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. 1996 Mar;16(3):1256–1266. doi: 10.1128/mcb.16.3.1256

Silkworm TFIIIB binds both constitutive and silk gland-specific tRNA Ala promoters but protects only the constitutive promoter from DNase I cleavage.

L S Young 1, N Ahnert 1, K U Sprague 1
PMCID: PMC231108  PMID: 8622670

Abstract

We have identified a complex between TFIIIB and the upstream promoter of silkworm tRNA Ala genes that is detectable by gel retardation and DNase I footprinting. Formation of this complex depends on the integrity of previously identified upstream promoter elements and on the presence of other silkworm transcription factors, either TFIIID or a fraction that contains both TFIIIC and TFIIID. We have used this complex to compare the interactions of TFIIIB with two kinds of tRNA Ala genes whose different in vitro transcription properties are conferred by the upstream segments of their promoters. These are the tRNA C Ala genes, which are transcribed constitutively, and the tRNA SG Ala genes, which are transcribed only in the silk gland. We find that TFIIIB binds tRNA SG Ala genes with lower affinity than it binds tRNA C Ala genes. In addition, the TFIIIB complex formed on tRNA SG Ala genes differ qualitatively from those formed on tRNA C Ala genes. Both the transcriptional activity of tRNA SG Ala complexes and the ability of the complexes to protect upstream DNA from DNase I digestion are reduced.

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Selected References

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  1. Bieker J. J., Martin P. L., Roeder R. G. Formation of a rate-limiting intermediate in 5S RNA gene transcription. Cell. 1985 Jan;40(1):119–127. doi: 10.1016/0092-8674(85)90315-0. [DOI] [PubMed] [Google Scholar]
  2. Bouvet P., Dimitrov S., Wolffe A. P. Specific regulation of Xenopus chromosomal 5S rRNA gene transcription in vivo by histone H1. Genes Dev. 1994 May 15;8(10):1147–1159. doi: 10.1101/gad.8.10.1147. [DOI] [PubMed] [Google Scholar]
  3. Burke D. J., Schaack J., Sharp S., Söll D. Partial purification of Drosophila Kc cell RNA polymerase III transcription components. Evidence for shared 5 S RNA and tRNA gene factors. J Biol Chem. 1983 Dec 25;258(24):15224–15231. [PubMed] [Google Scholar]
  4. Candelas G. C., Arroyo G., Carrasco C., Dompenciel R. Spider silkglands contain a tissue-specific alanine tRNA that accumulates in vitro in response to the stimulus for silk protein synthesis. Dev Biol. 1990 Jul;140(1):215–220. doi: 10.1016/0012-1606(90)90069-u. [DOI] [PubMed] [Google Scholar]
  5. Chavancy G., Chevallier A., Fournier A., Garel J. P. Adaptation of iso-tRNA concentration to mRNA codon frequency in the eukaryote cell. Biochimie. 1979;61(1):71–78. doi: 10.1016/s0300-9084(79)80314-4. [DOI] [PubMed] [Google Scholar]
  6. Chavancy G., Garel J. P., Daillie J. Functional adaptation of aminoacyl-tRNA synthetases to fibroin biosynthesis in the silkgland of Bombyx mori L. FEBS Lett. 1975 Jan 1;49(3):380–384. doi: 10.1016/0014-5793(75)80790-3. [DOI] [PubMed] [Google Scholar]
  7. Dieci G., Duimio L., Peracchia G., Ottonello S. Selective inactivation of two components of the multiprotein transcription factor TFIIIB in cycloheximide growth-arrested yeast cells. J Biol Chem. 1995 Jun 2;270(22):13476–13482. doi: 10.1074/jbc.270.22.13476. [DOI] [PubMed] [Google Scholar]
  8. Dignam S. S., Dignam J. D. Glycyl- and alanyl-tRNA synthetases from Bombyx mori. Purification and properties. J Biol Chem. 1984 Apr 10;259(7):4043–4048. [PubMed] [Google Scholar]
  9. Dingermann T., Werner H., Schütz A., Zündorf I., Nerke K., Knecht D., Marschalek R. Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene. Mol Cell Biol. 1992 Sep;12(9):4038–4045. doi: 10.1128/mcb.12.9.4038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ford P. J., Southern E. M. Different sequences for 5S RNA in kidney cells and ovaries of Xenopus laevis. Nat New Biol. 1973 Jan 3;241(105):7–12. doi: 10.1038/newbio241007a0. [DOI] [PubMed] [Google Scholar]
  11. Fournier A., Guérin M. A., Corlet J., Clarkson S. G. Structure and in vitro transcription of a glycine tRNA gene from Bombyx mori. EMBO J. 1984 Jul;3(7):1547–1552. doi: 10.1002/j.1460-2075.1984.tb02009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fournier A., Taneja R., Gopalkrishnan R., Prudhomme J. C., Gopinathan K. P. Differential transcription of multiple copies of a silk worm gene encoding tRNA(Gly1). Gene. 1993 Dec 8;134(2):183–190. doi: 10.1016/0378-1119(93)90092-h. [DOI] [PubMed] [Google Scholar]
  13. Garber M. E., Vilalta A., Johnson D. L. Induction of Drosophila RNA polymerase III gene expression by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is mediated by transcription factor IIIB. Mol Cell Biol. 1994 Jan;14(1):339–347. doi: 10.1128/mcb.14.1.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garber M., Panchanathan S., Fan R. S., Johnson D. L. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, induces specific transcription by RNA polymerase III in Drosophila Schneider cells. J Biol Chem. 1991 Nov 5;266(31):20598–20601. [PubMed] [Google Scholar]
  15. Garel J. P., Mandel P., Chavancy G., Daillie J. Functional adaptation of tRNAs to fibroin biosynthesis in the silkgland of Bombyx mori L. FEBS Lett. 1970 May 1;7(4):327–329. doi: 10.1016/0014-5793(70)80196-x. [DOI] [PubMed] [Google Scholar]
  16. Gottesfeld J. M., Wolf V. J., Dang T., Forbes D. J., Hartl P. Mitotic repression of RNA polymerase III transcription in vitro mediated by phosphorylation of a TFIIIB component. Science. 1994 Jan 7;263(5143):81–84. doi: 10.1126/science.8272869. [DOI] [PubMed] [Google Scholar]
  17. Gouilloud E., Clarkson S. G. A dispersed tyrosine tRNA gene from Xenopus laevis with high transcriptional activity in vitro. J Biol Chem. 1986 Jan 5;261(1):486–494. [PubMed] [Google Scholar]
  18. Hatfield D., Varricchio F., Rice M., Forget B. G. The aminoacyl-tRNA population of human reticulocytes. J Biol Chem. 1982 Mar 25;257(6):3183–3188. [PubMed] [Google Scholar]
  19. Henry R. W., Sadowski C. L., Kobayashi R., Hernandez N. A TBP-TAF complex required for transcription of human snRNA genes by RNA polymerase II and III. Nature. 1995 Apr 13;374(6523):653–656. doi: 10.1038/374653a0. [DOI] [PubMed] [Google Scholar]
  20. Huibregtse J. M., Engelke D. R. Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region. Mol Cell Biol. 1989 Aug;9(8):3244–3252. doi: 10.1128/mcb.9.8.3244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Joazeiro C. A., Kassavetis G. A., Geiduschek E. P. Identical components of yeast transcription factor IIIB are required and sufficient for transcription of TATA box-containing and TATA-less genes. Mol Cell Biol. 1994 Apr;14(4):2798–2808. doi: 10.1128/mcb.14.4.2798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kassavetis G. A., Braun B. R., Nguyen L. H., Geiduschek E. P. S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors. Cell. 1990 Jan 26;60(2):235–245. doi: 10.1016/0092-8674(90)90739-2. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. LUCAS F., SHAW J. T., SMITH S. G. The silk fibroins. Adv Protein Chem. 1958;13:107–242. doi: 10.1016/s0065-3233(08)60599-9. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Majima R., Kawakami M., Shimura K. The biosynthesis of transfer RNA in insects. I. Increase of amino acid acceptor activity of specific tRNA's utilized for silk protein biosynthesis in the silk gland of Bombyx mori. J Biochem. 1975 Aug;78(2):391–400. doi: 10.1093/oxfordjournals.jbchem.a130919. [DOI] [PubMed] [Google Scholar]
  27. Marschalek R., Brechner T., Amon-Böhm E., Dingermann T. Transfer RNA genes: landmarks for integration of mobile genetic elements in Dictyostelium discoideum. Science. 1989 Jun 23;244(4911):1493–1496. doi: 10.1126/science.2567533. [DOI] [PubMed] [Google Scholar]
  28. Matsuzaki K. Fractionation of amino acid-specific s-RNA from silkgland by methylated albumin column chromatography. Biochim Biophys Acta. 1966 Feb 21;114(2):222–226. doi: 10.1016/0005-2787(66)90303-0. [DOI] [PubMed] [Google Scholar]
  29. Matthews J. L., Zwick M. G., Paule M. R. Coordinate regulation of ribosomal component synthesis in Acanthamoeba castellanii: 5S RNA transcription is down regulated during encystment by alteration of TFIIIA activity. Mol Cell Biol. 1995 Jun;15(6):3327–3335. doi: 10.1128/mcb.15.6.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McBryant S. J., Kassavetis G. A., Gottesfeld J. M. Repression of vertebrate RNA polymerase III transcription by DNA binding proteins located upstream from the transcription start site. J Mol Biol. 1995 Jul 14;250(3):315–326. doi: 10.1006/jmbi.1995.0379. [DOI] [PubMed] [Google Scholar]
  31. Meza L., Araya A., Leon G., Krauskopf M. Specific alanine-tRNA species associated with fibroin biosynthesis in the posterior sild-gland of Bombyx mori L. FEBS Lett. 1977 May 15;77(2):255–260. doi: 10.1016/0014-5793(77)80246-9. [DOI] [PubMed] [Google Scholar]
  32. Nishio K., Kawakami M. Purification and properties of alanyl-tRNA synthetase from Bombyx mori: a monomeric enzyme. J Biochem. 1984 Dec;96(6):1867–1874. doi: 10.1093/oxfordjournals.jbchem.a135021. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Palida F. A., Hale C., Sprague K. U. Transcription of a silkworm tRNA(cAla) gene is directed by two AT-rich upstream sequence elements. Nucleic Acids Res. 1993 Dec 25;21(25):5875–5881. doi: 10.1093/nar/21.25.5875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Peck L. J., Bartilson M., DeRisi J. L. Bead-shift isolation of protein--DNA complexes on a 5S RNA gene. Nucleic Acids Res. 1994 Feb 11;22(3):443–449. doi: 10.1093/nar/22.3.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Reynolds W. F., Azer K. Sequence differences upstream of the promoters are involved in the differential expression of the Xenopus somatic and oocyte 5S RNA genes. Nucleic Acids Res. 1988 Apr 25;16(8):3391–3403. doi: 10.1093/nar/16.8.3391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reynolds W. F. Developmental stage-specific regulation of Xenopus tRNA genes by an upstream promoter element. J Biol Chem. 1995 May 5;270(18):10703–10710. doi: 10.1074/jbc.270.18.10703. [DOI] [PubMed] [Google Scholar]
  38. Reynolds W. F., Johnson D. L. Differential expression of oocyte-type class III genes with fraction TFIIIC from immature or mature oocytes. Mol Cell Biol. 1992 Mar;12(3):946–953. doi: 10.1128/mcb.12.3.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reynolds W. F. The tyrosine phosphatase cdc25 selectively inhibits transcription of the Xenopus oocyte-type tRNAtyrC gene. Nucleic Acids Res. 1993 Sep 11;21(18):4372–4377. doi: 10.1093/nar/21.18.4372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sadowski C. L., Henry R. W., Lobo S. M., Hernandez N. Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing complex activates transcription from snRNA promoters through the PSE. Genes Dev. 1993 Aug;7(8):1535–1548. doi: 10.1101/gad.7.8.1535. [DOI] [PubMed] [Google Scholar]
  41. Schultz P., Marzouki N., Marck C., Ruet A., Oudet P., Sentenac A. The two DNA-binding domains of yeast transcription factor tau as observed by scanning transmission electron microscopy. EMBO J. 1989 Dec 1;8(12):3815–3824. doi: 10.1002/j.1460-2075.1989.tb08559.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Segall J., Matsui T., Roeder R. G. Multiple factors are required for the accurate transcription of purified genes by RNA polymerase III. J Biol Chem. 1980 Dec 25;255(24):11986–11991. [PubMed] [Google Scholar]
  43. Seidel C. W., Peck L. J. Kinetic control of 5 S RNA gene transcription. J Mol Biol. 1992 Oct 20;227(4):1009–1018. doi: 10.1016/0022-2836(92)90517-n. [DOI] [PubMed] [Google Scholar]
  44. Setzer D. R., Brown D. D. Formation and stability of the 5 S RNA transcription complex. J Biol Chem. 1985 Feb 25;260(4):2483–2492. [PubMed] [Google Scholar]
  45. Shastry B. S., Ng S. Y., Roeder R. G. Multiple factors involved in the transcription of class III genes in Xenopus laevis. J Biol Chem. 1982 Nov 10;257(21):12979–12986. [PubMed] [Google Scholar]
  46. Sprague K. U., Hagenbüchle O., Zuniga M. C. The nucleotide sequence of two silk gland alanine tRNAs: implications for fibroin synthesis and for initiator tRNA structure. Cell. 1977 Jul;11(3):561–570. doi: 10.1016/0092-8674(77)90074-5. [DOI] [PubMed] [Google Scholar]
  47. Sprague K. U. The Bombyx mori silk proteins: characterization of large polypeptides. Biochemistry. 1975 Mar 11;14(5):925–931. doi: 10.1021/bi00676a008. [DOI] [PubMed] [Google Scholar]
  48. Stillman D. J., Better M., Geiduschek E. P. Electron-microscopic examination of the binding of a large RNA polymerase III transcription factor to a tRNA gene. J Mol Biol. 1985 Sep 20;185(2):451–455. doi: 10.1016/0022-2836(85)90417-6. [DOI] [PubMed] [Google Scholar]
  49. Stutz F., Gouilloud E., Clarkson S. G. Oocyte and somatic tyrosine tRNA genes in Xenopus laevis. Genes Dev. 1989 Aug;3(8):1190–1198. doi: 10.1101/gad.3.8.1190. [DOI] [PubMed] [Google Scholar]
  50. Sullivan H. S., Young L. S., White C. N., Sprague K. U. Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions. Mol Cell Biol. 1994 Mar;14(3):1806–1814. doi: 10.1128/mcb.14.3.1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Taneja R., Gopalkrishnan R., Gopinathan K. P. Regulation of glycine tRNA gene expression in the posterior silk glands of the silkworm Bombyx mori. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1070–1074. doi: 10.1073/pnas.89.3.1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tapping R. I., Syroid D. E., Capone J. P. Upstream interactions of functional mammalian tRNA gene transcription complexes probed using a heterologous DNA-binding protein. J Biol Chem. 1994 Aug 26;269(34):21812–21819. [PubMed] [Google Scholar]
  53. Tower J., Sollner-Webb B. Polymerase III transcription factor B activity is reduced in extracts of growth-restricted cells. Mol Cell Biol. 1988 Feb;8(2):1001–1005. doi: 10.1128/mcb.8.2.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Underwood D. C., Knickerbocker H., Gardner G., Condliffe D. P., Sprague K. U. Silk gland-specific tRNA(Ala) genes are tightly clustered in the silkworm genome. Mol Cell Biol. 1988 Dec;8(12):5504–5512. doi: 10.1128/mcb.8.12.5504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Waldschmidt R., Jahn D., Seifart K. H. Purification of transcription factor IIIB from HeLa cells. J Biol Chem. 1988 Sep 15;263(26):13350–13356. [PubMed] [Google Scholar]
  56. White R. J., Gottlieb T. M., Downes C. S., Jackson S. P. Mitotic regulation of a TATA-binding-protein-containing complex. Mol Cell Biol. 1995 Apr;15(4):1983–1992. doi: 10.1128/mcb.15.4.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. White R. J., Stott D., Rigby P. W. Regulation of RNA polymerase III transcription in response to F9 embryonal carcinoma stem cell differentiation. Cell. 1989 Dec 22;59(6):1081–1092. doi: 10.1016/0092-8674(89)90764-2. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Wolffe A. P., Brown D. D. Developmental regulation of two 5S ribosomal RNA genes. Science. 1988 Sep 23;241(4873):1626–1632. doi: 10.1126/science.241.4873.1626. [DOI] [PubMed] [Google Scholar]
  60. Wolffe A. P., Brown D. D. Differential 5S RNA gene expression in vitro. Cell. 1987 Dec 4;51(5):733–740. doi: 10.1016/0092-8674(87)90096-1. [DOI] [PubMed] [Google Scholar]
  61. Wolffe A. P. The role of transcription factors, chromatin structure and DNA replication in 5 S RNA gene regulation. J Cell Sci. 1994 Aug;107(Pt 8):2055–2063. doi: 10.1242/jcs.107.8.2055. [DOI] [PubMed] [Google Scholar]
  62. Wormington W. M., Bogenhagen D. F., Jordan E., Brown D. D. A quantitative assay for Xenopus 5S RNA gene transcription in vitro. Cell. 1981 Jun;24(3):809–817. doi: 10.1016/0092-8674(81)90106-9. [DOI] [PubMed] [Google Scholar]
  63. Yoshinaga S., Dean N., Han M., Berk A. J. Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration. EMBO J. 1986 Feb;5(2):343–354. doi: 10.1002/j.1460-2075.1986.tb04218.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Young L. S., Rivier D. H., Sprague K. U. Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors. Mol Cell Biol. 1991 Mar;11(3):1382–1392. doi: 10.1128/mcb.11.3.1382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Young L. S., Takahashi N., Sprague K. U. Upstream sequences confer distinctive transcriptional properties on genes encoding silkgland-specific tRNAAla. Proc Natl Acad Sci U S A. 1986 Jan;83(2):374–378. doi: 10.1073/pnas.83.2.374. [DOI] [PMC free article] [PubMed] [Google Scholar]

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