Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Sep 26;16(18):8753–8772. doi: 10.1093/nar/16.18.8753

Structure and sequence of the gene for the largest subunit of trypanosomal RNA polymerase III.

J Köck 1, R Evers 1, A W Cornelissen 1
PMCID: PMC338633  PMID: 3174432

Abstract

As the first step in the analysis of the transcription process in the African trypanosome, Trypanosoma brucei, we have started to characterise the trypanosomal RNA polymerases. We have previously described the gene encoding the largest subunit of RNA polymerase II and found that two almost identical RNA polymerase II genes are encoded within the genome of T. brucei. Here we present the identification, cloning and sequence analysis of the gene encoding the largest subunit of RNA polymerase III. This gene contains a single open reading frame encoding a polypeptide with a Mr of 170 kD. In total, eight encoding a polypeptide with a Mr of 170 kD. In total, eight highly conserved regions with significant homology to those previously reported in other eukaryotic RNA polymerase largest subunits were identified. Some of these domains contain functional sites, which are conserved among all eukaryotic largest subunit genes analysed thus far. Since these domains make up a large part of each polypeptide, independent of the RNA polymerase class, these data strongly support the hypothesis that these domains provide a major part of the transcription machinery of the RNA polymerase complex. The additional domains which are uniquely present in the largest subunit of RNA polymerase I and II, respectively, two large hydrophylic insertions and a C-terminal extension, might be a determining factor in specific transcription of the gene classes.

Full text

PDF
8753

Images in this article

8757Image
on p.8757
8765Image
on p.8765

Selected References

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

  1. Ahearn J. M., Jr, Bartolomei M. S., West M. L., Cisek L. J., Corden J. L. Cloning and sequence analysis of the mouse genomic locus encoding the largest subunit of RNA polymerase II. J Biol Chem. 1987 Aug 5;262(22):10695–10705. [PubMed] [Google Scholar]
  2. Alexandre S., Guyaux M., Murphy N. B., Coquelet H., Pays A., Steinert M., Pays E. Putative genes of a variant-specific antigen gene transcription unit in Trypanosoma brucei. Mol Cell Biol. 1988 Jun;8(6):2367–2378. doi: 10.1128/mcb.8.6.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allison L. A., Moyle M., Shales M., Ingles C. J. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell. 1985 Sep;42(2):599–610. doi: 10.1016/0092-8674(85)90117-5. [DOI] [PubMed] [Google Scholar]
  4. Allison L. A., Wong J. K., Fitzpatrick V. D., Moyle M., Ingles C. J. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function. Mol Cell Biol. 1988 Jan;8(1):321–329. doi: 10.1128/mcb.8.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  6. Bartolomei M. S., Corden J. L. Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II. Mol Cell Biol. 1987 Feb;7(2):586–594. doi: 10.1128/mcb.7.2.586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bartolomei M. S., Halden N. F., Cullen C. R., Corden J. L. Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II. Mol Cell Biol. 1988 Jan;8(1):330–339. doi: 10.1128/mcb.8.1.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bateman E., Paule M. R. Regulation of eukaryotic ribosomal RNA transcription by RNA polymerase modification. Cell. 1986 Nov 7;47(3):445–450. doi: 10.1016/0092-8674(86)90601-x. [DOI] [PubMed] [Google Scholar]
  9. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  10. Bernards A., Van der Ploeg L. H., Frasch A. C., Borst P., Boothroyd J. C., Coleman S., Cross G. A. Activation of trypanosome surface glycoprotein genes involves a duplication-transposition leading to an altered 3' end. Cell. 1981 Dec;27(3 Pt 2):497–505. doi: 10.1016/0092-8674(81)90391-3. [DOI] [PubMed] [Google Scholar]
  11. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Biggs J., Searles L. L., Greenleaf A. L. Structure of the eukaryotic transcription apparatus: features of the gene for the largest subunit of Drosophila RNA polymerase II. Cell. 1985 Sep;42(2):611–621. doi: 10.1016/0092-8674(85)90118-7. [DOI] [PubMed] [Google Scholar]
  13. Boothroyd J. C. Antigenic variation in African trypanosomes. Annu Rev Microbiol. 1985;39:475–502. doi: 10.1146/annurev.mi.39.100185.002355. [DOI] [PubMed] [Google Scholar]
  14. Borst P. Discontinuous transcription and antigenic variation in trypanosomes. Annu Rev Biochem. 1986;55:701–732. doi: 10.1146/annurev.bi.55.070186.003413. [DOI] [PubMed] [Google Scholar]
  15. Borst P., van der Ploeg M., van Hoek J. F., Tas J., James J. On the DNA content and ploidy of trypanosomes. Mol Biochem Parasitol. 1982 Jul;6(1):13–23. doi: 10.1016/0166-6851(82)90049-4. [DOI] [PubMed] [Google Scholar]
  16. Broyles S. S., Moss B. Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits. Proc Natl Acad Sci U S A. 1986 May;83(10):3141–3145. doi: 10.1073/pnas.83.10.3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Brun R., Schönenberger Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979 Sep;36(3):289–292. [PubMed] [Google Scholar]
  18. Corden J. L., Cadena D. L., Ahearn J. M., Jr, Dahmus M. E. A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7934–7938. doi: 10.1073/pnas.82.23.7934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Cornelissen A. W., Johnson P. J., Kooter J. M., Van der Ploeg L. H., Borst P. Two simultaneously active VSG gene transcription units in a single Trypanosoma brucei variant. Cell. 1985 Jul;41(3):825–832. doi: 10.1016/s0092-8674(85)80063-5. [DOI] [PubMed] [Google Scholar]
  20. De Lange T., Liu A. Y., Van der Ploeg L. H., Borst P., Tromp M. C., Van Boom J. H. Tandem repetition of the 5' mini-exon of variant surface glycoprotein genes: a multiple promoter for VSG gene transcription? Cell. 1983 Oct;34(3):891–900. doi: 10.1016/0092-8674(83)90546-9. [DOI] [PubMed] [Google Scholar]
  21. Dynan W. S., Tjian R. Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. 1985 Aug 29-Sep 4Nature. 316(6031):774–778. doi: 10.1038/316774a0. [DOI] [PubMed] [Google Scholar]
  22. Earnshaw D. L., Beebee T. J., Gutteridge W. E. Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of alpha-amanitin-resistant and -sensitive enzymes. Biochem J. 1987 Feb 1;241(3):649–655. doi: 10.1042/bj2410649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Earnshaw D. L., Beebee T. J., Gutteridge W. E. Unusual RNA polymerase content of Trypanosoma brucei nuclei. Biochem Biophys Res Commun. 1985 Sep 16;131(2):844–848. doi: 10.1016/0006-291x(85)91316-6. [DOI] [PubMed] [Google Scholar]
  24. Evans R. M., Hollenberg S. M. Zinc fingers: gilt by association. Cell. 1988 Jan 15;52(1):1–3. doi: 10.1016/0092-8674(88)90522-3. [DOI] [PubMed] [Google Scholar]
  25. Falkenburg D., Dworniczak B., Faust D. M., Bautz E. K. RNA polymerase II of Drosophila. Relation of its 140,000 Mr subunit to the beta subunit of Escherichia coli RNA polymerase. J Mol Biol. 1987 Jun 20;195(4):929–937. doi: 10.1016/0022-2836(87)90496-7. [DOI] [PubMed] [Google Scholar]
  26. Gibson W. C., Osinga K. A., Michels P. A., Borst P. Trypanosomes of subgenus Trypanozoon are diploid for housekeeping genes. Mol Biochem Parasitol. 1985 Sep;16(3):231–242. doi: 10.1016/0166-6851(85)90066-0. [DOI] [PubMed] [Google Scholar]
  27. Guyaux M., Cornelissen A. W., Pays E., Steinert M., Borst P. Trypanosoma brucei: a surface antigen mRNA is discontinuously transcribed from two distinct chromosomes. EMBO J. 1985 Apr;4(4):995–998. doi: 10.1002/j.1460-2075.1985.tb03729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hoeijmakers J. H., Borst P., van den Burg J., Weissmann C., Cross G. A. The isolation of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosoma brucei. Gene. 1980 Mar;8(4):391–417. doi: 10.1016/0378-1119(80)90043-8. [DOI] [PubMed] [Google Scholar]
  29. Jay G., Nomura S., Anderson C. W., Khoury G. Identification of the SV40 agnogene product: a DNA binding protein. Nature. 1981 May 28;291(5813):346–349. doi: 10.1038/291346a0. [DOI] [PubMed] [Google Scholar]
  30. Jeffreys A. J., Flavell R. A. The rabbit beta-globin gene contains a large large insert in the coding sequence. Cell. 1977 Dec;12(4):1097–1108. doi: 10.1016/0092-8674(77)90172-6. [DOI] [PubMed] [Google Scholar]
  31. Johnson P. J., Borst P. Mapping of VSG genes on large expression-site chromosomes of Trypanosoma brucei separated by pulsed-field gradient electrophoresis. Gene. 1986;43(3):213–220. doi: 10.1016/0378-1119(86)90209-x. [DOI] [PubMed] [Google Scholar]
  32. Kooter J. M., Borst P. Alpha-amanitin-insensitive transcription of variant surface glycoprotein genes provides further evidence for discontinuous transcription in trypanosomes. Nucleic Acids Res. 1984 Dec 21;12(24):9457–9472. doi: 10.1093/nar/12.24.9457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kooter J. M., van der Spek H. J., Wagter R., d'Oliveira C. E., van der Hoeven F., Johnson P. J., Borst P. The anatomy and transcription of a telomeric expression site for variant-specific surface antigens in T. brucei. Cell. 1987 Oct 23;51(2):261–272. doi: 10.1016/0092-8674(87)90153-x. [DOI] [PubMed] [Google Scholar]
  34. Laird P. W., Kooter J. M., Loosbroek N., Borst P. Mature mRNAs of Trypanosoma brucei possess a 5' cap acquired by discontinuous RNA synthesis. Nucleic Acids Res. 1985 Jun 25;13(12):4253–4266. doi: 10.1093/nar/13.12.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lanham S. M., Godfrey D. G. Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp Parasitol. 1970 Dec;28(3):521–534. doi: 10.1016/0014-4894(70)90120-7. [DOI] [PubMed] [Google Scholar]
  36. Lenardo M. J., Dorfman D. M., Reddy L. V., Donelson J. E. Characterization of the Trypanosoma brucei 5S ribosomal RNA gene and transcript: the 5S rRNA is a spliced-leader-independent species. Gene. 1985;35(1-2):131–141. doi: 10.1016/0378-1119(85)90165-9. [DOI] [PubMed] [Google Scholar]
  37. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  38. Mémet S., Gouy M., Marck C., Sentenac A., Buhler J. M. RPA190, the gene coding for the largest subunit of yeast RNA polymerase A. J Biol Chem. 1988 Feb 25;263(6):2830–2839. [PubMed] [Google Scholar]
  39. Nonet M., Sweetser D., Young R. A. Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II. Cell. 1987 Sep 11;50(6):909–915. doi: 10.1016/0092-8674(87)90517-4. [DOI] [PubMed] [Google Scholar]
  40. Osinga K. A., Swinkels B. W., Gibson W. C., Borst P., Veeneman G. H., Van Boom J. H., Michels P. A., Opperdoes F. R. Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei. EMBO J. 1985 Dec 30;4(13B):3811–3817. doi: 10.1002/j.1460-2075.1985.tb04152.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ovchinnikov YuA, Monastyrskaya G. S., Gubanov V. V., Guryev S. O., Salomatina I. S., Shuvaeva T. M., Lipkin V. M., Sverdlov E. D. The primary structure of E. coli RNA polymerase, Nucleotide sequence of the rpoC gene and amino acid sequence of the beta'-subunit. Nucleic Acids Res. 1982 Jul 10;10(13):4035–4044. doi: 10.1093/nar/10.13.4035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Queen C., Korn L. J. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):581–599. doi: 10.1093/nar/12.1part2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  44. Riva M., Memet S., Micouin J. Y., Huet J., Treich I., Dassa J., Young R., Buhler J. M., Sentenac A., Fromageot P. Isolation of structural genes for yeast RNA polymerases by immunological screening. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1554–1558. doi: 10.1073/pnas.83.6.1554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sentenac A. Eukaryotic RNA polymerases. CRC Crit Rev Biochem. 1985;18(1):31–90. doi: 10.3109/10409238509082539. [DOI] [PubMed] [Google Scholar]
  46. Shea C., Lee M. G., Van der Ploeg L. H. VSG gene 118 is transcribed from a cotransposed pol I-like promoter. Cell. 1987 Aug 14;50(4):603–612. doi: 10.1016/0092-8674(87)90033-x. [DOI] [PubMed] [Google Scholar]
  47. Shea C., Van der Ploeg L. H. Stable variant-specific transcripts of the variant cell surface glycoprotein gene 1.8 expression site in Trypanosoma brucei. Mol Cell Biol. 1988 Feb;8(2):854–859. doi: 10.1128/mcb.8.2.854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  49. Struhl K. Promoters, activator proteins, and the mechanism of transcriptional initiation in yeast. Cell. 1987 May 8;49(3):295–297. doi: 10.1016/0092-8674(87)90277-7. [DOI] [PubMed] [Google Scholar]
  50. Sweetser D., Nonet M., Young R. A. Prokaryotic and eukaryotic RNA polymerases have homologous core subunits. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1192–1196. doi: 10.1073/pnas.84.5.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tait A. Evidence for diploidy and mating in trypanosomes. Nature. 1980 Oct 9;287(5782):536–538. doi: 10.1038/287536a0. [DOI] [PubMed] [Google Scholar]
  52. Tittawella I. Variable RNA polymerase populations in the life cycle of Trypanosoma brucei. FEBS Lett. 1988 Jan 25;227(2):122–126. doi: 10.1016/0014-5793(88)80881-0. [DOI] [PubMed] [Google Scholar]
  53. Van der Ploeg L. H. Discontinuous transcription and splicing in trypanosomes. Cell. 1986 Nov 21;47(4):479–480. doi: 10.1016/0092-8674(86)90608-2. [DOI] [PubMed] [Google Scholar]
  54. Van der Ploeg L. H., Liu A. Y., Michels P. A., De Lange T., Borst P., Majumder H. K., Weber H., Veeneman G. H., Van Boom J. RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes. Nucleic Acids Res. 1982 Jun 25;10(12):3591–3604. doi: 10.1093/nar/10.12.3591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Van der Ploeg L. H., Schwartz D. C., Cantor C. R., Borst P. Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules. Cell. 1984 May;37(1):77–84. doi: 10.1016/0092-8674(84)90302-7. [DOI] [PubMed] [Google Scholar]
  56. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zehring W. A., Lee J. M., Weeks J. R., Jokerst R. S., Greenleaf A. L. The C-terminal repeat domain of RNA polymerase II largest subunit is essential in vivo but is not required for accurate transcription initiation in vitro. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3698–3702. doi: 10.1073/pnas.85.11.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES