperation between an upstream 0.6-kilobase pair (kb) fragment and far upstream sequences. In the 0.6-kb region, a 28-bp DNA element is shown to include overlapping in vitro binding sites for POU domain regulatory proteins and for the Winged Helix hepatocyte nuclear factor-3beta factor. An hepatocyte nuclear factor-3beta-binding site previously described in the short proximal promoter fragment is also shown to interact in vitro with POU proteins, although with a lower affinity than the 28-bp motif. Additional binding sites for POU factors were detected in the upstream 0.6-kb sequences. Progressive deletion in this region resulted in decreased expression levels of the transgenes in mice, suggesting synergistic interactions between these multiple POU-binding sites. We propose that DNA elements characterized by a dual binding specificity for both POU domain and Winged Helix transcription factors could play an essential role in the brain-specific expression of the aldolase C gene and other neuronal genes.

On the Relationship between Genomic Regulatory Element Organization and Gene Regulatory Dynamics

In this project we study the relationship betweengenomic regulatory element organizationandgene regulatory dynamics. This paper illustrates an approach to investigating this relationship based on the application of classical nonlinear system analysis techniques to a transcription level, statistical thermodynamical model like that used in Shea & Ackers (1985). Preliminary ideas presented at the ICMCM conference (Wolf & Eeckman, 1998) are developed in this manuscript. We show that, for prokaryotic gene circuits dominated by local promoter control, dynamical system behavior descriptors like the number and stability of equilibrium point steady states and their bifurcation potential can be largely determined from genomic organization (e.g. the number, type, and placement of regulatory protein binding sites). Concepts are illustrated on hypothetical gene regulation systems with one or two genes and varying numbers of regulatory protein binding sites (operators). Gene regulatory systems with a single gene and an arbitrary number of operator sites are shown to be globally stable, with the potential for having multiple equilibrium points and capable of bifurcating. A monomer-controlled gene regulation system withnoperator sites is proven to have a maximum of 1+n/2 stable equilibria for evenn, and (n+1)/2 for oddn, while a multimer-controlled,noperator site system is shown to have a maximum of 2+n/2 stable equilibria for evenn, and (n+3)/2 for oddn. These results are applied to the design of a two-state switch using a gene regulation system with two operator sites. The question “what is the simplest possible gene regulation system capable of acting like a switch?” is answered. The paper ends with an analysis of a two-gene regulation system, the results of which point to the existence of a “soft-switching” mechanism that may account for the “on-off” hypothesized behavior of some gene networks.

Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change.

Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.

FlyNets and GIF-DB, two internet databases for molecular interactions in Drosophila melanogaster.

GIF-DB and FlyNets are two WWW databases describing molecular (protein-DNA, protein-RNA and protein-protein) interactions occuring in the fly Drosophila melanogaster (http://gifts.univ-mrs.fr/GIFTS_home_page.html ). GIF-DB is a specialised database which focuses on molecular interactions involved in the process of embryonic pattern formation, whereas FlyNets is a new and more general database, the long-term goal of which is to report on any published molecular interaction occuring in the fly. The information content of both databases is distributed in specific lines arranged into an EMBL- (or GenBank-) like format. These databases achieve a high level of integration with other databases such as FlyBase, EMBL, GenBank and SWISS-PROT through numerous hyperlinks. In addition, we also describe SOS-DGDB, a new collection of annotated Drosophila gene sequences, in which binding sites for regulatory proteins are directly visible on the DNA primary sequence and hyperlinked both to GIF-DB and TRANSFAC database entries.

Identification of regulatory protein-binding sites in cytochrome P450 genes by means of the gel-retardation assay.

The gel-retardation, band-shift, or electromobility shift assay is used to investigate protein—DNA interactions. Nuclear protein from the desired source is incubated with a radiolabeled DNA fragment. The products of the reaction are then analyzed by electrophoresls through a nondenaturing polyacrylamide gel. Free DNA will migrate rapidly through the gel, but DNA bound to protein will move more slowly, i.e., the migration of bound DNA ts retarded. The protein-DNA complexes formed can be detected by autoradiography.

CpG islands from the alpha-globin gene cluster increase gene expression in an integration-dependent manner.

In contrast to other globin genes, the human and rabbit alpha-globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer. This enhancer-independent expression of the alpha-globin gene requires extensive sequences not only from the 5' flanking sequence but also from the intragenic region. However, the features of these internal sequences that are responsible for their positive effect are unclear. We tested several possible determinants of this activity. One possibility is that a previously identified array of discrete binding sites for known and potential regulatory proteins within the alpha-globin gene comprise an intragenic enhancer specific for the alpha-globin promoter, but directed rearrangements of the sequences show that this is not the case. Alternatively, the promoter may extend into the gene, with the function of the discrete binding sites being dependent on maintenance of their proper positions and orientations relative to the 5' flanking sequence. However, the positive effects observed in gene fusions do not localize to a discrete region of the alpha-globin gene and the results of internal deletions and point mutations argue against a required role of the targeted discrete binding sites. A third possibility is that the CpG island, which includes both the 5' flanking and intragenic regions associated with the positive activity, may itself have a more general effect on expression in transfected cells. Indeed, we show that the size of the CpG island in constructs correlates with the level of gene expression. Furthermore, the alpha-globin promoter is more active in the context of a previously inactive CpG island than in an A+T-rich context, showing that the CpG island provides an environment more permissive for expression. These effects are seen only after integration, suggesting a possible mechanism at the level of chromatin structure.

Upstream Stimulatory Factor Binding to the E-box at −65 Is Required for Insulin Regulation of the Fatty Acid Synthase Promoter

Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. We have shown that FAS transcription rate is induced dramatically when fasted animals are refed with a high carbohydrate diet or when streptozotocin-diabetic mice are given insulin. We also reported that FAS gene transcription was up-regulated by insulin through the proximal promoter region from -71 to -50 and that upstream stimulatory factors (USFs), including USF1 and USF2, interact with this region in vitro. In the present study, by using site-directed mutagenesis of the -71/-50 region and correlating functional assays of the mutated promoter with USF binding activities, we demonstrate that the -65/-60 E-box motif (5'-CATGTG-3') is functionally required for insulin regulation and that USFs are in vivo components of the insulin response complex. Mutation of the -65/-60 E-box sequence abolished insulin response in both transiently and stably transfected 3T3-L1 adipocytes in the -2. 1 kb promoter context, which contains all the necessary regulatory elements of the promoter based on our previous transgenic mice studies, and in the minimal -67 promoter context. Gel mobility shift assays demonstrated that USFs can no longer bind to the -71/-50 promoter region when the E-box is mutated. Cotransfection of USF1 and USF2 expression vectors with the FAS promoter-luciferase reporter constructs increased insulin-stimulated FAS promoter activity. Moreover, cotransfection of dominant negative USF1 and USF2 mutants lacking the DNA binding domain inhibited the insulin stimulation of the FAS promoter activity. On the other hand, site-directed mutagenesis of the -65/-60 E-box surrounding sequences within the overlapped tandem copies of sterol regulatory element-binding protein (SREBP) binding sites prevented SREBP from binding to -71/-50 promoter region in vitro but had no effect on insulin regulation of the FAS promoter in vivo. When rat liver nuclear extracts were used in gel mobility shift assays, only USF-containing protein-DNA complexes that can be supershifted by specific USF antibodies were observed. These results demonstrate that upstream stimulatory factor binding to the E-box at -65 is required for insulin regulation of the fatty acid synthase promoter.

Sterol regulation of acetyl coenzyme A carboxylase promoter requires two interdependent binding sites for sterol regulatory element binding proteins

The sterol regulatory element binding proteins (SREBPs) are central regulators of lipid homeostasis in mammalian cells. Their activity is controlled by a sterol-regulated two-step proteolytic process that releases the nuclear targeted amino-terminal domain from the membrane anchored carboxyl-terminal remnant. This ensures that transcriptional stimulation of the appropriate genes occurs only when increased intracellular sterol accumulation is required. Gene targets for SREBP encode key proteins of cholesterol metabolism as well as essential proteins of fatty acid biosynthesis, providing a mechanism for coordinate control of these two major lipid pathways when sterols and fatty acids need to accumulate together. However, the regulatory mechanism must provide a way to uncouple these two pathways to allow separate regulation when sterol or fat levels need to increase independently of each other. We compared the similarities and differences for how SREBP activates the promoter for the low density lipoprotein (LDL) receptor, which is the key protein involved in cholesterol uptake, relative to how it activates promoters for acetyl coenzyme A carboxylase (ACC) and fatty acid synthase (FAS), which are both key enzymes of fatty acid biosynthesis. In the current studies we show there are two distinct sites for SREBP binding that control activation of the ACC PII promoter whereas previous work has shown there is only a single SREBP site in the LDL receptor. Additionally, disruption of either ACC site results in a total loss in promoter function and a severe decrease in SREBP binding even to the neighboring unaltered site. Thus, the two sites are equally important and dependent on one another for optimal function. This is in contrast to the FAS promoter where SREBP binds to two adjacent sites independently and the one located closer to the binding site for the Sp1 co-regulator is more critical for sterol regulation and activation by SREBP over-expression.

An operon containing fumC and sodA encoding fumarase C and manganese superoxide dismutase is controlled by the ferric uptake regulator in Pseudomonas aeruginosa: fur mutants produce elevated alginate levels.

The activities of fumarase- and manganese-cofactored superoxide dismutase (SOD), encoded by the fumC and sodA genes in Pseudomonas aeruginosa, are elevated in mucoid, alginate-producing bacteria and in response to iron deprivation (D. J. Hassett, M. L. Howell, P. A. Sokol, M. L. Vasil, and G. E. Dean, J. Bacteriol. 179:1442-1451, 1997). In this study, a 393-bp open reading frame, fagA (Fur-associated gene), was identified immediately upstream of fumC, in an operon with orfX and sodA. Two iron boxes or Fur (ferric uptake regulatory protein) binding sites were discovered just upstream of fagA. Purified P. aeruginosa Fur caused a gel mobility shift of a PCR product containing these iron box regions. DNA footprinting analysis revealed a 37-bp region that included the Fur binding sites and was protected by Fur. Primer extension analysis and RNase protection assays revealed that the operon is composed of at least three major iron-regulated transcripts. Four mucoid fur mutants produced 1.7- to 2.6-fold-greater fumarase activity and 1.7- to 2.3-greater amounts of alginate than wild-type organisms. A strain devoid of the alternative sigma factor AlgT(U) produced elevated levels of one major transcript and fumarase C and manganase-cofactored SOD activity, suggesting that AlgT(U) may either play a role in regulating this transcript or function in some facet of iron metabolism. These data suggest that the P. aeruginosa fagA, fumC, orfX, and sodA genes reside together on a small operon that is regulated by Fur and is transcribed in response to iron limitation in mucoid, alginate-producing bacteria.

Two Tandem Binding Sites for Sterol Regulatory Element Binding Proteins Are Required for Sterol Regulation of Fatty-acid Synthase Promoter

We previously reported that sterol regulation of the rat fatty-acid synthase was lost when the DNA sequence between -73 and -43 of the promoter was deleted from a luciferase reporter construct (Bennett, M. K., Lopez, J. M., Sanchez, H. B., and Osborne, T. F. (1995) J. Biol. Chem. 270, 25578-25583). We also showed that there was a binding site for sterol regulatory element binding protein-1 (SREBP-1) in this region that contains a palindromic E-box motif (5'-CANNTG-3'). This is the consensus recognition element for basic-helix-loop-helix leucine zipper containing proteins such as the SREBPs. However, the SREBPs are unique basic-helix-loop-helix leucine zipper proteins that not only bind to a subset of E-boxes but also to the direct repeat SRE-1 element of the low density lipoprotein receptor promoter as well as to variant sites present in the promoters for key enzymes of both cholesterol and fatty acid biosynthesis. Based on the sequence of the variant SREBP recognition sites in these other promoters, we noted there was more than one potential recognition site for SREBP within the -73 to -43 interval of the fatty-acid synthase promoter. In the present studies we have systematically mutated these potential SREBP sites and have analyzed the consequences on sterol regulation, activation by exogenously supplied SREBPs, and binding by SREBPs in vitro. The results clearly show that the E-box element is not the SREBP recognition site in this region. Rather, there are two independent SREBP binding sites that flank the E-box, and both are required for maximal sterol regulation and activation by transfected SREBP protein.

Tempo and mode ofeven-skippedstripe 2 enhancer evolution inDrosophila

Abstract Regulatory sequences controlling important developmental genes are widely viewed as being functionally and evolutionarily constrained by temporal and cell-specific demands on expression. However, the evolution of gene regulation is also viewed as being an important factor in morphogenetic change. To address this issue, we compare sequences of theeven-skipped (eve)stripe 2 enhancer and promoter region within and between species ofDrosophila. The analysis reveals a large number of nucleotide substitutions in regulatory protein binding sites (bicoid, hunchback, kruppelandgiant), as well as a systematic change in the size of the enhancer. We argue that natural selection must be playing a positive role in the evolution of the stripe 2 enhancer.

DNA-stacking interactions Determine the Sequence Specificity of the Deoxyribonuclease Activity of 1,10-Phenanthroline-copper Ion

Bis(1,10-phenanthroline)-copper(I) ion (OP 2Cu +) binds reversibly to B-DNA and makes single-stranded cuts by oxidative attack on the deoxyribose moiety. The deoxyribonuclease activity is sequence-depen dent yet not nucleotide-specific at the cutting site. OP 2Cu +sequence specificity was analysed in terms of local variations of DNA stability. Kinetic constants of strand cleavage were measured at sequence positions on the two strands and converted into activation free energies of the cleavage reaction. DNA unwinding free energies were calculated from the base sequence using B-DNA stacking parameters for calculations. The two free-energy variations were statistically compared for a series of DNA restriction fragments bearing the binding sites of regulatory proteins and representing a total of 345 DNA base positions. This study shows that the mean activation free energy of strand cleavage at a pair of opposing sugars across the DNA minor groove varies like the unwinding free energy of the DNA sequence delimited by opposing sugars (3 to 4 bp). A statistical equality between the two free-energy variations is demonstrated when considering the sum of the two cleavage events at the opposing sugars. Systematic deviations between the two free-energy distributions were observed at specific sequences, including polypurine-polypyrimidine tracts (A n T m /A m T n , C n T m C p/G pA m G n ), alternating purine- pyrimidine tracts ((TA) n /(TA) n , (TG) n /(CA) n ) and at certain G + C-rich triplets (GGC, GCC and CGC). The physical significance of these observations is discussed and a model of OP 2Cu +binding and cleavage specificity based on the free-energy equality is proposed.

Differences in the RNA binding sites of iron regulatory proteins and potential target diversity.

Posttranscriptional regulation of genes of mammalian iron metabolism is mediated by the interaction of iron regulatory proteins (IRPs) with RNA stem-loop sequence elements known as iron-responsive elements (IREs). There are two identified IRPs, IRP1 and IRP2, each of which binds consensus IREs present in eukaryotic transcripts with equal affinity. Site-directed mutagenesis of IRP1 and IRP2 reveals that, although the binding affinities for consensus IREs are indistinguishable, the contributions of arginine residues in the active-site cleft to the binding affinity are different in the two RNA binding sites. Furthermore, although each IRP binds the consensus IRE with high affinity, each IRP also binds a unique alternative ligand, which was identified in an in vitro systematic evolution of ligands by exponential enrichment procedure. Differences in the two binding sites may be important in the function of the IRE-IRP regulatory system.

Characterization of a 1.8-kb DNA segment located upstream from the human Ha-ras protooncogene and possibly regulating its function

We have cloned a 1.8-kb segment of DNA just adjacent to the 5 ′ end of the 6.4-kb BamHI fragment which contains the Ha-ras oncogene ( rasl; GenBank notation HUMRASH). Electrophoretic mobility shift assays (EMSA) indicate the presence of multiple nuclear protein-binding sites within the 1.8-kb segment. At least three putative regulatory protein-binding sites have been identified in a 206-bp subfragment by DNase I footprint analysis. Within the subfragment containing the DNase-protected regions, there is also a segment containing a number of consensus sequences for DNA-binding proteins and two sub-sequences that exhibit strong sequence homology to at least two previously characterized enhancers. These data suggest that a novel set of regulatory elements may lie as far as 2 kb upstream from the normal Ha-ras transcription start points.

Using in vitro nucleic acid selections for conventional drug design

AbstractIn vitro selection of nucleic acids has typically been used for the generation of high molecular weight inhibitors of enzymes. However, this technique can also be used for more conventional drug design in several different ways. First, the binding sites of regulatory proteins on DNA or RNA can be precisely delimited using in vitro selection. Second, “artificial” phylogenices generated by this technique can be used to model the three‐dimensional structure of nucleic acids. And, finally, the technique can be used to screen large chemical libraries for new lexitropsins, compounds that can specifically recognize nucleic acid sequence or structure. © 1994 Wiley‐Less, Inc.

Transcription factor binding sites in the matrix attachment region (MAR) of the chicken alpha-globin gene.

Nuclear matrix is a nuclear protein-DNA superstructure believed to be the exclusive site of DNA replication, transcription, repair, and recombination. The attachment regions of chromatin loops to the nuclear matrix, called MARs, nest origins of replication, have transcriptional enhancer activity, and via their interaction with protein transcription factors may govern gene switch during development and tissue-specific gene expression. In this study the 967 bp MAR of the chicken alpha-globin gene is analyzed for the presence of hexanucleotides from a number (83 in total) of vertebrate protein transcription factors and core origins of replication. A total number of 760 hexanucleotides from factor sites or origins of replication were used for this search. We found that: (1) The occurrence of protein transcription factor binding sites overall on the MAR fragment as well as on the enhancer and promoter regions of other genes is only about 1.2-1.5 times higher than in random DNA, something consistent for all MAR and enhancer sequences examined. However, a high concentration (up to 2.7 times over random sequences) of hexanucleotide factor sites is observed on small stretches of the alpha-globin gene MAR. (2) Some regulatory protein binding sites are underrepresented whereas others are overrepresented, giving to an MAR a particular transcription factor flavor. (3) The DNA curvature map of the MAR sequence and the potential sites of positioned nucleosomes suggest the sites where a competition between core histone octamers and protein transcription factors for DNA might be found. This approach might provide a novel technique to diagnose for the regulatory or nonregulatory function of a stretch of DNA. Furthermore, MARs are proposed to constitute important regulatory elements of genes in addition to enhancers, promoters, silencers, locus control regions, and origins of replication. Additional parameters such as interaction of a transcription factor with other transcription factors fixed at vicinal sites, DNA methylation, intrinsic DNA curvature torsional strain, and nucleosome positioning might also determine the high-affinity binding of a transcription factor to its functional sites and its exclusion from or low affinity binding to other nonregulatory regions.

Interaction of Proteins with a Cytochrome P450 2B2 Gene Promoter: Identification of Two DNA Sequences That Bind Proteins That Are Enriched or Activated in Response to Phenobarbital

Cytochromes P450 (CYPs) are of central importance in the metabolism of foreign hydrophobic compounds. Members of the CYP2B subfamily are inducible at the transcriptional level by the barbiturate, phenobarbital. Owing to the lack of a suitable phenobarbital-responsive cell line, very little is known regarding the mechanisms by which phenobarbital induces the expression of these genes. We report the use of gel retardation and DNase I footprinting to investigate the presence of regulatory protein binding sites within a CYP2B2 gene promoter. Two DNA sequences, located between -183 to -199 and -31 to -72, have been identified that bind rat liver nuclear proteins that are enriched or activated in vivo by phenobarbital. Gel retardation competition experiments demonstrated that the two sequences bound different proteins. In vitro transcription competition experiments demonstrated that the sequences and the proteins with which they interact are involved in regulating CYP2B2 gene transcription. These two DNA sequences and their cognate binding proteins may play a role in the induction of CYP2B2 gene expression in response to phenobarbital.

Regulation of protein synthesis by mRNA structure

In addition to the m7G cap structure, the length of the 5' UTR and the position and context of the AUG initiator codon (which have been discussed elsewhere in this volume), higher order structures within mRNA represent a critical parameter for translation. The role of RNA structure in translation initiation will be considered primarily, although structural elements have also been found to affect translation elongation and termination. We will first describe the different effects of higher order RNA structures per se, and then consider specific examples of RNA structural elements which control translation initiation by providing binding sites for regulatory proteins.