Transcription of adenovirus RNA polymerase III genes.
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Adenoviridae↗
Biomedical subjects
S J Flint
Publications and source records attributed to S J Flint.
Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein.
The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies. More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified. All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein. Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS. Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides. Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes. Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III. These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells.
Animals↗
Mutational analysis of the adenovirus 2 IVa2 initiator and downstream elements.
The initiator element of the adenovirus type 2 IVa2 promoter is sufficient to direct accurate initiation by RNA polymerase II. Analysis of the effects of substitution of specific base pairs on initiator activity in in vitro transcription systems indicated that specific sequences between positions -4 and +5 were essential for initiator activity. Mutations that impaired or eliminated initiator activity altered both base pairs that are conserved in sequence-related initiators and nonconserved sequences. Neither the downstream TA-rich sequence of the IVa2 promoter, nor the adenovirus 2 major late TATA element placed at the same downstream site could overcome the severe inhibitory effects of initiator mutations, indicating that the initiator is the primary determinant of the specificity and direction of IVa2 transcription. By contrast, when the ML TATA element was placed 31 nucleotides upstream of the IVa2 initiator, the precise specificity, but neither the efficiency nor direction of transcription, depended on the presence of a functional initiator. Activity of the IVa2 promoter was relatively insensitive to changes in the orientation or nature of the TA-rich sequence. Furthermore, only a promoter containing the ML TA-TAAAA sequence downstream of the IVa2 initiator was competent to direct both IVa2 transcription and transcription from the opposite strand. The implications of this functional difference for recognition of the downstream element are discussed.
Adenoviruses, Human↗
Specific transcription from the adenovirus E2E promoter by RNA polymerase III requires a subpopulation of TFIID.
The early E2 (E2E) promoter of adenovirus type 2 possesses a TATA-like element and binding sites for the factors E2F and ATF. This promoter is transcribed by RNA polymerase II in high salt nuclear extracts, but by RNA polymerase III in standard nuclear extracts, as judged by sensitivity to low and high, respectively, concentrations of alpha-amanitin. Transcription by the two RNA polymerases initiated at the same site and depended, in both cases, on the TATA-like sequence and upstream elements. However, RNA polymerase III transcripts, unlike those synthesized by RNA polymerase II, terminated at two runs of Ts downstream of the initiation site. Although they are not essential, sequences downstream of the initiation site increased the efficiency of E2E transcription by RNA polymerase III. Such RNA polymerase III dependent transcription required a subpopulation of the general transcription factor, TFIID: TFIID that binds weakly to phosphocellulose (0.3 M eluate) complemented a TFIID-depleted extract to restore RNAp III transcription, whereas TFIID tightly associated with phosphocellulose (1 M eluate) was unable to do so.
Adenovirus E2 Proteins↗
The downstream regulatory sequence of the adenovirus type 2 major late promoter is functionally redundant.
Mutagenesis of promoter sequences and oligonucleotide competition assays have been used to demonstrate the late-phase-specific stimulation of the adenovirus type 2 major late promoter is mediated by functionally redundant elements located between positions +75 and +125. These octamer motif-related sequences are recognized by multiple factors.
Adenoviruses, Human↗
Anatomy of an unusual RNA polymerase II promoter containing a downstream TATA element.
The adenovirus type 2 IVa2 promoter lacks a conventional TATA element yet directs transcription from two closely spaced initiation sites. To define elements required for in vitro transcription of this promoter, IVa2 templates carrying 5' deletions or linker-scanning mutations were transcribed in HeLa whole-cell extracts and the transcripts were analyzed by primer extension. Mutation of the sequence centered on position -47, which is specifically recognized by a cellular factor, reduced the efficiency of IVa2 transcription two- to threefold, whereas mutation of the sequence centered on position -30 selectively impaired utilization of the minor in vivo initiation site. Utilization of the major in vivo site was decreased no more than fivefold by deletion of all sequences upstream of position -15. By contrast, mutation of the region from +13 to +19 or of the initiation region severely impaired IVa2 transcription. The sequence spanning the initiation sites was sufficient to direct accurate initiation by RNA polymerase II from the major in vivo site. Thus, the two initiation sites of the IVa2 promoter are specified by independent elements, and a downstream element is the primary determinant of efficient transcription from both of these sites. The downstream element identified by mutational analysis altered the TATA element-like sequence TATAGAAA lying at positions +21 to +14 in the coding strand. Transcription from the wild-type IVa2 promoter was severely inhibited when endogenous TFIID was inactivated by mild heat treatment. Exogenous human TATA-binding protein (TBP) synthesized in Escherichia coli restored specific IVa2 transcription from both initiation sites when added to such heat-treated extracts. Although efficient IVa2 transcription requires both the downstream TATA sequence and active TFIID, bacterially synthesized TBP also stimulated the low level of IVa2 transcription observed when the TATA sequence was mutated to a sequence that failed to bind TBP.
Adenoviruses, Human↗
Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels.
A synthetic 27-base-long oligodeoxyribonucleotide, termed PU1, has been shown to bind to duplex DNA to form a triplex at a single site within the human c-myc P1 promoter. PU1 has been administered to HeLa cells in culture to examine the feasibility of influencing transcription of the c-myc gene in vivo. It is shown that uptake of PU1 into the nucleus of HeLa cells is efficient and that the compound remains intact for at least 4 hr. In nuclei extracted from PU1-treated cells, inhibition of DNase I cleavage is detected within the c-myc P1 promoter at the target site for triplex formation. The inhibition is shown to be both site and oligodeoxyribonucleotide specific. After cellular uptake of PU1, it is shown that steady-state mRNA arising from the c-myc P1 initiation site is selectively reduced relative to total mRNA, relative to mRNA from the alternative c-myc P2 initiation site, and relative to mRNA derived from the beta-actin promoter. Significant mRNA repression is not seen upon treating cells with oligodeoxyribonucleotides that fail to bind to the P1 promoter target. Taken together, these data suggest that triplex formation can occur between an exogenous oligodeoxyribonucleotide and duplex DNA in the nucleus of treated cells.
Base Sequence↗
The adenovirus L4 100-kilodalton protein is necessary for efficient translation of viral late mRNA species.
When screening a number of adenovirus type 5 (Ad5) temperature-sensitive mutants for defects in viral gene expression, we observed that H5ts1-infected 293 cells accumulated reduced levels of newly synthesized viral late proteins. Pulse-labeling and pulse-chase experiments were used to establish that the late proteins synthesized in H5ts1-infected cells under nonpermissive conditions were as stable as those made in Ad5-infected cells. H5ts1-infected cells contained normal levels of viral late mRNAs. Because these observations implied that translation of viral mRNA species was defective in mutant virus-infected cells, the association of viral late mRNAs with polyribosomes was examined during the late phase of infection at a nonpermissive temperature. In Ad5-infected cells, the majority of the viral L2, L3, L4, pIX, and IVa2 late mRNA species were polyribosome bound. By contrast, these same mRNA species were recovered from H5ts1-infected cells in fractions nearer the top of polyribosome gradients, suggesting that initiation of translation was impaired. During the late phase of infection, neither the polyribosome association nor the translation of most viral early mRNA species was affected by the H5ts1 mutation. This lesion, mapped by marker rescue to the L4 100-kilodalton (kDa) nonstructural protein, has been identified as a single base pair substitution that replaces Ser-466 of the Ad5 100-kDa protein with Pro. A set of temperature-independent revertants of H5ts1 was isolated and characterized. Either true reversion of the H5ts1 mutation or second-site mutation of Pro-466 of the H5ts1 100-kDa protein to Thre, Leu, or His restored both temperature-independent growth and the efficient synthesis of viral late proteins. We therefore conclude that the Ad5 L4 100-kDa protein is necessary for efficient initiation of translation of viral late mRNA species during the late phase of infection.
Adenoviruses, Human↗
Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein.
The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells.
Adenovirus Early Proteins↗
Characterization of the adenovirus 2 virion protein, mu.
Adenovirus 2 virions contain a small, highly basic protein known as mu (mu). Partial sequence analysis of mu labeled with radioactive amino acids showed that it is derived from an 11-kDa virion precursor protein, L2-79R. Amino acid analysis, direct microsequence analysis, time-of-flight mass spectrometer analysis, and chemical synthesis demonstrated that mu is the unmodified, 19 amino acid peptide obtained from the 79-residue precursor by adenovirus-encoded proteinase-mediated cleavage after glycine31 and glycine50. Mu bound tightly to DNA and was located in the virion core. In vitro, mu could precipitate DNA fragments, suggesting that it may have a role in viral chromosome condensation.
Adenoviruses, Human↗
A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor.
Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.
Base Composition↗
Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro.
A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.
Electrophoresis↗
DNA-binding properties of an adenovirus 289R E1A protein.
An adenovirus 2 289 amino acid (289R) E1A protein purified from Escherichia coli has been shown to interact with DNA by two independent methods. UV-crosslinking of complexes containing unmodified, uniformly 32P-labelled DNA and purified E1A protein induced efficient labelling of the protein with covalently attached oligonucleotides, indicating that the E1A protein itself contacts DNA. Discrete nucleoprotein species were also observed when E1A protein--DNA complexes were analysed by gel electrophoresis. Although the 289R E1A protein exhibited no significant binding to single-stranded DNA or to RNA, no evidence for its sequence-specific binding to double-stranded DNA was obtained with either assay. Identification of the sites of covalent attachment of 32P-labelled oligonucleotides by partial proteolysis of the crosslinked E1A protein indicated that the interaction of this protein with DNA is mediated via domain(s) in the C-terminal half of the protein. Such previously unrecognized DNA-binding activity is likely to contribute to the regulatory activities of this important adenoviral protein.
Adenovirus Early Proteins↗
Isolation and characterization of monoclonal antibodies against the adenovirus core proteins.
Monoclonal antibodies have been prepared that recognize adenovirus core proteins V, VII, and mu in ELISA and Western blot assays. Antibodies produced by all of 87 positive hybridoma colonies obtained from a mouse injected with the precursor to protein VII, pVII, produced antibodies that also reacted with purified protein VII in an ELISA assay and all tested recognized denatured protein VII immobilized on nitrocellulose. Such failure to recover antibodies that specifically recognized only protein pVII suggests that epitopes common to the 174 amino acid protein VII and its 197 amino acid precursor were more effective antigenic determinants than the N-terminal 23 amino acid segment unique to pVII. All antibodies raised against protein mu cross-reacted with protein VII in both assays, but only a small fraction of the anti-protein VII or pVII antibodies recognized protein mu. Such cross-reactivity is discussed in relation to an unusual, arginine-rich sequence present in both protein VII and protein mu.
Adenoviruses, Human↗
Adenovirus type 2 endopeptidase: an unusual phosphoprotein enzyme matured by autocatalysis.
A 19-kDa protein, present in low copy number in purified adenovirus type 2, has been characterized. Several criteria were used to establish that this protein is neither a degradation product of the known structural proteins of the virion nor a minor, unusually modified, form of protein VII. This 19-kDa protein, unlike other virion proteins, possesses alkali-resistant phosphoamino acids. Analysis by partial proteolysis indicated that it is related to a 23-kDa phosphoprotein present in H2ts-1 virions assembled in infected cells maintained at 39 degrees C. Affinity labeling with [3H]diisopropyl fluorophosphate showed that the 19-kDa protein contains the active site for a serine protease. We, therefore, conclude that the 19-kDa protein is the active form of the adenovirus-encoded endopeptidase, defined by the H2ts-1 mutation, and is synthesized as a 23-kDa precursor that appears to mature by autocatalysis.
Adenoviruses, Human↗
Isolation and characterization of adenovirus core nucleoprotein subunits.
Digestion of adenovirus type 2 (Ad2) or Ad5 cores with micrococcal nuclease generated four nucleoprotein species that could be resolved by electrophoresis in low-ionic-strength polyacrylamide gels: these nucleoproteins displayed mobilities equivalent to those of DNA fragments of 900 to 1,025, 775 to 850, 650 to 725, and 525 to 600 base pairs (bp) and thus were readily distinguishable from HeLa cell mononucleosomes. The DNA fragments associated with the core nucleoprotein species were more than 250 to 90 bp long. Nucleoproteins containing 150, 120, or 90 bp of DNA were the most stable. Polypeptide VII was associated with each of the nucleoprotein species liberated from Ad2 cores. These data suggest that polypeptide VII and viral DNA of 90 to 150 bp comprise the unit particle of the Ad2 or Ad5 core nucleoproteins.
Adenoviruses, Human↗
Comparison of the interactions of the adenovirus type 2 major core protein and its precursor with DNA.
The interactions of the major core protein of adenovirus type 2 (Ad2) protein VII, and its precursor, protein pre-VII, with viral DNA, were studied using UV light induced crosslinking of 32P-labelled oligonucleotides to the proteins. Proteolytic fragments of these two proteins that contain DNA-binding domains were identified by virtue of their covalently attached, alkali-resistant 32P-radioactivity. The overall efficiency of crosslinking of protein pre-VII to DNA, in H2ts1 virions assembled at 39 degrees C, was comparable to that of the crosslinking of protein VII to DNA in Ad2 virions. However, a protease V8 fragment comprising the N-terminal half of protein pre-VII crosslinked to DNA at least ten times more efficiently than the corresponding N-terminal fragment of protein VII, which is truncated by the removal of 23 amino acids from the N-terminus of protein pre-VII during virion maturation.
Adenoviruses, Human↗
Identification of proteins and protein domains that contact DNA within adenovirus nucleoprotein cores by ultraviolet light crosslinking of oligonucleotides 32P-labelled in vivo.
A new approach to the identification of DNA binding proteins has been developed and used to study the DNA-protein interactions within the nucleoprotein core of subgroup C adenoviruses. Virions labelled in vivo with [32P]orthophosphate were exposed to ultraviolet light and the DNA digested by chemical or enzymatic methods. Labelled phosphoamino acids of the virion phosphoproteins were selectively hydrolysed by alkali, permitting proteins crosslinked to DNA to be identified by virtue of their covalently attached, 32P-labelled nucleotides. In parallel experiments, [3H]arginine-labelled virions were crosslinked by exposure to ultraviolet light and analysed by more conventional methods. The results indicate that proteins VII and V lie in close contact with viral DNA within the core. The compact arrangement of the nucleoprotein core appears to be capable of trapping protein VII molecules that are not covalently attached to DNA after exposure to ultraviolet light, suggesting that viral DNA might be wrapped around clusters of protein VII molecules. The domains of protein VII that lie in contact with DNA were identified by partial proteolytic mapping of the sites of covalent-attachment of the 32P-labelled oligonucleotides. The implications of these data for the nature of the interactions that mediate the packaging of viral DNA within the nucleoprotein core of adenovirions are discussed.
Adenoviridae↗