Roles of TATA and initiator elements in determining the start site location and direction of RNA polymerase II transcription.
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Base Sequence↗
Biomedical subjects
S T Smale
Publications and source records attributed to S T Smale.
Similar mechanisms for transcription initiation mediated through a TATA box or an initiator element.
Previous studies have identified several biochemical parameters that resolve promoter-specific transcription initiation by RNA polymerase II into distinct steps. We have employed these parameters to compare the mechanisms of initiation mediated through either a TATA box or a transcriptional initiator element. Time course experiments revealed that initiator-mediated transcription in a cell-free extract does not display the lag period typically found with TATA-mediated transcription. Titration experiments with increasing amounts of extract revealed further differences between TATA- and initiator-mediated transcription, again suggesting that different steps are rate-limiting. In contrast, experiments with the detergent Sarkosyl, which is believed to define later steps in the initiation process, revealed a striking degree of similarity between TATA- and initiator-mediated transcription. Additional similarities were found using dinucleotide priming experiments, which are believed to measure the flexibility of initiation after RNA polymerase II is positioned over the transcription start site region. Taken together, these results suggest that the location of a preinitiation complex can be determined by core promoter recognition through either a TATA box or an initiator element. However, following these distinct template recognition events, the steps leading to initiation may be very similar and may proceed in a DNA sequence-independent manner.
Base Sequence↗
Roles of TATA and initiator elements in determining the start site location and direction of RNA polymerase II transcription.
Transcriptional initiator elements and TATA elements are functionally similar, in that both can act in concert with a Sp1-dependent activator element to direct high levels of accurately initiated transcription. In this study, we have used in vitro transcription experiments to elucidate the relative activities of TATA and initiator. By varying the locations of TATA and initiator elements within a simple promoter, we compared their abilities to localize transcription start sites and to mediate transcriptional activation by Sp1. In addition, we addressed the contributions of initiator and TATA towards determining the direction of transcription from a promoter. Finally, we addressed the prevalence of initiator elements by analyzing the start site regions of several genes. We found that many of these regions possess initiator activity, although an initiator consensus sequence could not be defined. Taken together, the data presented suggest that an initiator is a common element that can influence the direction of transcription as well as the location of a transcription start site. However, in general, an initiator was incapable of altering these activities of a TATA box within a promoter containing both core elements.
Base Sequence↗
Alternative pathway for induction of human immunodeficiency virus gene expression: involvement of the general transcription machinery.
Human immunodeficiency virus type 1 (HIV-1) is viable and mitogen inducible in the absence of its binding sites for the inducible transcription factor NF-kappa B. We have investigated alternative mechanisms for induction of HIV-1 transcription. Using transient transfection assays, we found that transcription from an HIV-1 LTR containing mutant kappa B sites was activated 10- to 20-fold in a variety of human cell types by the phorbol ester phorbol myristate acetate (PMA). The promoter elements conferring this inducibility were localized to the region downstream of nucleotide -70, which contains the TATA and TAR elements and binding sites for transcription factors Sp1 and LBP-1. Synthetic promoters containing only Sp1 sites and a TATA element were also induced in transfection experiments as well as in in vitro transcription experiments with T-cell nuclear extracts. Moreover, promoters containing a TATA box in the absence of Sp1 sites or Sp1 sites in the absence of a TATA box were equally inducible in vitro, as was an RNA polymerase III promoter. The activities of RNA polymerases II and III and of the 38-kDa TATA-binding protein transcription factor IID (TFIID), were not induced by PMA, but electrophoretic mobility shift assays revealed a highly inducible protein-DNA complex that interacted specifically with the TATA sequence. This protein-DNA complex appeared to be much larger than that found with the 38-kDa human TFIID expressed in bacteria. Taken together, these data suggest that a component of the general transcription machinery, and possibly a TFIID-associated protein, is induced in T cells by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-specific genes.
We have studied transcriptional control of the murine terminal deoxynucleotidyltransferase (TdT) gene, which is activated specifically in immature B and T lymphocytes. This analysis has led to the identification and purification of a 50-kDa sequence-specific DNA-binding protein, LyF-1, that interacts with the approximate consensus sequence PyPyTGGGAGPu and is enriched in cells at most stages of B- and T-cell differentiation. LyF-1 binds tightly to an element in the TdT promoter that we show is required for transcription in lymphocytes. LyF-1 also interacts with an element in the immunoglobulin mu enhancer, called microB, that was recently shown to be important for lymphocyte-specific enhancer activity. Moreover, LyF-1 binds to the promoters for the lymphocyte-specific genes lambda 5, VpreB, and lck, all of which we speculate have additional features in common with the TdT promoter. Thus, LyF-1 may be a general transcriptional activator for genes whose expression is restricted to the B- and/or T-lymphocyte lineages.
Animals↗
Transcriptional activation by Sp1 as directed through TATA or initiator: specific requirement for mammalian transcription factor IID.
Transcription of mammalian genes by RNA polymerase II often begins at a specific nucleotide, whose location is determined either by an upstream DNA element known as a TATA box or by an element positioned at the transcription start site called an initiator (Inr). By in vitro analysis of synthetic promoters, we demonstrate here that the TATA and Inr elements are functionally similar and that the Inr is contained between nucleotides -3 and +5 relative to the initiation site. Moreover, we found that a mammalian transcription factor IID (TFIID) protein fraction is required for transcriptional stimulation by an Sp1-dependent activating element placed upstream of either TATA or Inr elements. However, in these assays, the yeast TATA-binding protein, which previously was shown to function similarly to mammalian TFIID, could not efficiently substitute for the mammalian TFIID fraction. These results demonstrate that mammalian TFIID is functionally distinct from the yeast TATA-binding protein and may contain additional subunits or domains that are important for transcriptional activation from some promoters.
Adenovirus Early Proteins↗
The "initiator" as a transcription control element.
Transcription of the lymphocyte-specific terminal deoxynucleotidyltransferase gene begins at a single nucleotide, but no TATA box is present. We have identified a 17 bp element that is sufficient for accurate basal transcription of this gene both in vitro and in vivo. This motif, the initiator (Inr), contains within itself the transcription start site. Homology to the Inr is found in many TATA-containing genes, and specific mutagenesis influences both the efficiency and accuracy of initiation. Moreover, in the presence of either a TATA box or the SV40 21 bp repeats, a greatly increased level of transcription initiates specifically at the Inr. Thus, the Inr constitutes the simplest functional promoter that has been identified and provides one explanation for how promoters that lack TATA elements direct transcription initiation.
Animals↗
Inhibition of simian virus 40 DNA replication in vitro by poly(ADP-ribosyl)ated diadenosine tetraphosphate.
Poly(ADP-ribosyl)ated diadenosine tetraphosphate was found to inhibit the in vitro replication of SV40 DNA. This inhibition was sensitive to preincubation of the polymer with either poly(ADP-ribose) glycohydrolase, diadenosine tetraphosphate (Ap4A):ADP phosphohydrolase, or an excess of free Ap4A. In contrast, the general catalytic activity of DNA polymerase was not inhibited by the poly(ADP-ribosyl)ated Ap4A when activated salmon sperm DNA was used as a template. These data suggest that inhibition of SV40 DNA replication by poly(ADP-ribosyl)ated Ap4A requires both the intact polymer and intact Ap4A moiety and is specific to events occurring during the initiation or elongation of a double-stranded template. Since both poly(ADP-ribose) and Ap4A accumulate in cultured mammalian cells following stresses which are accompanied by DNA strand breaks, these data are consistent with a model in which poly(ADP-ribosyl)ated Ap4A inhibits DNA replication following DNA damage.
Adenine Nucleotides↗
Inhibition of simian virus 40 DNA replication by specific modification of T-antigen with oxidized ATP.
We covalently bound periodate-oxidized ATP (oATP) to purified simian virus 40 (SV40) large T-antigen and determined the effect of this modification on viral DNA replication and three other biochemical activities of T-antigen. The oATP bound specifically to T-antigen, inhibiting the ATPase activity and preventing T-antigen from activating SV40 DNA replication in vitro. In contrast, binding of oATP had no effect on the DNA-binding activity of T-antigen nor on its ability to form a complex with DNA polymerase alpha. These results provide direct biochemical evidence suggesting that the T-antigen ATPase activity is necessary for viral DNA replication.
Adenosine Triphosphatases↗
Two distinct promoter elements in the human rRNA gene identified by linker scanning mutagenesis.
A cell-free RNA polymerase I transcription system was used to evaluate the transcription efficiency of 21 linker scanning mutations that span the human rRNA gene promoter. Our analysis revealed the presence of two major control elements, designated the core and upstream elements, that affect the level of transcription initiation. The core element extends from -45 to +18 relative to the RNA start site, and transcription is severely affected (up to 100-fold) by linker scanning mutations in this region. Linker scanning and deletion mutations in the upstream element, located between nucleotides -156 and -107, cause a three- to fivefold reduction in transcription. Under certain reaction conditions, such as the presence of a high ratio of protein to template or supplementation of the reaction with partially purified protein fractions, sequences upstream of the core element can have an even greater effect (20- to 50-fold) on RNA polymerase I transcription. Primer extension analysis showed that RNA synthesized from all of these mutant templates is initiated at the correct in vivo start site. To examine the functional relationship between the core and the upstream region, mutant promoters were constructed that alter the orientation, distance, or multiplicity of these control elements relative to each other. The upstream control element appears to function in only one orientation, and its position relative to the core is constrained within a fairly narrow region. Moreover, multiple core elements in close proximity to each other have an inhibitory effect on transcription.
Base Sequence↗
T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.
We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.
Antibodies, Monoclonal↗
Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters.
We studied RNA polymerase I transcription in cells transfected with a plasmid, prHuTK, containing the herpes simplex virus tk gene fused to a human rRNA promoter. Primer extension analysis of tk RNA isolated from COS cells transfected with prHuTK reveals that transcription from the RNA polymerase I promoter is highly efficient and initiates at the same position used for the synthesis of endogenous rRNA in HeLa cells. The RNA products derived from prHuTK are distinguishable from normal RNA polymerase II transcripts of tk in that they are not polyadenylated, are extremely unstable, and are found predominantly in the nucleus. Moreover, the transcription observed is resistant to 300 micrograms of alpha-amanitin per ml. These results strongly suggest that prHuTK transcription is under the control of the human rRNA promoter and RNA polymerase I. To further characterize the activity of the human rDNA promoter in vivo, a series of 5' and 3' deletion mutants was tested in this transfection assay. The deletion analysis indicates that a core region of ca. 40 base pairs overlapping the initiation site is critical for transcription. In addition, a region between nucleotides -234 and -131 upstream from the core sequence serves to modulate the efficiency of transcription. Insertion into prHuTK of additional ribosomal nontranscribed spacer DNA or the simian virus 40 enhancer element has no apparent effect on the promoter activity. Surprisingly, RNA polymerase II transcripts synthesized at low levels from two start sites within the core control element of the wild-type RNA polymerase I promoter are activated upon deletion of upstream RNA polymerase I promoter sequences. However, these RNA polymerase II transcripts are not expressed from the endogenous rRNA promoter.
Amanitins↗
Regulation of human ribosomal RNA transcription.
We have used a cell-free polymerase I transcription system derived from HeLa cells to study the regulation of human rRNA synthesis. Analysis of deletion mutants spanning the start site of transcription at nucleotide +1 indicates that the control region affecting initiation of human rRNA synthesis is contained within sequences from nucleotides -158 to +18. This promoter region can be subdivided into (i) a central segment of approximately 40 base pair that is required for transcription and (ii) flanking sequences that influence the efficiency of transcription in vitro. We have examined the in vitro transcriptional activity of the human extract under various conditions that are thought to modulate rRNA synthesis in vivo. Cell-free extracts prepared from HeLa cells infected with adenovirus 2 synthesize human rRNA at levels greatly decreased relative to uninfected cell extracts. By contrast, in vitro transcription of human rRNA is stimulated 2- to 3-fold by the addition of purified simian virus 40 large tumor antigen to the transcription reaction. Moreover, a mutant tumor antigen known to be defective for rRNA activation in vivo is incapable of stimulating rRNA synthesis in vitro. The ability to detect these different regulatory phenomena in vitro provides us with an experimental basis for investigating the molecular mechanisms that control rRNA synthesis.
Cell Transformation, Neoplastic↗