RNA Polymerase Density Research Articles

Oligomerization of the transcription termination factor TTF-I: implications for the structural organization of ribosomal transcription units.

Mammalian ribosomal genes are flanked at their 5'and 3'ends by terminator sequences which are recognized by the transcription termination factor TTF-I. The occurrence of the same binding site upstream and downstream of the gene raises the possibility that TTF-I can interact with both sequences simultaneously and thus brings the terminator in the vicinity of the gene promoter by looping out the pre-rRNA coding sequence. To test this model, we have examined the ability of TTF-I to oligomerize and found that both full-length and N-terminally truncated versions of TTF-I form stable oligomeric structures. At least two domains of TTF-I located within the 184 N-terminal and 445 C-terminal amino acids, respectively, mediate the self-association of several TTF-I molecules. In support of the looping model, TTF-I is capable of linking two separate DNA fragments via binding to the target sites. This result indicates that in addition to its function in transcription termination, TTF-I may serve a role in the structural organization of the ribosomal genes which may be important for maintaining the high loading density of RNA polymerase I on active rRNA genes.

Positive supercoiling of DNA greatly diminishes mRNA synthesis in yeast.

In Saccharomyces cerevisiae cells harboring a GAL1 promoter-linked beta-galactosidase gene, the simultaneous expression of Escherichia coli DNA topoisomerase I and inactivation of yeast DNA topoisomerases I and II reduces the cellular level of beta-galactosidase to an undetectable level. Analysis of intracellular mRNA level and the density of RNA polymerase along DNA indicates that this reduction is due to the suppression of transcription and that both plasmid-borne and chromosomally located genes are affected. These results are interpreted in terms of inhibition of transcription in vivo due to positive supercoiling of the DNA template: preferential removal of transcription-generated negative supercoils by E. coli DNA topoisomerase I in the absence of both yeast DNA topoisomerases I and II results in the accumulation of positive supercoils in intracellular DNA. In normal prokaryotic or eukaryotic cells, accumulation of positive supercoils is presumably avoided through the balanced actions of DNA topoisomerases.

New insights into the mechanism of HIV-1 trans-activation

acting factors were found sub- sequently in all other lentiviruses, including HIV-2 and simian im- munodeficiency virus (SIV). These findings stimulated intensive re- search into the mechanism of action of Tat and a search for antiviral compounds 1. Now, nearly seven years later, a coherent pic- ture of how Tat works is emerging. Transcription from the HIV LTR is initiated by the binding of cellu- lar DNA-transcription factors and RNA polymerase to the proviral LTR. In the absence of Tat, most of the viral transcripts produced are short RNA molecules of 60-80 nucleotides, and most of the RNA polymerases engaged in transcrip- tion appear to stall near the pro- moter 2-4. In the presence of Tat, there is a large increase in the efficiency of elongation, and the density of RNA polymerases down- stream of the promoter is markedly increased2,3.

Chapter 14 Protein—DNA Cross-Linking as a Means to Determine the Distribution of Proteins on DNA in Vivo

The protein-DNA cross-linking can be used to determine the distribution of proteins on DNA in vivo. The temporal and spatial distributions of particular proteins on specific DNA sequences provide key insights into the regulation and mechanics of transcription and chromosome replication and basic information on chromatin structure. The chapter describes a method for directly mapping the distribution of a protein on DNA in living cells by ultraviolet (UV)-induced cross-linking. UV irradiation of cells cross-links protein to DNA at the points of contact. The protein–DNA adducts are purified from cells, and then the protein of interest is immunoprecipitated with an appropriate antibody. The distribution of the protein on DNA in the cell is revealed by identifying specific restriction fragments that coprecipitate with the protein via their UV-induced covalent attachment to the protein. The UV-cross-linking method provides a unique opportunity to evaluate mechanistic questions in vivo. The method has been used to demonstrate that Drosophila topoisomerase I is recruited during heat shock to activated transcription units. It has also provided a direct measure of the relative in vivo density of RNA polymerase II on a variety of Drosophila genes.

Identification and characterization of transcriptional arrest sites in exon 1 of the human adenosine deaminase gene.

Analysis of human adenosine deaminase (ADA) gene transcription in four different cell lines indicated that a high density of RNA polymerase II complexes is present at the 5' end of the gene and that the extent of transcription elongation beyond the promoter-proximal region governs gene expression. To determine the sequence requirements for a potential transcription arrest site in the promoter-proximal region, genomic clones containing the ADA promoter, exon 1, and various lengths of intron 1 were injected into Xenopus laevis oocyte germinal vesicles. Transcription analysis indicated that nascent ADA transcripts were highly represented at the promoter-proximal region of the injected templates, suggesting that transcription arrest occurred in the oocyte transcription system. Analysis of the transcription products indicated that ADA transcription initiated at the authentic start site and that the most prominent, short ADA transcripts were 105 nucleotides in length. The 3' end of these transcripts mapped within exon 1, 10 nucleotides downstream of the translation initiation codon. Deletion analysis demonstrated that sequences within exon 1 were sufficient to specify the synthesis of the 105-nucleotide transcripts. Taken together, these data suggest that a transcription arrest mechanism operates in the promoter-proximal region of the human ADA gene and that regulation of elongation beyond this point plays a major role in regulating ADA gene expression.

Identification and Characterization of Transcriptional Arrest Sites in Exon 1 of the Human Adenosine Deaminase Gene

Analysis of human adenosine deaminase (ADA) gene transcription in four different cell lines indicated that a high density of RNA polymerase II complexes is present at the 5' end of the gene and that the extent of transcription elongation beyond the promoter-proximal region governs gene expression. To determine the sequence requirements for a potential transcription arrest site in the promoter-proximal region, genomic clones containing the ADA promoter, exon 1, and various lengths of intron 1 were injected into Xenopus laevis oocyte germinal vesicles. Transcription analysis indicated that nascent ADA transcripts were highly represented at the promoter-proximal region of the injected templates, suggesting that transcription arrest occurred in the oocyte transcription system. Analysis of the transcription products indicated that ADA transcription initiated at the authentic start site and that the most prominent, short ADA transcripts were 105 nucleotides in length. The 3' end of these transcripts mapped within exon 1, 10 nucleotides downstream of the translation initiation codon. Deletion analysis demonstrated that sequences within exon 1 were sufficient to specify the synthesis of the 105-nucleotide transcripts. Taken together, these data suggest that a transcription arrest mechanism operates in the promoter-proximal region of the human ADA gene and that regulation of elongation beyond this point plays a major role in regulating ADA gene expression.

A transcriptional terminator between enhancer and promoter does not affect remote transcriptional control

Enhancers stimulate transcription of RNA polymerase II-transcribed genes in an orientation-independent manner and over long distances. This stimulation is known to be associated with an increased polymerase density over the linked gene. However, many aspects of the exact mechanism of remote gene control remain to be elucidated. Based on some reports on RNA polymerase I transcription, we wanted to test whether RNA polymerase II enters at the enhancer and from there proceeds towards the promoter while synthesizing unstable transcripts ("scanning/readthrough transcription" model). For this, we have inserted a complete terminator region from the mouse beta-globinmaj gene between the SV40 enhancer and the rabbit beta-globin promoter. In contrast to what the model predicts, insertion of the terminator had no affect on remote enhancer action. Furthermore, we have determined the RNA polymerase density over the spacer DNA between enhancer and promoter, and over the reporter gene, by means of the so-called run-on transcription assay. We find very low transcription of the spacer, but high transcription of the globin reporter gene. Thus, our data are not consistent with a scanning/readthrough transcription mechanism where RNA polymerase II would move from the enhancer to the promoter while transcribing the intervening spacer DNA. These and other findings are compatible with a model where enhancer and promoter are brought into close proximity, perhaps with concomitant looping out of the intervening DNA.

High initiation rates at the ribosomal gene promoter do not depend upon spacer transcription.

We report experiments that test the model that in Xenopus laevis, RNA polymerase I is "handed over" in a conservative fashion from the T3 terminator to the adjacent gene promoter. We have introduced transcription-terminating lesions into the ribosomal DNA repeat by irradiating cultured cells with ultraviolet light. We used isolated nuclei to measure the effect of such lesions on transcription. UV damage sufficient to prevent all elongating RNA polymerase from reaching T3 from upstream had no adverse effect on the density of RNA polymerase at the very 5' end of the gene. We conclude that high rates of transcription initiation at the gene promoter do not depend upon polymerase passing from one repeat to the next or on polymerase initiating at the spacer promoters.

Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes.

Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role.

Transcriptional control of μ- and и-Gene expression in resting and bacterial lipopolysaccharide-activated normal B cells

When murine resting B cells are polyclonally stimulated by bacterial lipopolysaccharide(LPS) in vitro for a short period of 4 days, they are activated to DNA synthesis and cell division, and they also differentiate to immunoglobulin (Ig)-secreting plasma cells. These two events are accompanied with several qualitative changes at the Ig mRNA level:1) the disappearance of delta mRNA after stimulation, 2) the switch from membrane to secretory form of \\gm-mRNA, and 3) the late appearance of IgM joining chain (J-chain) mRNA. There is also a quantitative increase of Ig-gene expression at the level of: a) Ig gene transcription, b) \\gm-, \\qi- and J-chain mRNA accumulation, and c) Ig translation and secretion. A comparison of Ig transcription rates before and in the course of LPS stimulation, asdetermined by in vitro transcriptional run-on assays, has shown that there is a large increase of the RNA polymerase density on both μ- and и-loci (30–60-fold), which is quantitatively comparable with the accumulation of both μ- and и-mRNAs at the steady state mRNA level. These data therefore suggest that former results obtained with tumor cells regarding post-transcriptionalcontrol of Ig gene expression do not reflect the physiological behavior of normal B cells with respect to the molecular events of B cell triggering. We also propose that additional molecular events such as RNA processing and the transcriptional activation of J-chain gene might be essential for controlling the maximal transcriptional rate across the Ig loci.

During B-cell differentiation enhancer activity and transcription rate of immunoglobulin heavy chain genes are high before mRNA accumulation

During differentiation of B lymphocytes, a low level of immunoglobulin heavy chain gene transcripts is apparent at the pre-B-cell stage, and a dramatic increase in immunoglobulin mRNA level is seen after stimulated B cells have matured into immunoglobulin-secreting plasma cells. We have measured the transcription rate of endogenous heavy chain genes using cell lines representative of various stages of murine B-lymphocyte differentiation. We observe a good correlation between RNA polymerase density, as determined by nuclear run-on transcription experiments, and the activity of the heavy chain gene enhancer, as assayed by transfection experiments. Both enhancer activity and heavy chain gene transcription are very high in pre-B-cell lines. Thus we conclude that the increased accumulation of immunoglobulin heavy chain mRNA in immunoglobulin-secreting plasma cells is regulated mainly by posttranscriptional processes.

In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.

We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.

In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.

We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.

Electron microscopic visualization of trp operon expression in Salmonella typhimurium.

Transcriptional activity of plasmids carrying wild-type and mutant trp operons was visualized in cell lysates of Salmonella typhimurium. Plasmid and transcription-unit sizes varied with the size of the cloned operon. Following 3-(3-indolyl)acrylic acid derepression, all operons of a particular type exhibited the same high level of transcriptional activity. An estimated 11-14 transcripts must be initiated each minute to maintain the 190-base-pair spacing of RNA polymerases observed on the promoter-proximal half of the wild-type trp operon. A decline in RNA polymerase density was observed on promoter-distal portions of cloned trp operons, which may be attributable to premature transcription termination accompanying translation inhibition due to indolylacrylic acid's interference with normal tryptophanyl-tRNA synthetase activity.

Simian virus 40 enhancer increases RNA polymerase density within the linked gene.

Enhancers are regulatory DNA elements, usually about 200 base pairs (bp) long, which are able to stimulate transcription of linked genes in eukaryotic cells. This activation can be exerted over large distances, and from a position 5' or 3' to the gene. Enhancers have been identified in viral genomes and cellular genes. Using a transient expression assay, we have analysed transcription of the rabbit beta-globin gene and the thymidine kinase gene from herpes simplex virus with and without a simian virus 40 (SV40) enhancer. S1 nuclease mapping shows a high level of specific transcripts when the genes are linked to the enhancer. To determine whether this increased number of transcripts is due to a higher transcription rate, or perhaps to a shift from nonspecific to specific initiation, we have performed run-on transcription assays with isolated nuclei. Our results, presented here, demonstrate that the SV40 enhancer increases the RNA polymerase density within the linked gene. Therefore, enhancers apparently increase the rate of transcription initiation without influencing the specificity of initiation.

Chromatin structure of a hyperactive secretory protein gene (in Balbiani ring 2) of Chironomus.

We examined the chromatin structure of a Balbiani ring (secretory protein gene) in the salivary glands of Chironomus larvae in its hyperactive state after stimulation with pilocarpine. For the inactive state of the gene an established tissue culture cell line, not expressing the gene, was used. Electron microscopy showed an RNA polymerase density of approximately 38/microns. Micrococcal nuclease digestion of purified nuclei followed by DNA transfer and hybridization revealed a smear with no recognizable discrete DNA fragments. Without pilocarpine stimulation a faint nucleosomal repeat was superimposed upon the smear, and in tissue culture cells a clear nucleosomal repeat was revealed. The restriction enzyme XbaI, which has a 6-bp recognition sequence, cut the gene in the hyperactive chromatin state, but not in its inactive conformation. The combined results are best explained by the absence of most of the nucleosomes in this hyperactive RNA polymerase II transcribed gene.

Transcription of tandemly repeated genes in embryonal and larval tissues of Calliphora erythrocephala

The transcription of rRNA genes in Calliphora erythrocephala embryonal and larval fat bodies and guts was analyzed by electron microscopy. A gradual increase in the number of active genes and in the RNA polymerase density was observed in both tissues in the hours preceding hatching. Despite this increase, the rRNA genes at hatching are not fully loaded with RNA polymerase molecules. An additional increase in rRNA synthesis is characteristic of the first two days of larval development. This increase in rRNA synthesis is accomplished by an increase in RNA polymerase density, by activation of previously inactive rRNA genes, and by activation of newly replicated rRNA genes. The rRNA seems to be generated exclusively on genes not interrupted by a 6.1 kb intervening sequence. After the first 2 days, the RNA polymerase densities decline. Synthesis of rRNA seems to be coordinately regulated in these two tissues. — Two classes of tandemly repeated nonribosomal transcription units were detected in the fat bodies. The first class has an estimated DNA length of 3.3 kb and cannot be assigned to any of the known genes. The second class appears to represent very short transcription units with no visible RNP fibrils and is a candidate for 5S or tRNA transcription units.

A quantitative electron microscopic analysis of transcription in sea urchin embryos

In an effort to define stage-specific embryonic patterns of sea urchin transcription, we have examined by electron microscopy the distribution of nascent RNP fibrils in dispersed chromatin from nuclei of Strongylocentrotus purpuratus gastrulae. Detailed analysis of individual embryonic nuclei has revealed several new features of nuclear RNA production. Most (82%) of the active chromatin regions observed were represented by only a single fibril. 11% of the active regions contained multiple RNP fibril arrays with an average RNA polymerase density of 1.7 +/- 1.0 polymerases/micron of chromatin and an average contour length of 4.7 +/- 2.8 microns chromatin. An analysis of the lengths of RNP fibrils in single vs. multiple fibril arrays indicates that the differential distribution of RNA polymerases is due to different rates of initiation rather than to different lengths of transcription units (assuming the rate of RNA chain elongation to be constant). We discuss these data in relation to various transcriptional parameters measured by other workers and to EM analyses of other embryonic nuclei. Elucidation of transcriptional patterns in gastrula embryos can provide the basis for further comparative studies of transcription at other stages of sea urchin development in which rates of total genomic transcription vary but the rate at individual loci is as yet unknown.