Transcription Elongation Block Research Articles

Abstract PR04: Transcriptional regulation of mitochondrial metabolism by TIF1γ drives erythroid progenitor differentiation

Abstract Understanding in-vivo mechanisms of erythropoiesis is critical for directed differentiation approaches to treat blood disorders such as leukemias. Zebrafish moonshine (mon) mutant embryos defective for the conserved transcriptional intermediary factor 1 gamma (tif1γ) do not specify enough erythroid progenitors due to a transcription elongation block characterized by aberrantly paused RNA polymerase II. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we performed a chemical suppressor screen using 3,100 compounds and identified inhibitors of the essential mitochondrial pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH). Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar rescue the formation of erythroid progenitors in 61% (38/62) and 68% (50/74) of mon embryos, respectively. Beyond changes in nucleotide metabolism, in-vivo metabolic analyses revealed low levels of TCA cycle metabolites which were functionally linked to a reduced oxygen consumption rate. In addition, an increased 2HG/αKG ratio was associated with higher histone methylation states at H3K27, H3K36 and H4K20 as assessed by quantitative targeted mass spectrometry, which may contribute to the erythroid differentiation block upon tif1γ loss. DHODH is the only pyrimidine de novo synthesis enzyme located in mitochondria and its activity is coupled to that of the electron transport chain (ETC) via coenzyme Q (CoQ). Rotenone, a potent ETC complex I inhibitor reversed the rescue by DHODH inhibition in mon embryos. Through parallel genome-wide transcriptome and chromatin immunoprecipitation analyses, we found that genes encoding CoQ metabolic enzymes are direct TIF1γ targets. Treatment with the CoQ analog decylubiquinone rescued erythroid progenitors in 26% (33/126) of mon embryos. These results demonstrate a tight coordination of nucleotide and mitochondrial metabolism as a key function of tif1γ-dependent transcription and reveal that TIF1γ activity regulates a metabolic program that drives cell fate decisions in the early blood lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases. Citation Format: Marlies P. Rossmann, Karen Hoi, Victoria Chan, Brian J. Abraham, Song Yang, James Mullahoo, Malvina Papanastasiou, Ilaria Elia, Sejal Vyas, Partha P. Nag, Lucas B. Sullivan, Julie R. Perlin, Elliott J. Hagedorn, Sara Hetzel, Raha Weigert, Curtis R. Warren, Bilguujin Dorjsuren, Eugenia Custo Greig, Chad A. Cowan, Stuart L. Schreiber, Richard A. Young, Alexander Meissner, Marcia Haigis, Steven A. Carr, Leonard I. Zon. Transcriptional regulation of mitochondrial metabolism by TIF1γ drives erythroid progenitor differentiation [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR04.

Transcriptional Regulation of Coenzyme Q Biosynthesis By TIF1γ Drives Erythropoiesis

Understanding the cell-autonomous as well as niche contributions governing erythropoiesis is critical for directed differentiation approaches of hematopoietic stem cells into differentiated red blood cells (RBCs) to treat blood disorders such as anemias and leukemias. Transcriptional intermediary factor 1 gamma (TIF1γ) is essential for erythropoiesis from zebrafish to mammals. Zebrafish moonshine mutant embryos defective for tif1γ do not make red blood cells (RBCs) due to a transcription elongation block characterized by aberrantly paused RNA polymerase II. Loss of factors involved in transcription elongation control, PAF1 and spt5, rescues the moonshine RBC defect. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we have performed a high-content chemical suppressor screen in the bloodless moonshine mutant using 3,500 compounds. Among the suppressors, we identified leflunomide, an inhibitor of dihydroorotate dehydrogenase (DHODH), an essential enzyme for de novo pyrimidine synthesis. Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar both rescue the formation of primitive erythroid cells in 61% (38/62) and 68% (50/74) of moonshine embryos, respectively. Blastula transplant experiments revealed that tif1γ, in addition to its cell-autonomous role, plays a role in the hematopoietic niche for RBC development. Through in-vivo metabolomics analyses we have identified nucleotide metabolism as the most significantly altered process in moonshine mutants, including elevated levels of uridine monophosphate and low levels of nicotinamide adenine dinucleotide (NAD+). Low NAD+ levels are accompanied by a reduced oxygen consumption rate in tif1γ-depleted embryos by Seahorse analysis. In support, genome-wide transcriptome analysis coupled with chromatin immunoprecipitation studies revealed genes encoding coenzyme Q (CoQ) metabolic enzymes as direct TIF1γ targets. DHODH is the only enzyme of the pyrimidine de novo synthesis pathway located on the inner mitochondrial membrane and its activity is coupled to that of the electron transport chain (ETC). Rotenone, a potent ETC complex I inhibitor reverses the rescue of the blood defect by DHODH inhibition in moonshine embryos. Since DHODH function is linked to mitochondrial oxidative phosphorylation via CoQ activity, we asked whether alterations in mitochondrial metabolism might be causal for the RBC defect in moonshine mutants. Indeed, treatment with the CoQ analog decylubiquinone results in rescue of βe3 globin expression in 26% (33/126) of moonshine embryos. These results demonstrate a tight coordination of nucleotide and mitochondrial metabolism as a key function of tif1γ-dependent transcription and reveal that TIF1γ activity regulates a metabolic program that drives cell fate decisions in the early blood lineage. Our work highlights the importance of the plasticity achieved by transcription regulatory processes such as transcription elongation for metabolic processes during lineage differentiation and could have therapeutic potential for blood diseases and consequences for erythroid differentiation protocols. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

The union of transcription and mRNA processing: 20 years of coupling.

The union of transcription and mRNA processing: 20 years of coupling.

Degradation of DNA damage-independently stalled RNA polymerase II is independent of the E3 ligase Elc1.

Transcription elongation is a highly dynamic and discontinuous process, which includes frequent pausing of RNA polymerase II (RNAPII). RNAPII complexes that stall persistently on a gene during transcription elongation block transcription and thus have to be removed. It has been proposed that the cellular pathway for removal of these DNA damage-independently stalled RNAPII complexes is similar or identical to the removal of RNAPII complexes stalled due to DNA damage. Here, we show that—consistent with previous data—DNA damage-independent stalling causes polyubiquitylation and proteasome-mediated degradation of Rpb1, the largest subunit of RNAPII, using Saccharomyces cerevisiae as model system. Moreover, recruitment of the proteasome to RNAPII and transcribed genes is increased when transcription elongation is impaired indicating that Rpb1 degradation takes place at the gene. Importantly, in contrast to the DNA damage-dependent pathway Rpb1 degradation of DNA damage-independently stalled RNAPII is independent of the E3 ligase Elc1. In addition, deubiquitylation of RNAPII is also independent of the Elc1-antagonizing deubiquitylase Ubp3. Thus, the pathway for degradation of DNA damage-independently stalled RNAPII is overlapping yet distinct from the previously described pathway for degradation of RNAPII stalled due to DNA damage. Taken together, we provide the first evidence that the cell discriminates between DNA damage-dependently and -independently stalled RNAPII.

Identification of Flavopiridol Analogues that Selectively Inhibit Positive Transcription Elongation Factor (P‐TEFb) and Block HIV‐1 Replication

The positive transcription elongation factor (P-TEFb; CDK9/cyclin T1) regulates RNA polymerase II-dependent transcription of cellular and integrated viral genes. It is an essential cofactor for HIV-1 Tat transactivation, and selective inhibition of P-TEFb blocks HIV-1 replication without affecting cellular transcription; this indicates that P-TEFb could be a potential target for developing anti-HIV-1 therapeutics. Flavopiridol, a small molecule CDK inhibitor, blocks HIV-1 Tat transactivation and viral replication by inhibiting P-TEFb kinase activity, but it is highly cytotoxic. In the search for selective and less cytotoxic P-TEFb inhibitors, we prepared a series of flavopiridol analogues and evaluated their kinase inhibitory activity against P-TEFb and CDK2/cyclin A, and tested their cellular antiviral potency and cytotoxicity. We identified several analogues that selectively inhibit P-TEFb kinase activity in vitro and show antiviral potency comparable to that of flavopiridol, but with significantly reduced cytotoxicity. These compounds are valuable molecular probes for understanding P-TEFb-regulated cellular and HIV-1 gene transcription and provide potential anti-HIV-1 therapeutics.

Cover Picture: Identification of Flavopiridol Analogues that Selectively Inhibit Positive Transcription Elongation Factor (P‐TEFb) and Block HIV‐1 Replication (ChemBioChem 12/2009)

Cover Picture: Identification of Flavopiridol Analogues that Selectively Inhibit Positive Transcription Elongation Factor (P‐TEFb) and Block HIV‐1 Replication (ChemBioChem 12/2009)

Glucocorticoid resistance in a multiple myeloma cell line is regulated by a transcription elongation block in the glucocorticoid receptor gene (NR3C1)

Glucocorticoid (GC) effects are mediated by the glucocorticoid receptor (GR). Several studies have demonstrated that a lower number of receptors per cell were associated with poor GC response. The regulation of GR expression is complex; the levels of GR can be autologously regulated by its ligand and also by transcriptional, post-transcriptional and post-translational mechanisms. Using three human myeloma cell lines that parallel the development of GC resistance, this work describes the mechanism involved in the downregulation of GR expression. The decreased expression was neither due to mutations in the gene encoding GR, NR3C1, nor due to methylation of the promoters. A gradual decrease in NR3C1 transcripts was seen during the development of resistance, the level of expression of exon 1 to 2 RNA fragments remained the same in sensitive and resistant cell lines but a chromatin immunoprecipitation assay demonstrated that RNA polymerase II, detectable throughout exon 2 to 3 in the sensitive cells, was undetectable on exon 3 in the resistant variant, suggesting lower or no transcription at this site. These studies demonstrated that downregulation of NR3C1 mRNA in a resistant cell line involves a block to transcriptional elongation within intron B of NR3C1. This block may represent an important element in the regulation of GR expression.

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.

Purification and characterization of FBI-1, a cellular factor that binds to the human immunodeficiency virus type 1 inducer of short transcripts.

The human immunodeficiency virus (HIV-1) promoter directs the synthesis of two classes of RNA molecules, short transcripts and full-length transcripts. The synthesis of short transcripts depends on a bipartite DNA element, the inducer of short transcripts (IST), located in large part downstream of the HIV-1 start site of transcription. IST does not require any viral product for function and is thought to direct the assembly of transcription complexes that are incapable of efficient elongation. Nothing is known, however, about the biochemical mechanisms that mediate IST function. Here, we report the identification and purification of a factor that binds specifically to the IST. This factor, FBI-1, recognizes a large bipartite binding site that coincides with the bipartite IST element. It is constituted at least in part by an 86-kDa polypeptide that can be specifically cross-linked to IST. FBI-1 also binds to promoter and attenuation regions of a number of cellular and viral transcription units that are regulated by a transcription elongation block. This observation, together with the observation that the binding of FBI-1 to IST mutants correlates with the ability of these mutants to direct IST function, suggests that FBI-1 may be involved in the establishment of abortive transcription complexes.

Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein-DNA interactions.

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to region 5' and 3' of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3' end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

Initiation of transcription from the minute virus of mice P4 promoter is stimulated in rat cells expressing a c-Ha-ras oncogene.

Transformation of FR3T3 rat fibroblasts by a c-Ha-ras oncogene but not by bovine papillomavirus type 1 is associated with an increase in the abundance of mRNAs from prototype strain MVMp of infecting minute virus of mice, an oncosuppressive parvovirus. This differential parvovirus gene expression correlates with the reported sensitization of ras- but not bovine papillomavirus type 1-transformed cells to the killing effect of MVMp (N. Salomé, B. van Hille, N. Duponchel, G. Meneguzzi, F. Cuzin, J. Rommelaere, and J. Cornelis, Oncogene 5:123-130, 1990). Experiments were performed to determine at which level parvovirus expression is up-regulated in ras transformants. An MVMp "attenuation" sequence responsible for the premature arrest of RNA elongation was either placed or not placed in front of the chloramphenicol acetyltransferase gene and brought under the control of MVMp early promoter P4. Although the MVMp attenuator reduced P4-driven chloramphenicol acetyltransferase expression, the extent of attenuation was similar in normal and ras-transformed cells. Moreover, the analysis of P4-directed viral RNAs in MVMp-infected cultures by RNase protection and nuclear run-on assays also revealed a transcription elongation block of a similar amplitude in both types of cells. In addition, the stabilities of the three major parvoviral mRNAs did not vary significantly between normal and ras-transformed cells. Hence, it is concluded that the ras-induced increase in the accumulation of parvoviral mRNAs is mainly controlled at the level of transcription. Consistently, the TATA motif of the P4 promoter proved to have a differential photoreactivity when tested by in vivo UV footprinting assays in ras-transformed versus normal cells.

Molecular genetic evidence for a differentiation-proliferation coupling during DMSO-induced myeloid maturation of HL-60 cells: Role of the transcription elongation block in the c- myc gene

Proliferation-differentiation coupling was studied during dimethyl sulfoxide (DMSO)-induced myeloid maturation of HL-60 cells using transcription of the myeloperoxidase ( MPO) and c- myc genes as indicators of differentiation and proliferation, respectively. Concomitant cell cycle kinetic analysis correlated the proliferation and transcription patterns. Transcription, cell cycle phases and rate of DNA synthesis were examined for up to 5 days of induction and, at 1-day intervals, analyzed during a 24-h reculture without the inducer. DMSO suppressed transcription of the c- myc and MPO genes with a t 1 2 of 16 min and 7 h, respectively. The ability to recover transcription following reculture diminished with the progression of the induction and ultimately was lost; concomitantly, the cells irreversibly lost the capacity to divide. This indicated that the differentiation and proliferation processes are inseparable and that terminal differentiation accompanies irreversible proliferation arrest in HL-60 cells. We also studied the kinetics of the block to transcription elongation at the exon 1-intron 1 boundary of the c- myc gene. This block produces a 0.38 kb truncated transcript that is constitutively expressed in somatic cells (Re , Oncogene 5, 1247, 1990). During induction the level of the 0.38 kb RNA increased, while that of the complete c- myc mRNA decreased, indicating that this truncated RNA is generated instead of message through a monotonously initiated transcriptional process. Transcription initiation and synthesis of the 0.3 kb RNA persisted in terminally differentiated cells, suggesting a role for this RNA in non-proliferating cells.

Molecular Analysis of the c‐myc Transcription Elongation Block

Molecular Analysis of the c‐myc Transcription Elongation Block

The block to transcription elongation is promoter dependent in normal and Burkitt's lymphoma c-myc alleles.

Aberrant c-myc expression patterns occur in human Burkitt's lymphoma cells, which consistently exhibit c-myc chromosomal translocations, mutations within and flanking the translocated allele, a loss of the block to transcription elongation in exon 1, and a promoter shift to use of the upstream P1 promoter. To define the mechanism responsible for the loss of transcription elongation blockage and resulting c-myc deregulation in Burkitt's lymphoma, we analyzed transcription patterns after transfer of normal and Burkitt's lymphoma c-myc alleles into murine cells and Xenopus oocyte germinal vesicles. We have determined that although the mutations within and surrounding several Burkitt's lymphoma c-myc alleles are not sufficient, in themselves, to abrogate the transcription elongation block, transcription initiation from the P2 promoter may be necessary to obtain the block to transcription elongation. To test directly the role of c-myc promoters in programming transcription elongation blockage, we analyzed transcription patterns from in vitro mutagenized c-myc genes containing deletions of either the P1 or P2 promoter. These data confirm that P1-initiated c-myc transcripts do not terminate at discrete sites near the 3' end of exon 1, whereas P2-initiated transcripts either terminate or read through the transcription block signals. Therefore, overexpression and/or constitutive expression from the c-myc P1 promoter may contribute to increased readthrough transcription in Burkitt's lymphoma cells and, hence, to aberrant expression patterns or levels of c-myc steady-state transcripts. In addition, the ability of normal cells to modulate c-myc P2-initiated transcription to either read through or to block elongation provides a fine control mechanism over c-myc steady-state RNA levels.

Tyrosine kinase oncogenes abrogate interleukin-3 dependence of murine myeloid cells through signaling pathways involving c-myc: conditional regulation of c-myc transcription by temperature-sensitive v-abl.

Retroviral expression vectors carrying the tyrosine kinase oncogenes abl, fms, src, and trk abrogate the requirements of murine myeloid FDC-P1 cells for interleukin-3 (IL-3). Factor-independent clones constitutively express c-myc in the absence of IL-3, whereas in parental cultures c-myc transcription requires the presence of the ligand. To directly test the effect of a tyrosine kinase oncogene on c-myc expression, retroviral constructs containing three different temperature-sensitive mutants of v-abl were introduced into myeloid IL-3-dependent FDC-P1 and 32D cells. At the permissive temperature, clones expressing temperature-sensitive abl behaved like wild-type abl-containing cells in their growth properties and expressed c-myc constitutively. Temperature shift experiments demonstrated that both IL-3 abrogation and the regulation of c-myc expression correlated with the presence of functional v-abl. Induction of c-myc expression by reactivation of temperature-sensitive v-abl mimicked c-myc induction by IL-3 in that it did not require protein synthesis and occurred at the level of transcription, with effects on both initiation and a transcription elongation block. However, v-abl-regulated FDC-P1 cell growth differed from IL-3-regulated growth in that c-fos and junB, which are normally induced by IL-3, were not induced by activation of v-abl.

Tyrosine Kinase Oncogenes Abrogate Interleukin-3 Dependence of Murine Myeloid Cells through Signaling Pathways Involving c-myc: Conditional Regulation of c-myc Transcription by Temperature-Sensitive v-abl

Retroviral expression vectors carrying the tyrosine kinase oncogenes abl, fms, src, and trk abrogate the requirements of murine myeloid FDC-P1 cells for interleukin-3 (IL-3). Factor-independent clones constitutively express c-myc in the absence of IL-3, whereas in parental cultures c-myc transcription requires the presence of the ligand. To directly test the effect of a tyrosine kinase oncogene on c-myc expression, retroviral constructs containing three different temperature-sensitive mutants of v-abl were introduced into myeloid IL-3-dependent FDC-P1 and 32D cells. At the permissive temperature, clones expressing temperature-sensitive abl behaved like wild-type abl-containing cells in their growth properties and expressed c-myc constitutively. Temperature shift experiments demonstrated that both IL-3 abrogation and the regulation of c-myc expression correlated with the presence of functional v-abl. Induction of c-myc expression by reactivation of temperature-sensitive v-abl mimicked c-myc induction by IL-3 in that it did not require protein synthesis and occurred at the level of transcription, with effects on both initiation and a transcription elongation block. However, v-abl-regulated FDC-P1 cell growth differed from IL-3-regulated growth in that c-fos and junB, which are normally induced by IL-3, were not induced by activation of v-abl.

A block of transcription elongation by RNA polymerase II at synthetic sites in vitro

We have previously suggested that transcription elongation by RNA polymerase II can be blocked when the nascent RNA is folded into a stem-and-loop structure followed by polyuridines. As an approach to test this suggestion in vitro, several GC-rich deoxyoligonucleotides with dyad symmetries were chemically synthesized and inserted following the adenovirus 2 major late promoter. These constructs were transcribed in vitro using HeLa whole cell extract. The transcripts of the synthetic inserts can potentially form stem-and-loop structures with destabilization energy from 0 to -48 kcal followed by 3, 5, and 8 U residues. The results obtained show that transcription elongation is blocked by these synthetic inserts and that the extent of the elongation block is directly correlated to the stabilities of the potential stem-and-loop structure and the proceeding number of U residues. Three levels of elongation blocks were observed: a brief pause of the polymerase occurs when the RNA could be folded into a secondary structure or when there were 5-6 T residues on the sense DNA strand. An extended pause occurred when the number of T residues on the sense DNA strand was increased to 8. Transcription termination, with a partial release of the attenuated transcript occurred when a stable RNA secondary structure (delta G = -48 kcal) was followed by 8 U residues. The relevancy of these in vitro results to the in vivo mechanism of a transcription elongation block is discussed.

Intrinsic sites of transcription termination and pausing in the c-myc gene.

We have studied transcription elongation and termination in the human c-myc gene. Transcription of c-myc gene sequences with purified mammalian RNA polymerase II revealed several sites of transcription termination and pausing in the vicinity of the exon 1-intron 1 junction. This region previously has been shown to block transcription elongation in vivo by nuclear run-on analysis (D. Bentley and M. Groudine, Nature [London] 321:702-706, 1986). These sites were recognized by purified RNA polymerase II, and we therefore designated them intrinsic sites of termination and pausing. Two of these sites cause termination of RNA polymerase III transcription as well. RNA polymerase II terminated transcription in a cluster of seven consecutive T residues in the nontranscribed strand and paused during transcription at three additional sites in this region. The intrinsic sites of transcription termination and pausing described here correspond closely to the 3' ends of transcripts synthesized in Xenopus oocytes injected with plasmids containing the c-myc termination region (D. Bentley and M. Groudine, Cell 53:245-256, 1988). This correspondence suggests that the intrinsic recognition of these termination and pause sites by purified RNA polymerase II may play a role in the transcription elongation block observed in vivo.

Intrinsic sites of transcription termination and pausing in the c-myc gene.

We have studied transcription elongation and termination in the human c-myc gene. Transcription of c-myc gene sequences with purified mammalian RNA polymerase II revealed several sites of transcription termination and pausing in the vicinity of the exon 1-intron 1 junction. This region previously has been shown to block transcription elongation in vivo by nuclear run-on analysis (D. Bentley and M. Groudine, Nature [London] 321:702-706, 1986). These sites were recognized by purified RNA polymerase II, and we therefore designated them intrinsic sites of termination and pausing. Two of these sites cause termination of RNA polymerase III transcription as well. RNA polymerase II terminated transcription in a cluster of seven consecutive T residues in the nontranscribed strand and paused during transcription at three additional sites in this region. The intrinsic sites of transcription termination and pausing described here correspond closely to the 3' ends of transcripts synthesized in Xenopus oocytes injected with plasmids containing the c-myc termination region (D. Bentley and M. Groudine, Cell 53:245-256, 1988). This correspondence suggests that the intrinsic recognition of these termination and pause sites by purified RNA polymerase II may play a role in the transcription elongation block observed in vivo.