Regulation Of Transcription Initiation Research Articles

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha-amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.

Theoretical and experimental analysis of the phage lambda genetic switch implies missing levels of co-operativity

The behavior of the cro-repressor switch in phage lambda, a temperate phage of Escherichia coli, is described at the organismal level within a dynamical system framework. The molecular biology of the switch has been well characterized up to the level of the regulation of transcription initiation. In this paper we construct a description of a lysogen whose prophage is mutated in certain genes, so that the switch is functionally isolated from the rest of the phage genome. Such a lysogen has two stable epigenetic states, and comparison of the theory with measurements of intracellular cro concentration corresponding to one of these states suggest that additional levels of regulation, not included in the current description of the switch, should exist for this system. In addition, we suggest a new method for the measurement of intracellular concentrations.

A cis downstream element participates in regulation of in vitro transcription initiation from the P38 promoter of minute virus of mice

We report the use of a HeLa whole cell extract (WCE) runoff transcription system for the study of cis- and trans-acting elements, participating in the regulation of transcription initiation from the P38 promoter of the parvovirus minute virus of mice (MVM). Our initial studies with HeLa WCE indicated that transcription from the P38 promoter is very inefficient, compared with transcription from the P4 promoter. Supplementation of the HeLa WCE with WCE prepared from uninfected Ehrlich ascites cells enhanced transcription from the P38 promoter twofold, indicating a role for a cellular factor in transcription from the P38 promoter. Furthermore, supplementation with WCE prepared from MVM-infected Ehrlich ascites cells enhanced transcription from the P38 promoter about sixfold, indicating a role for a virally encoded or induced factor. Analyses of runoffs produced by transcription of DNA templates digested with various restriction enzymes defined a downstream promoter element (DPE) necessary for efficient transcription initiation from the P38 promoter. This element resides 282 to 647 base pairs 3' to the transcription initiation site, between the NarI site and the HindIII site (2287 to 2652, MVM numbering system). The virally encoded NS1 protein was shown by DNA precipitation to bind directly or indirectly through a cellular factor to the DPE. This interaction is suggested to be involved in the up regulation of the P38 promoter of MVM. Finally, with a DNase I protection assay performed on a fragment containing the DPE, we estimated the sequence involved in the binding of a factor present in uninfected and infected extracts. The correlation between the binding and transcription activation is discussed.

A sequence pattern that occurs at the transcription initiation region of yeast RNA polymerase II Promoters

Saccharomyces cerevisiae mRNA 5'-ends map at a variable distance from the TATA element. The mechanism for the choice of the transcription Initiation Region (IR) over other neighbouring sequences is not clearly understood. Sequences on the coding strand flanking the IR of 95 yeast RNA polymerase II promoters have been compared. They indicate the following pattern: statistically, a preponderance of T residues beginning as far as 30 nucleotides upstream and ending approximately 10 nucleotides upstream of the IR, and a preponderance of A residues from approximately 8 nucleotides upstream of the transcription initiation-site onward. The switch in base composition noted above thus occurs over a short region that is centered typically -9 nucleotides with respect to the major transcription start-site. We call this overall sequence pattern the locator. It is more evident among strong promoters than weak ones, suggesting a role in transcription initiation. The promoter of the TCM1 gene (coding for ribosomal protein L3) has a typical locator in the region of its IR. In an attempt to confirm the role of this sequence motif in defining the IR, deletions were introduced between the TATA element and the IR of the TCM1 gene. In most deletions, the new transcription start-sites are found within a recognizable locator, supporting the suggestion that this sequence pattern is important in defining the IR. These data appear to indicate that in yeast the IR is defined by a pattern of base composition situated at a suitable distance from the TATA element.

AbrB, a regulator of gene expression in Bacillus, interacts with the transcription initiation regions of a sporulation gene and an antibiotic biosynthesis gene.

The abrB gene of Bacillus subtilis is believed to encode a repressor that controls the expression of genes involved in starvation-induced processes such as sporulation and the production of antibiotics and degradative enzymes. Two such genes, spoVG, a sporulation gene of B. subtilis, and tycA, which encodes tyrocidine synthetase I of the tyrocidine biosynthetic pathway in Bacillus brevis, are negatively regulated by abrB in B. subtilis. To examine the role of abrB in the repression of gene transcription, the AbrB protein was purified and then tested for its ability to bind to spoVG and tycA promoter DNA. In a gel mobility shift experiment, AbrB was found to bind to a DNA fragment containing the sequence from -95 to +61 of spoVG. AbrB protein exhibited reduced affinity for DNA of two mutant forms of the spoVG promoter that had been shown to be insensitive to abrB-dependent repression in vivo. These studies showed that an upstream A + T-rich sequence from -37 to -95 was required for optimal AbrB binding. AbrB protein was also observed to bind to the tycA gene within a region between the transcription start site and the tycA coding sequence as well as to a region containing the putative tycA promoter. These findings reinforce the hypothesis that AbrB represses gene expression through its direct interaction with the transcription initiation regions of genes under its control.

Novel regulation of transcription initiation of the peptide IX gene of adenovirus 2.

cis-Acting elements involved in transcription of the peptide IX (pIX) gene of adenovirus 2 were identified by using in vivo transient expression assays and two in vitro transcription systems. Deletion of either the sequence between positions -45 and -70 or the TATA box abolished the initiation of pIX gene transcription in vivo and transcription with HeLa cell nuclear extracts in vitro. These results initially suggested the presence of a positive factor acting on the upstream element. However, when proteins in the nuclear extract were fractionated by column chromatography and analyzed by reconstitution of transcription in vitro, it was found that a certain fraction could direct TATA box-dependent transcription initiation even in the absence of the upstream element. Furthermore, activity inhibiting TATA box-dependent transcription was found in the nuclear extract. In contrast, inhibition of TATA box-dependent transcription was suppressed by deletion of a downstream sequence between positions +33 and +122. These results indicate that the TATA box of the pIX gene by itself has the ability to direct initiation of constitutive transcription but that the function of this element is under negative control by a repressor acting on a downstream sequence. Thus, the upstream element of the pIX gene appears to have a novel function: suppression of the transcriptional repression exerted by a downstream sequence, leading to a net transcription activation. Possible mechanisms for transcription initiation of pIX DNA are discussed.

Novel regulation of transcription initiation of the peptide IX gene of adenovirus 2.

cis-Acting elements involved in transcription of the peptide IX (pIX) gene of adenovirus 2 were identified by using in vivo transient expression assays and two in vitro transcription systems. Deletion of either the sequence between positions -45 and -70 or the TATA box abolished the initiation of pIX gene transcription in vivo and transcription with HeLa cell nuclear extracts in vitro. These results initially suggested the presence of a positive factor acting on the upstream element. However, when proteins in the nuclear extract were fractionated by column chromatography and analyzed by reconstitution of transcription in vitro, it was found that a certain fraction could direct TATA box-dependent transcription initiation even in the absence of the upstream element. Furthermore, activity inhibiting TATA box-dependent transcription was found in the nuclear extract. In contrast, inhibition of TATA box-dependent transcription was suppressed by deletion of a downstream sequence between positions +33 and +122. These results indicate that the TATA box of the pIX gene by itself has the ability to direct initiation of constitutive transcription but that the function of this element is under negative control by a repressor acting on a downstream sequence. Thus, the upstream element of the pIX gene appears to have a novel function: suppression of the transcriptional repression exerted by a downstream sequence, leading to a net transcription activation. Possible mechanisms for transcription initiation of pIX DNA are discussed.

Interferons as Gene Activators: Indications for repeated gene duplication during the evolution of a cluster of interferon-activatable genes on murine chromosome 1

We have described earlier a gene cluster, including at least six interferon-activatable genes closely linked to the erythroid α spectrin locus and the serum amyloid P-component locus on murine chromosome 1. Here, we report that sequences of three genes from the cluster (the 201, 202, and 204 genes) are very similar in a segment extending from at least 550 nucleotides upstream of the 3′ end of the transcription initiation region to beyond the first exon intron border (96% similarity between the 202 and 204 genes and 89% similarity between the 201 and 204 genes). This region contains the following two types of interferon-responsive enhancers: a GA box and a Friedman Stark sequence. The proteins coded for by the 202 gene (51 kDa) and the 204 gene (72 kDa) are hydrophilic. The amino acids have been conserved in the two proteins in 47% of the sequence. Each protein includes two apparently contiguous, approximately 200-amino acid long segments with much sequence similarity (27% in the 202 protein and 34% in the 204 protein). These segments are preceded in the 204 protein only by a segment including four perfect and three imperfect repeats of a 7 amino acid sequence. These and other data suggest that the evolution of the gene cluster involved the duplication of a DNA segment generating a double length transcription unit and subsequent divergence and duplication of this unit giving rise to at least two interferon-activatable genes (the 202 and 204 genes).

Mutational analysis of IS10′s outside end.

We present the genetic analysis of a large number of mutations in the outside end of insertion sequence IS10. (i) The terminal inverted repeat sequence is probably the primary site of transposase binding. Mutations in this region fall into phenotypic classes which correspond to their map locations, suggesting that this region may consist of several distinct functional segments. Similarities between the organization of IS10's inverted repeat and those of other transposable elements are discussed. (ii) Base pairs 23-42 include a consensus binding sequence for one of the IS10 transposition host factors, IHF. The phenotypes of mutations in this region suggest that IHF is the major host factor for outside-end transposition activity in vivo and that base pairs throughout this region are important for the IHF interaction. (iii) Mutations in bp 43-61 do not affect outside-end transposition activity but do affect, in expected ways, previously identified determinants involved in expression and regulation of transposase. (iv) Some mutations in bp 23-42 also affect transposase expression; the possibility that IHF negatively regulates transcription initiation is discussed.

Clones containing variant forms of complete human rRNA genes characterization and sequence of their transcription initiation region

In order to assess the extent of structural heterogeneity among ribosomal genes in the human genome, several cosmid clones, each containing an entire transcription unit, have been isolated and analyzed. Five cloned genes were recognized as structurally different from each other to some extent, either within the transcription unit or in its immediate vicinity. The sequence of a 1.2 kb region encompassing the transcription start site has been determined for the five cloned genes. Point differences among the genes are observed at five nucleotide positions within the analyzed portion of the 5′ external transcribed spacer of the ribosomal gene. Moreover, upstream from the transcription start site, a unique difference is observed with the absence from the three genes of a 19-nucleotide long stretch which is present in the two other variant gene forms.

Changing the RNA polymerase specificity of U snRNA gene promoters

The promoter of a Xenopus tropicalis U6 gene can be transcribed by both RNA polymerases II and III. Two distinct elements, a TATA-like sequence and the region of transcription initiation, are only required for transcription by RNA polymerase III, while further common elements are required for transcription by both polymerases. Based on the unusually stringent requirement for a purine at the normal position of polymerase III transcription initiation and on the properties of mutants in this region, we suggest that RNA polymerase III itself may recognize the site of transcription initiation and thus be directly involved in efficient promoter selection. We have used the information obtained on U6 promoter structure to manufacture a U6 promoter that is RNA polymerase II-specific and to change the Xenopus U2 gene promoter specificity from RNA polymerase II to RNA polymerase III.

A conserved CATTCCT motif is required for skeletal muscle-specific activity of the cardiac troponin T gene promoter.

Transcription of the cardiac troponin T (cTNT) gene is restricted to cardiac and embryonic skeletal muscle tissue. A DNA segment containing 129 nucleotides upstream from the cTNT transcription initiation site (cTNT-129) directs expression of a heterologous marker gene in transfected embryonic skeletal muscle cells but is inactive in embryonic cardiac or fibroblast cells. By using chimeric promoter constructions, in which distal and proximal segments of cTNT-129 are fused to reciprocal segments of the herpes simplex virus thymidine kinase (HSV tk) gene promoter, the DNA segment responsible for this cell specificity can be localized to the cTNT distal promoter region, located between 50 and 129 nucleotides upstream of the transcription initiation site. The ability of the cTNT distal promoter region to confer skeletal muscle-specific activity upon a heterologous promoter is abolished when it is displaced 60 nucleotides upstream, indicating that its ability to direct skeletal muscle-specific transcription probably requires proximity to other components of the transcription initiation region. Two copies of the heptamer, CATTCCT ("muscle-CAT" or "M-CAT" motif), reside within the 80-nucleotide cTNT distal promoter region. A 3-nucleotide mutation in one of these copies inactivates the cTNT promoter in skeletal muscle cells. Therefore, the M-CAT motif is a distal promoter element required for expression of the cTNT promoter in embryonic skeletal muscle cells. Since the M-CAT motif is found in other contractile protein gene promoters, it may represent one example of a muscle-specific promoter element.

In Vitro Sequence-Specific Cleavage in Transcribed Spacer of Mouse Precursor Ribosomal RNA1

When we compared the sequences near four terminal sites of transcripts including two endoribonucleolytic cleavage sites in external transcribed spacer 1 upstream of the 5' end of the mouse 18S rRNA (Mishima, Y., Mitsuma, T., & Ogata, K. (1985) EMBO J. 4, 3879-3886), a seven-nucleotide consensus sequence, GGPyUUGPy (Py is C or U), was obtained. The results of both in vitro pulse-chase experiments and S1 nuclease mappings using the mouse rDNA fragment of the transcription initiation region indicated that ribonucleolytic cleavages take place in the sequence matching the consensus sequence at more than five nucleotides out of the seven positions. Furthermore, effective ribonucleolytic cleavages in vitro were observed in a sequence, GGCUUGU, in the internal transcribed spacer 2 located between 5.8S and 28S rRNA. These results demonstrate that the ribonucleolytic cleavages occur preferentially in the sequence of GGPyUUGPy in the transcribed spacer regions of the mouse pre-rRNA. From this information, we infer the existence of processing steps for the pre-rRNA maturation involving the sequence-specific ribonucleolytic cleavages.

YAP1 encodes a yeast homolog of mammalian transcription factor AP-1.

The regulation of transcription initiation in eukaryotes is an essential control point in gene expression. In recent years, many trans-acting factors have been identified that bind to regulatory promoter elements of mammals, Drosophila, and yeast (for reviews, see McKnight and Tjian 1986; Jones et al. 1988). The biochemical purification of some trans-activating factors and the subsequent molecular cloning of the gene that encodes a given activating factor have been achieved in several instances. This has allowed a detailed biochemical and physiological characterization of the functional domains present in the activator proteins. In addition to the biochemical characterization of a given transcription factor, an important issue that remains to be addressed is how signals from the outside of the cell manifest themselves as alterations in gene transcription.

Methods to define and locate patterns of motifs in sequences.

A method to define and search for complex patterns of motifs in nucleic acid and protein sequences is described. With this method nucleic acid motifs can be defined in eight different ways and protein motifs in six. A pattern is defined by a list of motifs. The motifs in a list are combined using the logical operators AND, OR and NOT. The list also defines the ranges of allowed separations of the motifs in the pattern. Programs to search for patterns in individual sequences and libraries of sequences are described. Patterns are defined by users and stored as annotated disk files. Hence the programming to define and locate new structures can be performed by users and fewer specific novel algorithms should be required. Examples are given of searches for transcription initiation regions, nematode mitochondrial tRNA genes and for members of the globin sequence family.

Conserved elements in the transcription initiation regions preceding highly expressed structural genes of methanogenic archaebacteria.

The sequences of the intergenic regions of the strongly expressed genes encoding methyl CoM reductase in three different methanogenic archaebacteria were determined and the 5'-ends of the transcripts were mapped. After alignment, consensus sequences were found which are located both upstream and downstream of the transcription starts. They correspond, in part, to those previously characterized as putative elements of archaebacterial promoter sequences. In addition, bending of the DNA in front of the transcription start sites was shown in two cases and a characteristic common DNA structure immediately downstream of the 5'-end of the transcript was discovered. This structure was also found in the corresponding regions of previously described genes in methanogens. Our results suggest that both sequence and structural information may have roles in the initiation of transcription of protein encoding genes of these archaebacteria.

Variation among mouse ribosomal RNA genes within and between chromosomes.

We compared nucleotide sequences of the transcription-initiation region of five mouse ribosomal DNA (rDNA) clones. Two clones were isolated from Japanese wild mice, Mus musculus molossinus, and the other three from BALB/c mice originating in M. m. domesticus. Two BALB/c clones that are derived from the same chromosome are very similar, suggesting the occurrence of intrachromosomal homogenization of rDNA repeats. However, the other clone of BALB/c was approximately as different from the former two clones as from the rDNA clones of M. m. molossinus. These results suggest that, compared with intrachromosomal homogenization, homogenization among rDNA repeats on nonhomologous chromosomes occurs relatively infrequently after the mouse subspecies separation.

Regions upstream from the core promoter of the rat ribosomal gene are required for the formation of a stable transcription initiation complex by RNA polymerase I in vitro

The sites required for the formation of a stable transcription initiation complex and for the initiation of transcription of rat rDNA in vitro were examined. A series of 5′ deletion mutants of the rat transcription initiation region (−167 through +638) were constructed. These mutants were examined for their ability to support the faithful initiation of transcription in vitro. Mutants which contain less than 31 nucleotides upstream of the initiation site (+1) were unable to support detectable initiation of transcription. In this transcription system a series of deletion mutants from −167 to −31 were transcribed with equal efficiency when assayed individually. On the other hand, when the wild-type and mutant templates were compared in order-of-addition assays, they were found to be unequal. The incubation of an extract with a wild-type template, prior to the addition of nucleotides, precluded transcription of any second template added after the preincubation step. However, the preincubation of extract with mutants of the region upstream of the core promoter, from −122 to −31, did not preclude transcription of a wild-type template added after the preincubation step. Formation of the stable preinitiation complex was found to require the region between −122 and −167.

Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis.

An approximately 16-kilobase pair region of the Bacillus subtilis chromosome at 55 degrees containing genes for de novo purine nucleotide synthesis (Piggot, P. J., and Hoch, J. A. (1985) Microbiol. Rev. 49, 158-179) was cloned. The nucleotide sequence of over 13 kilobase pairs indicates that this region contains a cluster of 12 genes, 11 of which encode enzymes that catalyze the 10 reactions for de novo purine nucleotide synthesis from 5-phosphoribosyl 1-pyrophosphate to IMP. The genes were identified by complementation of Escherichia coli pur mutants and by sequence comparisons with homologous enzymes. The cluster is likely an operon and is organized into three groups of overlapping genes followed by the last gene: purEKB-purC(orf)QLF-purMNH(J)-purD. Sequence comparisons provide evidence for homology of monofunctional purine nucleotide biosynthetic enzymes from B. subtilis with the corresponding multifunctional enzymes from yeast and Drosophila. Sequence alignment of the phosphoribosylaminoimidazole carboxylase heterodimer from B. subtilis with the monomeric enzyme from Methanobrevibacter smithii indicates an evolutionary relationship between these two enzymes. S1 nuclease analysis was used to map the mRNA 5' and 3' ends and to estimate levels of mRNA. These experiments indicate that synthesis of purine nucleotides is regulated independently by adenine and guanine nucleotides. Adenine nucleotides regulate transcription initiation. Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide 5' untranslated mRNA leader region. Groups of overlapping genes, regulated at least in part by transcription termination-antitermination is likely to be a common theme for genetic organization and regulation of biosynthetic genes in this Gram-positive organism.

Long-range cooperativity between gene regulatory sequences in a prokaryote.

Regulation of transcription initiation by proteins binding at DNA sequences some distance from the promoter region itself seems to be a general phenomenon in both eukaryotes and prokaryotes. Proteins bound to an enhancer site in eukaryotes can turn on a distant gene, whereas efficient repression of some prokaryotic genes such as the gal, ara and deo operons of Escherichia coli, requires the presence of two operator sites, separated by 110, 200 and 600 base pairs (bp) respectively. In the deo operon, which encodes nucleoside catabolizing enzymes, we have shown that efficient and cooperative repression can be obtained when the distance between the two sites ranges from 224 to 997 bp. Here, we report that transcription initiation can be regulated from an operator site placed 1 to 5 kilobases (kb) downstream of the deoP2 promoter (and downstream of the transcribed gene), and present the first experimental data for prokaryotic regulation at distances greater than 1 kb. Our results support the model of DNA loop formation as a common regulatory mechanism explaining both some prokaryotic regulation and the action of eukaryotic enhancers.