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. 1991 Mar;11(3):1382–1392. doi: 10.1128/mcb.11.3.1382

Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

L S Young 1, D H Rivier 1, K U Sprague 1
PMCID: PMC369409  PMID: 1996100

Abstract

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker R. E., Camier S., Sentenac A., Hall B. D. Gene size differentially affects the binding of yeast transcription factor tau to two intragenic regions. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8768–8772. doi: 10.1073/pnas.84.24.8768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker R. E., Hall B. D. Structural features of yeast tRNA genes which affect transcription factor binding. EMBO J. 1984 Dec 1;3(12):2793–2800. doi: 10.1002/j.1460-2075.1984.tb02211.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartholomew B., Kassavetis G. A., Braun B. R., Geiduschek E. P. The subunit structure of Saccharomyces cerevisiae transcription factor IIIC probed with a novel photocrosslinking reagent. EMBO J. 1990 Jul;9(7):2197–2205. doi: 10.1002/j.1460-2075.1990.tb07389.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Brow D. A., Guthrie C. Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position. Genes Dev. 1990 Aug;4(8):1345–1356. doi: 10.1101/gad.4.8.1345. [DOI] [PubMed] [Google Scholar]
  6. Carey M. F., Gerrard S. P., Cozzarelli N. R. Analysis of RNA polymerase III transcription complexes by gel filtration. J Biol Chem. 1986 Mar 25;261(9):4309–4317. [PubMed] [Google Scholar]
  7. Ciliberto G., Castagnoli L., Melton D. A., Cortese R. Promoter of a eukaryotic tRNAPro gene is composed of three noncontiguous regions. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1195–1199. doi: 10.1073/pnas.79.4.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cromlish J. A., Roeder R. G. Human transcription factor IIIC (TFIIIC). Purification, polypeptide structure, and the involvement of thiol groups in specific DNA binding. J Biol Chem. 1989 Oct 25;264(30):18100–18109. [PubMed] [Google Scholar]
  9. Dean N., Berk A. J. Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography. Nucleic Acids Res. 1987 Dec 10;15(23):9895–9907. doi: 10.1093/nar/15.23.9895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dingermann T., Sharp S., Schaack J., Söll D. Stable transcription complex formation of eukaryotic tRNA genes is dependent on a limited separation of the two intragenic control regions. J Biol Chem. 1983 Sep 10;258(17):10395–10402. [PubMed] [Google Scholar]
  11. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fuhrman S. A., Engelke D. R., Geiduschek E. P. HeLa cell RNA polymerase III transcription factors. Functional characterization of a fraction identified by its activity in a second template rescue assay. J Biol Chem. 1984 Feb 10;259(3):1934–1943. [PubMed] [Google Scholar]
  13. Gabrielsen O. S., Marzouki N., Ruet A., Sentenac A., Fromageot P. Two polypeptide chains in yeast transcription factor tau interact with DNA. J Biol Chem. 1989 May 5;264(13):7505–7511. [PubMed] [Google Scholar]
  14. Galibert F., Sedat J., Ziff E. Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA. J Mol Biol. 1974 Aug 15;87(3):377–407. doi: 10.1016/0022-2836(74)90093-x. [DOI] [PubMed] [Google Scholar]
  15. Galli G., Hofstetter H., Birnstiel M. L. Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements. Nature. 1981 Dec 17;294(5842):626–631. doi: 10.1038/294626a0. [DOI] [PubMed] [Google Scholar]
  16. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Geiduschek E. P., Tocchini-Valentini G. P. Transcription by RNA polymerase III. Annu Rev Biochem. 1988;57:873–914. doi: 10.1146/annurev.bi.57.070188.004301. [DOI] [PubMed] [Google Scholar]
  18. Hagenbüchle O., Larson D., Hall G. I., Sprague K. U. The primary transcription product of a silkworm alanine tRNA gene: identification of in vitro sites of initiation, termination and processing. Cell. 1979 Dec;18(4):1217–1229. doi: 10.1016/0092-8674(79)90234-4. [DOI] [PubMed] [Google Scholar]
  19. Hoeffler W. K., Kovelman R., Roeder R. G. Activation of transcription factor IIIC by the adenovirus E1A protein. Cell. 1988 Jun 17;53(6):907–920. doi: 10.1016/s0092-8674(88)90409-6. [DOI] [PubMed] [Google Scholar]
  20. Kassavetis G. A., Riggs D. L., Negri R., Nguyen L. H., Geiduschek E. P. Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes. Mol Cell Biol. 1989 Jun;9(6):2551–2566. doi: 10.1128/mcb.9.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kondo K., Hodgkin J., Waterston R. H. Differential expression of five tRNA(UAGTrp) amber suppressors in Caenorhabditis elegans. Mol Cell Biol. 1988 Sep;8(9):3627–3635. doi: 10.1128/mcb.8.9.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Larson D., Bradford-Wilcox J., Young L. S., Sprague K. U. A short 5' flanking region containing conserved sequences is required for silkworm alanine tRNA gene activity. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3416–3420. doi: 10.1073/pnas.80.11.3416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lassar A. B., Martin P. L., Roeder R. G. Transcription of class III genes: formation of preinitiation complexes. Science. 1983 Nov 18;222(4625):740–748. doi: 10.1126/science.6356356. [DOI] [PubMed] [Google Scholar]
  24. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  25. Newman A. J., Ogden R. C., Abelson J. tRNA gene transcription in yeast: effects of specified base substitutions in the intragenic promoter. Cell. 1983 Nov;35(1):117–125. doi: 10.1016/0092-8674(83)90214-3. [DOI] [PubMed] [Google Scholar]
  26. Ottonello S., Rivier D. H., Doolittle G. M., Young L. S., Sprague K. U. The properties of a new polymerase III transcription factor reveal that transcription complexes can assemble by more than one pathway. EMBO J. 1987 Jul;6(7):1921–1927. doi: 10.1002/j.1460-2075.1987.tb02452.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parsons M. C., Weil P. A. Purification and characterization of Saccharomyces cerevisiae transcription factor TFIIIC. Polypeptide composition defined with polyclonal antibodies. J Biol Chem. 1990 Mar 25;265(9):5095–5103. [PubMed] [Google Scholar]
  28. Ruet A., Camier S., Smagowicz W., Sentenac A., Fromageot P. Isolation of a class C transcription factor which forms a stable complex with tRNA genes. EMBO J. 1984 Feb;3(2):343–350. doi: 10.1002/j.1460-2075.1984.tb01809.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schaack J., Sharp S., Dingermann T., Söll D. Transcription of eukaryotic tRNA genes in vitro. II. Formation of stable complexes. J Biol Chem. 1983 Feb 25;258(4):2447–2453. [PubMed] [Google Scholar]
  31. Sharp S., DeFranco D., Dingermann T., Farrell P., Söll D. Internal control regions for transcription of eukaryotic tRNA genes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6657–6661. doi: 10.1073/pnas.78.11.6657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sharp S., Dingermann T., Schaack J., DeFranco D., Söll D. Transcription of eukaryotic tRNA genes in vitro. I. Analysis of control regions using a competition assay. J Biol Chem. 1983 Feb 25;258(4):2440–2446. [PubMed] [Google Scholar]
  33. Sprague K. U., Larson D., Morton D. 5' flanking sequence signals are required for activity of silkworm alanine tRNA genes in homologous in vitro transcription systems. Cell. 1980 Nov;22(1 Pt 1):171–178. doi: 10.1016/0092-8674(80)90165-8. [DOI] [PubMed] [Google Scholar]
  34. Wilson E. T., Condliffe D. P., Sprague K. U. Transcriptional properties of BmX, a moderately repetitive silkworm gene that is an RNA polymerase III template. Mol Cell Biol. 1988 Feb;8(2):624–631. doi: 10.1128/mcb.8.2.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wilson E. T., Larson D., Young L. S., Sprague K. U. A large region controls tRNA gene transcription. J Mol Biol. 1985 May 25;183(2):153–163. doi: 10.1016/0022-2836(85)90209-8. [DOI] [PubMed] [Google Scholar]
  36. Yoshinaga S. K., Boulanger P. A., Berk A. J. Resolution of human transcription factor TFIIIC into two functional components. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3585–3589. doi: 10.1073/pnas.84.11.3585. [DOI] [PMC free article] [PubMed] [Google Scholar]

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