Abstract
In previous studies to characterize basal and activated transcription from the early promoter of the gp64 envelope fusion protein (efp) gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus, the TATA box was identified as a functional element, essential for basal transcription from a minimal promoter construct. In the current study, we used discrete deletions and multiple point mutations that removed the functional TATA box from larger promoter constructs of the gp64 efp gene to reveal an overlapping TATA-independent transcriptional activity. TATA-independent transcriptional activity was inhibited in vitro by alpha-amanitin but not by tagetitoxin, demonstrating that like the TATA-dependent activity, the TATA-independent activity is mediated by RNA polymerase II. Using constructs in which the TATA box (TATATAA) was destroyed by substitution mutations, we identified four elements that are required for the TATA-independent activity. Two of the required elements, GATA (at -114) and CACGTG (at -104), were previously shown to specifically bind host transcription factors and activate transcription from the TATA-dependent wild-type gp64 efp promoter. The role of the early start site consensus CAGT sequence in TATA-independent transcription was also examined. Single-nucleotide substitution mutations in the CAGT sequence indicated that certain nucleotides within the CAGT start site were essential. In addition to the start site sequence and two upstream elements, a fourth essential element was identified in the 5' untranslated leader region (5'UTR). While the 5'UTR was not necessary for TATA-dependent transcription, deletion of a 10-bp 5'UTR sequence resulted in the loss of TATA-independent transcriptional activity. Although necessary, neither the GATA, CACGTG, start site region, nor 5'UTR element was alone sufficient for accurately initiated TATA-independent transcription from the consensus CAGT start site. Thus, the gp64 efp early promoter contains overlapping TATA-dependent and TATA-independent transcriptional activities. A number of common sequence elements (GATA, CACGTG, and start site CAGT) are involved in both of these activities, while one element (in the 5'UTR) is required only in the TATA-independent context.
Full Text
The Full Text of this article is available as a PDF (555.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ayres M. D., Howard S. C., Kuzio J., Lopez-Ferber M., Possee R. D. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology. 1994 Aug 1;202(2):586–605. doi: 10.1006/viro.1994.1380. [DOI] [PubMed] [Google Scholar]
- Blissard G. W., Kogan P. H., Wei R., Rohrmann G. F. A synthetic early promoter from a baculovirus: roles of the TATA box and conserved start site CAGT sequence in basal levels of transcription. Virology. 1992 Oct;190(2):783–793. doi: 10.1016/0042-6822(92)90916-d. [DOI] [PubMed] [Google Scholar]
- Blissard G. W., Quant-Russell R. L., Rohrmann G. F., Beaudreau G. S. Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding p39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata. Virology. 1989 Feb;168(2):354–362. doi: 10.1016/0042-6822(89)90276-6. [DOI] [PubMed] [Google Scholar]
- Blissard G. W., Rohrmann G. F. Baculovirus diversity and molecular biology. Annu Rev Entomol. 1990;35:127–155. doi: 10.1146/annurev.en.35.010190.001015. [DOI] [PubMed] [Google Scholar]
- Blissard G. W., Rohrmann G. F. Baculovirus gp64 gene expression: analysis of sequences modulating early transcription and transactivation by IE1. J Virol. 1991 Nov;65(11):5820–5827. doi: 10.1128/jvi.65.11.5820-5827.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blissard G. W., Rohrmann G. F. Location, sequence, transcriptional mapping, and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology. 1989 Jun;170(2):537–555. doi: 10.1016/0042-6822(89)90445-5. [DOI] [PubMed] [Google Scholar]
- Carson D. D., Summers M. D., Guarino L. A. Molecular analysis of a baculovirus regulatory gene. Virology. 1991 May;182(1):279–286. doi: 10.1016/0042-6822(91)90671-w. [DOI] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Cochran M. A., Faulkner P. Location of Homologous DNA Sequences Interspersed at Five Regions in the Baculovirus AcMNPV Genome. J Virol. 1983 Mar;45(3):961–970. doi: 10.1128/jvi.45.3.961-970.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dickson J. A., Friesen P. D. Identification of upstream promoter elements mediating early transcription from the 35,000-molecular-weight protein gene of Autographa californica nuclear polyhedrosis virus. J Virol. 1991 Aug;65(8):4006–4016. doi: 10.1128/jvi.65.8.4006-4016.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guarino L. A. Identification of a viral gene encoding a ubiquitin-like protein. Proc Natl Acad Sci U S A. 1990 Jan;87(1):409–413. doi: 10.1073/pnas.87.1.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guarino L. A., Smith M. W. Nucleotide sequence and characterization of the 39K gene region of Autographa californica nuclear polyhedrosis virus. Virology. 1990 Nov;179(1):1–8. doi: 10.1016/0042-6822(90)90266-t. [DOI] [PubMed] [Google Scholar]
- Guarino L. A., Smith M. Regulation of delayed-early gene transcription by dual TATA boxes. J Virol. 1992 Jun;66(6):3733–3739. doi: 10.1128/jvi.66.6.3733-3739.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guarino L. A., Summers M. D. Nucleotide sequence and temporal expression of a baculovirus regulatory gene. J Virol. 1987 Jul;61(7):2091–2099. doi: 10.1128/jvi.61.7.2091-2099.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoopes R. R., Jr, Rohrmann G. F. In vitro transcription of baculovirus immediate early genes: accurate mRNA initiation by nuclear extracts from both insect and human cells. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4513–4517. doi: 10.1073/pnas.88.10.4513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huh N. E., Weaver R. F. Identifying the RNA polymerases that synthesize specific transcripts of the Autographa californica nuclear polyhedrosis virus. J Gen Virol. 1990 Jan;71(Pt 1):195–201. doi: 10.1099/0022-1317-71-1-195. [DOI] [PubMed] [Google Scholar]
- Kogan P. H., Blissard G. W. A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements. J Virol. 1994 Feb;68(2):813–822. doi: 10.1128/jvi.68.2.813-822.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kool M., Vlak J. M. The structural and functional organization of the Autographa californica nuclear polyhedrosis virus genome. Arch Virol. 1993;130(1-2):1–16. doi: 10.1007/BF01318992. [DOI] [PubMed] [Google Scholar]
- Kool M., Voeten J. T., Goldbach R. W., Tramper J., Vlak J. M. Identification of seven putative origins of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus DNA replication. J Gen Virol. 1993 Dec;74(Pt 12):2661–2668. doi: 10.1099/0022-1317-74-12-2661. [DOI] [PubMed] [Google Scholar]
- Kool M., Voeten J. T., Goldbach R. W., Vlak J. M. Functional mapping of regions of the Autographa californica nuclear polyhedrosis viral genome required for DNA replication. Virology. 1994 Feb;198(2):680–689. doi: 10.1006/viro.1994.1080. [DOI] [PubMed] [Google Scholar]
- Kool M., van den Berg P. M., Tramper J., Goldbach R. W., Vlak J. M. Location of two putative origins of DNA replication of Autographa californica nuclear polyhedrosis virus. Virology. 1993 Jan;192(1):94–101. doi: 10.1006/viro.1993.1011. [DOI] [PubMed] [Google Scholar]
- Krappa R., Behn-Krappa A., Jahnel F., Doerfler W., Knebel-Mörsdorf D. Differential factor binding at the promoter of early baculovirus gene PE38 during viral infection: GATA motif is recognized by an insect protein. J Virol. 1992 Jun;66(6):3494–3503. doi: 10.1128/jvi.66.6.3494-3503.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leisy D. J., Rohrmann G. F. Characterization of the replication of plasmids containing hr sequences in baculovirus-infected Spodoptera frugiperda cells. Virology. 1993 Oct;196(2):722–730. doi: 10.1006/viro.1993.1529. [DOI] [PubMed] [Google Scholar]
- Lu A., Carstens E. B. Immediate-early baculovirus genes transactivate the p143 gene promoter of Autographa californica nuclear polyhedrosis virus. Virology. 1993 Aug;195(2):710–718. doi: 10.1006/viro.1993.1422. [DOI] [PubMed] [Google Scholar]
- Lu A., Carstens E. B. Nucleotide sequence of a gene essential for viral DNA replication in the baculovirus Autographa californica nuclear polyhedrosis virus. Virology. 1991 Mar;181(1):336–347. doi: 10.1016/0042-6822(91)90500-b. [DOI] [PubMed] [Google Scholar]
- Lu A., Carstens E. B. Transcription analysis of the EcoRI D region of the baculovirus Autographa californica nuclear polyhedrosis virus identifies an early 4-kilobase RNA encoding the essential p143 gene. J Virol. 1992 Feb;66(2):655–663. doi: 10.1128/jvi.66.2.655-663.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mackett M., Smith G. L., Moss B. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol. 1984 Mar;49(3):857–864. doi: 10.1128/jvi.49.3.857-864.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nissen M. S., Friesen P. D. Molecular analysis of the transcriptional regulatory region of an early baculovirus gene. J Virol. 1989 Feb;63(2):493–503. doi: 10.1128/jvi.63.2.493-503.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Passarelli A. L., Miller L. K. Identification and characterization of lef-1, a baculovirus gene involved in late and very late gene expression. J Virol. 1993 Jun;67(6):3481–3488. doi: 10.1128/jvi.67.6.3481-3488.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Passarelli A. L., Miller L. K. Identification and transcriptional regulation of the baculovirus lef-6 gene. J Virol. 1994 Jul;68(7):4458–4467. doi: 10.1128/jvi.68.7.4458-4467.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pearson M., Bjornson R., Pearson G., Rohrmann G. The Autographa californica baculovirus genome: evidence for multiple replication origins. Science. 1992 Sep 4;257(5075):1382–1384. doi: 10.1126/science.1529337. [DOI] [PubMed] [Google Scholar]
- Purnell B. A., Emanuel P. A., Gilmour D. S. TFIID sequence recognition of the initiator and sequences farther downstream in Drosophila class II genes. Genes Dev. 1994 Apr 1;8(7):830–842. doi: 10.1101/gad.8.7.830. [DOI] [PubMed] [Google Scholar]
- Purnell B. A., Gilmour D. S. Contribution of sequences downstream of the TATA element to a protein-DNA complex containing the TATA-binding protein. Mol Cell Biol. 1993 Apr;13(4):2593–2603. doi: 10.1128/mcb.13.4.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ray F. A., Nickoloff J. A. Site-specific mutagenesis of almost any plasmid using a PCR-based version of unique site elimination. Biotechniques. 1992 Sep;13(3):342–348. [PubMed] [Google Scholar]
- Rodems S. M., Friesen P. D. The hr5 transcriptional enhancer stimulates early expression from the Autographa californica nuclear polyhedrosis virus genome but is not required for virus replication. J Virol. 1993 Oct;67(10):5776–5785. doi: 10.1128/jvi.67.10.5776-5785.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Steinberg T. H., Mathews D. E., Durbin R. D., Burgess R. R. Tagetitoxin: a new inhibitor of eukaryotic transcription by RNA polymerase III. J Biol Chem. 1990 Jan 5;265(1):499–505. [PubMed] [Google Scholar]
- Theilmann D. A., Stewart S. Identification and characterization of the IE-1 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology. 1991 Feb;180(2):492–508. doi: 10.1016/0042-6822(91)90063-h. [DOI] [PubMed] [Google Scholar]
- Theilmann D. A., Stewart S. Molecular analysis of the trans-activating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology. 1992 Mar;187(1):84–96. doi: 10.1016/0042-6822(92)90297-3. [DOI] [PubMed] [Google Scholar]
- Thiem S. M., Miller L. K. A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper. J Virol. 1989 Nov;63(11):4489–4497. doi: 10.1128/jvi.63.11.4489-4497.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thiem S. M., Miller L. K. Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol. 1989 May;63(5):2008–2018. doi: 10.1128/jvi.63.5.2008-2018.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tomalski M. D., Eldridge R., Miller L. K. A baculovirus homolog of a Cu/Zn superoxide dismutase gene. Virology. 1991 Sep;184(1):149–161. doi: 10.1016/0042-6822(91)90831-u. [DOI] [PubMed] [Google Scholar]
- Tomalski M. D., Wu J. G., Miller L. K. The location, sequence, transcription, and regulation of a baculovirus DNA polymerase gene. Virology. 1988 Dec;167(2):591–600. [PubMed] [Google Scholar]
- Whitford M., Faulkner P. Nucleotide sequence and transcriptional analysis of a gene encoding gp41, a structural glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol. 1992 Aug;66(8):4763–4768. doi: 10.1128/jvi.66.8.4763-4768.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whitford M., Stewart S., Kuzio J., Faulkner P. Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol. 1989 Mar;63(3):1393–1399. doi: 10.1128/jvi.63.3.1393-1399.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu J. G., Miller L. K. Sequence, transcription and translation of a late gene of the Autographa californica nuclear polyhedrosis virus encoding a 34.8K polypeptide. J Gen Virol. 1989 Sep;70(Pt 9):2449–2459. doi: 10.1099/0022-1317-70-9-2449. [DOI] [PubMed] [Google Scholar]