Adenovirus Major Late Promoter Research Articles

The Sensitivity of RNA Polymerase II in Elongation Complexes to C-terminal Domain Phosphatase

The phosphorylation state of the carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit plays an important role in the regulation of transcript elongation. This report examines the sensitivity of RNAP II to dephosphorylation by CTD phosphatase (CTDP) and addresses factors that regulate its sensitivity. The CTDP sensitivity of RNAP IIO in paused elongation complexes on a dC-tailed template does not significantly differ from that of free RNAP IIO. RNAP IIO contained in elongation complexes that initiate transcription from the adenovirus-2 major late promoter in the presence of a nuclear extract is relatively resistant to dephosphorylation. Complexes treated with 1% Sarkosyl remain elongation-competent but demonstrate a 5-fold increase in CTDP sensitivity. Furthermore, the sensitivity of RNAP IIO in both control and Sarkosyl-treated elongation complexes is dependent on their position relative to the start site of transcription. Elongation complexes 11-24 nucleotides downstream are more sensitive to dephosphorylation than complexes 50-150 nucleotides downstream. The incubation of Sarkosyl-treated elongation complexes with nuclear extract restores the original resistance to dephosphorylation. These results suggest that a conformational change occurs in RNAP II as it clears the promoter, which results in an increased resistance to dephosphorylation. Furthermore, the sensitivity to dephosphorylation can be modulated by a factor(s) present in the nuclear extract.

Enhancement of the Basal-Level Activity of HIV-1 Long Terminal Repeat by HIV-1 Nucleocapsid Protein

Two HIV-1 proteins, Tat and NCp7 (NC), have zinc finger-like structures. NC is a virion protein and has been shown to accumulate in the nucleus 8 h postinfection. Since transcription factors with zinc fingers assist the transcriptional activity of both RNA polymerases II and III, we examined the effect of NC on HIV-1 LTR-directed gene expression. The HIV-1 NC binds to the HIV-1 LTR and results in a mobility shift in polyacrylamide gel electrophoresis. Competition assays with cold probes revealed that the binding of NC and formation of a DNA–protein complex could be prevented by the addition of excess unlabeled LTR self-probe, but not the HIV-1 V3 envelope gene. The DNase I footprint analysis showed that NC binds to six regions within HIV-1 LTR, four of which are near the transcription start site. The NC alone enhances LTR basal-level activity in RNA runoff experiments. When the general transcription factors (GTFs) were added in the assay, NC enhances NF-κB, Sp1, and TFIIB-induced HIV-1 LTR-directed RNA transcription. RNA transcription directed by the adenovirus major late promoter, however, is not significantly affected by NC in the cell-free system. Transient transfection of human T lymphocytes with the plasmids containing HIV-1 nc or gag showed enhancement of LTR-CAT activity. Moreover, transfection of HIV-1 provirus containing mutations in NC zinc-finger domains dramatically decreases the enhancement activity in human T cells, in which HIV-1 LTR is stably integrated into the cellular genome. These observations show that NC binds to HIV-1 LTR and cooperatively enhances GTFs and NF-κB induced HIV-1 LTR basal-level activity. NC may play the role of a nucleation protein, which binds to LTR and enhances basal-level transcription by recruiting cellular transcription factors to the HIV-1 promoter in competition with cellular promoters.

Structural basis of preinitiation complex assembly on human pol II promoters.

Transcription initiation requires the assembly of a preinitiation complex (PIC), which is nucleated through binding of the TATA-box binding protein (TBP) to the promoter. Biochemical studies have shown, however, that TBP recognizes the TATA-box in both orientations and, therefore, cannot account for the directionality of PIC assembly. Transcription factor IIB (TFIIB) is essential for transcription initiation from RNA polymerase II promoters. Recent functional studies have identified a specific 7 bp TFIIB recognition element (BRE) immediately upstream of the TATA-box. We present here the 2.65 A resolution crystal structure of a human TFIIBc-TBPc complex bound to an idealized and extended adenovirus major late promoter. This structure now reveals that human TFIIBc binds to the promoter asymmetrically through base-specific contacts in the major groove upstream and in the minor groove downstream of the TATA-box. Binding of TFIIBc is, therefore, synergistic with TBPc requiring the distortion of the TATA-box. Thus, the newly described TFIIBc-DNA interface is likely to be a key determinant for the unidirectional assembly of a functional PIC.

TATA element recognition by the TATA box-binding protein has been conserved throughout evolution.

Cocrystal structures of wild-type TATA box-binding protein (TBP) recognizing 10 naturally occurring TATA elements have been determined at 2.3-1.8 A resolution, and compared with our 1.9 A resolution structure of TBP bound to the Adenovirus major late promoter (AdMLP) TATA box (5'-TATAAAAG-3'). Minor-groove recognition by the saddle-shaped protein induces the same conformational change in each of these oligonucleotides, despite variations in promoter sequence that reduce the efficiency of transcription initiation. Three molecular mechanisms explain assembly of diverse TBP-TATA element complexes. (1) T --> A and A --> T transversions leave the minor-groove face unchanged, permitting formation of TBP-DNA complexes on many A/T-rich core promoter sequences. (2) Cavities in the interface between TBP and the minor-groove face of the AdMLP TATA box accommodate the exocyclic NH(2) groups of G in a TACA box and in a TATAAG box. (3) Formation of a C:G Hoogsteen basepair in a TATAAAC box eliminates steric clashes that would be produced by the Watson-Crick base pair. We conclude that the structure of the TBP-TATA box complex found at the heart of the polymerase II (pol II) transcription machinery has remained constant over the course of evolution, despite variations in TBP and its DNA targets.

Functional Analysis of a Mutation Occurring between the Two In-frame AUG Codons of Human Angiotensinogen

Angiotensinogen (ANG) is the specific substrate of the renin-angiotensin system, a major participant in blood pressure control. We have identified a natural mutation at the -30 amino acid position of the angiotensinogen signal peptide, in which an arginine is replaced by a proline (R-30P). Heterozygous individuals with R-30P showed a tendency to lowered plasma angiotensinogen level (1563 ng of ANG I/ml (range 1129-1941)) compared with normal individuals in the family (1892 ng of ANG I/ml (range 1603-2072)). Human angiotensinogen mRNA has two in-phase translation initiation codons (AUG) starting upstream 39 and 66 nucleotides from the cap site. R-30P occurs in a cluster of basic residues adjacent to the first AUG codon that may affect intracellular sorting of the nascent protein. Pulse-chase experiments in transiently transfected cultured cells revealed that the R-30P mutation was associated with reduced amounts of both intra- and extracellular protein. In a cell-free system, we found that two forms of native angiotensinogen were generated by alternative initiation of translation at either AUG codon. Alteration of either the first or second AUG codons abolished the synthesis of the longer and the shorter form of native angiotensinogen, respectively. Furthermore, the rate of secretion of the shorter form was lower than that of the longer form. By transplanting angiotensinogen signal peptide onto green fluorescence protein, however, we found that both forms of the signal peptide could target green fluorescence protein, normally localized in the cytoplasm, to the secretory pathway. Although the R-30P mutation may not affect intracellular sorting of angiotensinogen in a qualitative manner, it leads to a quantitative reduction in the net secretion of mature angiotensinogen through decreased translocation or increased residence time in the endoplasmic reticulum.

A region within the RAP74 subunit of human transcription factor IIF is critical for initiation but dispensable for complex assembly.

Human transcription factor IIF (TFIIF) is an alpha(2)beta(2) heterotetramer of RNA polymerase II-associating 74 (RAP74) and RAP30 subunits. Mutagenic analysis shows that the N-terminal region of RAP74 between L155 (leucine at codon 155) and M177 is important for initiation. Mutants in this region have reduced activity in transcription, but none are inactive. Single amino acid substitutions at hydrophobic residues L155, W164, I176, and M177 have similar activity to RAP74(1-158), from which all but three amino acids of this region are deleted. Residual activity can be explained because each of these mutants forms a complex with RAP30 and recruits RNA polymerase II into the preinitiation complex. Mutants are defective for formation of the first phosphodiester bond from the adenovirus major late promoter but do not appear to have an additional significant defect in promoter escape. Negative DNA supercoiling partially compensates for the defects of TFIIF mutants in initiation, indicating that TFIIF may help to untwist the DNA helix for initiation.

Negative regulation of the androgen receptor gene promoter by NFI and an adjacently located multiprotein-binding site.

The upstream promoter of the rat androgen receptor (AR) gene contains a strong negative regulatory region located at the -388 to -340 nucleotide position. The distal part (-388/-373) of this regulatory region binds NFI, a ubiquitous transcription factor, while the proximal portion (-372/-340) contains an overlapping binding site for two nuclear proteins. This composite regulatory region (-388/-340) was initially defined by deoxyribonuclease I footprinting as the continuous stretch of a nuclease-protected site. NFI specificity of the distal portion (-388/-373) of the footprint was established through cross-competition in electrophoretic mobility shift assay (EMSA) using the well characterized NFI element of the adenovirus major late promoter and by immunoreactivity to the NFI antibody. EMSA with oligonucleotide duplexes corresponding to the proximal domain (-372/-340) indicated multiple retarded bands with at least two major DNA-protein complexes. Further analysis with truncated oligonucleotide duplexes showed that these two major proteins bind to this domain in an overlapping manner. Within this overlapping area, the position spanning -359 to -347 is essential for the formation of either of these two complexes. Substitution of four G with T residues in the overlapping area totally abolished all protein binding at the downstream -372/-340 site. Point mutations that abolish specific binding at either the NFI or immediately downstream multiprotein-binding site caused about a 10-fold increase in AR promoter activity in transfected HepG2 cells. Double mutation involving both the NFI and proximal overlapping protein-binding sites failed to cause any additional increase in promoter function. From these results we conclude that the AR promoter contains a composite negative regulatory region at -388/-340, and the repressor function may involve a coordinate interaction between NFI and at least two other nuclear factors.

TATA Box DNA Deformation with and without the TATA Box-binding Protein

DNA ring closure methods have been applied to TATA box DNA and its complex with the TATA box-binding protein (TBP). The J factors for cyclization (effective concentrations of one DNA end about the other) have been measured using cyclization kinetics, with and without bound TBP, for 18 DNA constructs containing the adenovirus major late promoter TATA box (TATAAAAG) separated by a variable helical phasing adapter from sequence-induced A-tract DNA bends. Six phasing lengths were used at three overall DNA lengths each. Cyclization kinetics were also measured in the absence of protein for the same set of molecules bearing a mutant TATA box (TACAAAAG). The results suggest that the TATA box DNA itself is strongly bent and anisotropically flexible, in a direction opposite to the bend induced by TBP, and that the mutant TACA box is much less bent/flexible. The bending and flexibility of the free DNA may govern the energetics of recognition of different DNA sequences by TBP, and the intrinsic bend may act to repress transcription complex assembly in the absence of TBP. The cyclization kinetics of TBP-DNA complexes in solution predict a geometry generally consistent with crystal structures, which show dramatic bending and unwinding. The novel observation of TBP-induced topoisomers suggests that this minicircle approach is able to distinguish TBP-induced unwinding from writhe (these cancel out in larger DNA), and this in turn suggests that changes in supercoiling in small topological domains can control TBP binding.

The Werner syndrome protein is involved in RNA polymerase II transcription.

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

Upstream stimulatory factor regulates major histocompatibility complex class I gene expression: the U2DeltaE4 splice variant abrogates E-box activity.

The tissue-specific expression of major histocompatibility complex class I genes is determined by a series of upstream regulatory elements, many of which remain ill defined. We now report that a distal E-box element, located between bp -309 and -314 upstream of transcription initiation, acts as a cell type-specific enhancer of class I promoter activity. The class I E box is very active in a neuroblastoma cell line, CHP-126, but is relatively inactive in the HeLa epithelial cell line. The basic helix-loop-helix leucine zipper proteins upstream stimulatory factor 1 (USF1) and USF2 were shown to specifically recognize the class I E box, resulting in the activation of the downstream promoter. Fine mapping of USF1 and USF2 amino-terminal functional domains revealed differences in their abilities to activate the class I E box. Whereas USF1 contained only an extended activation domain, USF2 contained both an activation domain and a negative regulatory region. Surprisingly, the naturally occurring splice variant of USF2 lacking the exon 4 domain, U2DeltaE4, acted as a dominant-negative regulator of USF-mediated activation of the class I promoter. This latter activity is in sharp contrast to the known ability of U2DeltaE4 to activate the adenovirus major late promoter. Class I E-box function is correlated with the relative amount of U2DeltaE4 in a cell, leading to the proposal that U2DeltaE4 modulates class I E-box activity and may represent one mechanism to fine-tune class I expression in various tissues.

Identification of a General Transcription Factor TFIIAα/β Homolog Selectively Expressed in Testis

In this paper we describe the isolation of a cDNA that encodes a human TFIIAalpha/beta-like factor (ALF). The open reading frame of ALF predicts a protein of 478 amino acids that contains characteristic N- and C-terminal conserved domains separated by an internal nonconserved domain. In addition, a rare ALF-containing cDNA, which possesses an extended N terminus (Stoned B/TFIIAalpha/beta-like factor; SALF) has also been identified. The results of Northern and dot blot analyses show that ALF is expressed almost exclusively in testis; in contrast, TFIIAalpha/beta and TFIIAgamma are enriched in testis but are also widely expressed in other human tissues. Recombinant ALF (69 kDa) and TFIIAgamma (12 kDa) polypeptides produced in Escherichia coli form an ALF/gamma complex that can stabilize TBP-TATA interactions in an electrophoretic mobility shift assay. The ALF/gamma complex is also able to restore transcription from the adenovirus major late promoter using HeLa cell nuclear extracts that have been depleted of TFIIA. Overall, the data show that ALF is a functional homolog of human general transcription factor TFIIAalpha/beta that may be uniquely important to testis biology.

Tetracycline-Mediated Regulation of Gene Expression within the Human Cytomegalovirus Genome

To evaluate the utility of tetracycline gene regulation in the study of human cytomegalovirus gene functions, expression of luciferase under the control of tetracycline-regulatable promoters was studied following transient plasmid transfections and from within recombinant human cytomegalovirus genomes. The tetracycline-regulatable promoter PhCMV*-1 contains sequences from the human cytomegalovirus ie1/ie2 promoter and seven upstream tet operator sites which bind the activator protein tTA only in the absence of tetracycline (Gossen and Bujard (1992). Proc. Natl. Acad. Sci. USA 89, 5547–5551). Two modifications of PhCMV*-1 were also studied: P1129, in which the tet operator sites were reduced from seven to one; and P1125, in which human cytomegalovirus sequences were replaced by adenovirus major late promoter and terminal deoxynucleotidyltransferase initiator sequences. In transient assays, PhCMV*-1 and P1125 exhibited modest differential regulation but were strongly activated by viral infection. P1129 exhibited less viral activation and narrower regulation. In the viral genome, PhCMV*-1 exhibited regulation up to 7-fold during late times of infection, whereas P1125 displayed nearly 100-fold regulation. Regulation of P1125 was fully reversed within 12 to 24 h of adding or removing tetracycline. These results suggest that P1125 may provide sufficient conditional expression to effectively regulate human cytomegalovirus late genes.

Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex

Binding of the TATA-binding protein (TBP) to the “TATA” sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased.

Inhibition of RNA polymerase II transcription in human cell extracts by cisplatin DNA damage.

The anticancer drug cisplatin induces a spectrum of lesions in DNA. The effect of such DNA damage on transcription by RNA polymerase II (RNA pol II) in human cell extracts was investigated at the level of initiation and elongation. RNA pol II transcription directed from the adenovirus major late promoter was inhibited following treatment of the promoter-containing template with increasing concentrations of cisplatin. Furthermore, transcription from an undamaged promoter fragment was depleted in the presence of increasing amounts of cisplatin DNA damage on an exogenous plasmid, suggesting such damage may hijack an essential factor for transcription initiation. The effect of cisplatin damage on RNA pol II elongation was investigated using site-specifically-placed cisplatin adducts. The GTG adduct was an effective block to RNA pol II elongation, inhibiting the polymerase by 80%. In contrast, RNA pol II completely bypassed the cisplatin GG intrastrand adduct. These studies suggest that the inhibition of RNA pol II transcription observed following the treatment of cells with cisplatin is likely to reflect the combined effects of DNA damage at the level of both transcription initiation and elongation.

Initially Transcribed Sequences Strongly Affect the Extent of Abortive Initiation by RNA Polymerase II

We investigated transcript initiation and early elongation by RNA polymerase II using templates mismatched between -9 and +3 (bubble templates). Highly purified RNA polymerase II alone was able to initiate transcription specifically on these templates in the presence of dinucleotide primers. The length distribution of abortively initiated RNAs was similar for purified RNA polymerase II on bubble templates and polymerase II on double-stranded templates in HeLa nuclear extracts. Increasing the U content in the initial portion of the transcript caused similar increases in abortive initiation for transcription of bubble templates by pure polymerase and double-stranded templates in extracts. Thus, the level of abortive initiation by RNA polymerase II is at least partly determined by interactions of the polymerase with the transcript and/or the template, independent of the general transcription factors. Substitution of 5-bromo-UTP for UTP reduced abortive initiation on bubble templates, consistent with the idea that transcription complex stability during early elongation depends on the strength of the initial RNA-DNA hybrid. Interestingly, transcription of bubble templates in HeLa extracts gave very high levels of abortive initiation, suggesting that inability to reanneal the initially melted template segment inhibits transcript elongation in the presence of the initiation factors.

Characterization and Purification of Carbohydrate Response Element-Binding Protein of the Rat L-Type Pyruvate Kinase Gene Promoter

The L-III transcriptional regulatory element of the rat pyruvate kinase L gene is located between −170 and −150 base pairs upstream of the hepatocyte-specific transcription initiation site. As the L-III element is not only necessary for cell type-specific expression but also for transcriptional stimulation by carbohydrates, it is also referred to as a carbohydrate-response element. Electrophoretic mobility shift assays using rat liver nuclear extract showed that L-III element-binding protein (L-IIIBP) was observed as multiple bands. These bands disappeared when the nuclear extract was preincubated at 60°C for 5 min and were competed with unlabeled L-III oligonucleotide but not with unlabeled adenovirus major late promoter E box oligonucleotide. In addition, these bands were not affected in the presence of antiserum against upstream stimulating factor (USF). Thus, we conclude that L-IIIBP is different from USF. Then, heat-labile L-IIIBP was purified from rat liver nuclear extracts. Purified L-IIIBP exhibited two bands on sodium dodecyl sulfate/polyacrylamide gel electrophoresis by silver staining. Ultraviolet crosslinking experiment showed that both bands had binding activity to the L-III oligonucleotide.

The Adrenomedullin gene is a target for negative regulation by the Myc transcription complex.

The Myc family of transcription factors plays a central role in vertebrate growth and development although relatively few genetic targets of the Myc transcription complex have been identified. In this study, we used mRNA differential display to investigate gene expression changes induced by the overexpression of the MC29 v-Myc oncoprotein in C3H10T1/2 mouse fibroblasts. We identified the transcript of the adrenomedullin gene (AM) as an mRNA that is specifically down-regulated in v-Myc overexpressing C3H10T1/2 cell lines as well as in a Rat 1a cell line inducible for c-Myc. Nucleotide sequence analysis of the mouse AM promoter reveals the presence of consensus CAAT and TATA boxes as well as an initiator element (INR) with significant sequence similarity to the INR responsible for Myc-mediated repression of the adenovirus major late promoter (AdMLP). Reporter gene assays confirm that the region of the AM promoter containing the INR is the target of Myc-mediated repression. Exogenous application of AM peptide to quiescent C3H10T1/2 cultures does not stimulate growth, and constitutive expression of AM mRNA in C3H10T1/2 cells correlates with a reduced potential of the cells to be cotransformed by v-Myc and oncogenic Ras p21. Additional studies showing that AM mRNA is underrepresented in C3H10T1/2 cell lines stably transformed by Ras p21 or adenovirus E1A suggest that AM gene expression is incompatible with deregulated growth in this cell line. We propose a model in which the repression of AM gene expression by Myc is important to the role of this oncoprotein as a potentiator of cellular transformation in C3H10T1/2 and perhaps other cell lines.

The Adrenomedullin Gene Is a Target for Negative Regulation by the Myc Transcription Complex

The Myc family of transcription factors plays a central role in vertebrate growth and development although relatively few genetic targets of the Myc transcription complex have been identified. In this study, we used mRNA differential display to investigate gene expression changes induced by the overexpression of the MC29 v-Myc oncoprotein in C3H10T1/2 mouse fibroblasts. We identified the transcript of the adrenomedullin gene (AM) as an mRNA that is specifically down-regulated in v-Myc overexpressing C3H10T1/2 cell lines as well as in a Rat 1a cell line inducible for c-Myc. Nucleotide sequence analysis of the mouse AM promoter reveals the presence of consensus CAAT and TATA boxes as well as an initiator element (INR) with significant sequence similarity to the INR responsible for Myc-mediated repression of the adenovirus major late promoter (AdMLP). Reporter gene assays confirm that the region of the AM promoter containing the INR is the target of Myc-mediated repression. Exogenous application of AM peptide to quiescent C3H10T1/2 cultures does not stimulate growth, and constitutive expression of AM mRNA in C3H10T1/2 cells correlates with a reduced potential of the cells to be cotransformed by v-Myc and oncogenic Ras p21. Additional studies showing that AM mRNA is underrepresented in C3H10T1/2 cell lines stably transformed by Ras p21 or adenovirus E1A suggest that AM gene expression is incompatible with deregulated growth in this cell line. We propose a model in which the repression of AM gene expression by Myc is important to the role of this oncoprotein as a potentiator of cellular transformation in C3H10T1/2 and perhaps other cell lines.

Inhibition of Host RNA Polymerase II-Dependent Transcription by Vesicular Stomatitis Virus Results from Inactivation of TFIID

During infection with vesicular stomatitis virus (VSV), host-cell mRNA synthesis is inhibited due to shut off of host-cell transcription. The transcriptional activity of nuclear extracts prepared from VSV-infected cells was compared to the activity of nuclear extracts from uninfected cells. An exogenous DNA template was used which contained an adenovirus major late promoter (AdMLP) but lacked upstream activating sequences, so that only basal transcription activity was assayed in these experiments. AdMLP-initiated transcription was decreased by 75% in nuclear extracts from infected cells as early as 3 h p.i. and by >90% by 6 h p.i. Mixing nuclear extracts from uninfected and VSV-infected cells revealed that the inhibition was caused by lack of an active form of a host factor involved in basal transcription rather than by the presence of an excess of inhibitory factor. To determine which transcription factors were lacking from nuclear extracts of infected cells, host transcription initiation factors isolated from uninfected cells by ion-exchange chromatography were added separately to nuclear extracts inactivated by VSV infection. A phosphocellulose column fraction from uninfected cells eluted with 0.8 M KCl, which contained transcription factor IID (TFIID), overcame the inhibition. The corresponding fraction from infected cells had no detectable activity in a TFIID-dependent in vitrotranscription assay. TATA-binding protein (TBP) is the DNA-binding subunit of TFIID and has been shown previously to substitute for TFIID in basal transcription. Purified recombinant TBP also reconstituted the transcription activity of nuclear extracts from infected cells, supporting the idea that TFIID is the target of virus-induced inhibition. Western blot analysis showed that the level of TBP in nuclear extracts or in the 0.8 M KCl column fraction was not changed by VSV infection. These results indicated that VSV infection leads to an inhibition of host transcription by inactivation of TFIID rather than reduction in the level of TFIID.

Inherent DNA curvature and flexibility correlate with TATA box functionality.

Four 1.5 ns molecular dynamics (MD) simulations were performed on the d(GCTATAAAAGGG).d(CCCTTTTATAGC) double helix dodecamer bearing the Adenovirus major late promoter TATA element and three iso-composition mutants for which physical and biochemical data are available from the same laboratory. Three of these DNA sequences experimentally induce tight binding with the TATA box binding protein (TBP) and induce high transcription rates; the other DNA sequence induces much lower TBP binding and transcription. The x-ray crystal structures have previously shown that the duplex DNA in DNA-TBP complexes are highly bent. We performed and analyzed MD simulations for these four DNAs, whose experimental structures are not available, in order to address the issue of whether inherent DNA structure and flexibility play a role in establishing these observed preferences. A comparison of the experimental and simulated results demonstrated that DNA duplex sequence-dependent curvature and flexibility play a significant role in TBP recognition, binding, and transcriptional activation.