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. 1990 Mar 25;18(6):1541–1548. doi: 10.1093/nar/18.6.1541

Saturation mutagenesis of the Drosophila tRNA(Arg) gene B-Box intragenic promoter element: requirements for transcription activation and stable complex formation.

B A Gaëta 1, S J Sharp 1, T S Stewart 1
PMCID: PMC330523  PMID: 2109308

Abstract

Transcription of eukaryotic tRNA genes is dependent on the A- and B-Box internal control regions (ICRs) and the upstream transcription modulatory region. The B-Box ICR spans nucleotides 52 to 62 and directs the primary binding of transcription factor C as the first step in the formation of a transcription complex. The conservation of the sequence of the B-Box in all tRNA species reflects its importance in both the expression of the gene and the processing, structure and function of the gene product. In order to identify the nucleotides essential to the promoter function of the B-Box ICR, site-directed mutagenesis was used to generate all the possible single point mutations at positions 52 to 58, 61 and 62 of a Drosophila melanogaster tRNA(Arg) gene. The effect of these mutations on gene transcription was evaluated using in vitro transcription and template exclusion competition assays. Optimal activity was displayed by the wild type tDNA(Arg) B-Box sequence but several other sequences supported in vitro transcription at wild type levels. The majority of mutants, however, showed lower efficiency in the in vitro transcription assay. Of the single point mutations, those at positions 53, 55, and 56 had a critical effect on gene function in Drosophila and HeLa transcription extracts and transcription factor interaction most likely requires base contacts at these positions. Since the effect of several of the point mutations cannot be explained in terms of possible major or minor groove contributions the possibility is raised that local DNA geometry also is an important determinant in specifying B-Box function.

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Selected References

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

  1. Aggarwal A. K., Rodgers D. W., Drottar M., Ptashne M., Harrison S. C. Recognition of a DNA operator by the repressor of phage 434: a view at high resolution. Science. 1988 Nov 11;242(4880):899–907. doi: 10.1126/science.3187531. [DOI] [PubMed] [Google Scholar]
  2. Allison D. S., Goh S. H., Hall B. D. The promoter sequence of a yeast tRNAtyr gene. Cell. 1983 Sep;34(2):655–664. doi: 10.1016/0092-8674(83)90398-7. [DOI] [PubMed] [Google Scholar]
  3. Baker R. E., Gabrielsen O., Hall B. D. Effects of tRNATyr point mutations on the binding of yeast RNA polymerase III transcription factor C. J Biol Chem. 1986 Apr 25;261(12):5275–5282. [PubMed] [Google Scholar]
  4. 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]
  5. Burke D. J., Söll D. Functional analysis of fractionated Drosophila Kc cell tRNA gene transcription components. J Biol Chem. 1985 Jan 25;260(2):816–823. [PubMed] [Google Scholar]
  6. Camier S., Gabrielsen O., Baker R., Sentenac A. A split binding site for transcription factor tau on the tRNA3Glu gene. EMBO J. 1985 Feb;4(2):491–500. doi: 10.1002/j.1460-2075.1985.tb03655.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dean N., Berk A. J. Ordering promoter binding of class III transcription factors TFIIIC1 and TFIIIC2. Mol Cell Biol. 1988 Aug;8(8):3017–3025. doi: 10.1128/mcb.8.8.3017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Folk W. R., Hofstetter H. A detailed mutational analysis of the eucaryotic tRNAmet1 gene promoter. Cell. 1983 Jun;33(2):585–593. doi: 10.1016/0092-8674(83)90439-7. [DOI] [PubMed] [Google Scholar]
  10. Folk W. R., Hofstetter H., Birnstiel M. L. Some bacterial tRNA genes are transcribed by eukaryotic RNA polymerase III. Nucleic Acids Res. 1982 Nov 25;10(22):7153–7162. doi: 10.1093/nar/10.22.7153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Jordan S. R., Pabo C. O. Structure of the lambda complex at 2.5 A resolution: details of the repressor-operator interactions. Science. 1988 Nov 11;242(4880):893–899. doi: 10.1126/science.3187530. [DOI] [PubMed] [Google Scholar]
  16. Koudelka G. B., Harrison S. C., Ptashne M. Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro. 1987 Apr 30-May 6Nature. 326(6116):886–888. doi: 10.1038/326886a0. [DOI] [PubMed] [Google Scholar]
  17. Kramer B., Kramer W., Fritz H. J. Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell. 1984 Oct;38(3):879–887. doi: 10.1016/0092-8674(84)90283-6. [DOI] [PubMed] [Google Scholar]
  18. Kramer W., Fritz H. J. Oligonucleotide-directed construction of mutations via gapped duplex DNA. Methods Enzymol. 1987;154:350–367. doi: 10.1016/0076-6879(87)54084-8. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lofquist A. K., Garcia A. D., Sharp S. J. A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes. Mol Cell Biol. 1988 Oct;8(10):4441–4449. doi: 10.1128/mcb.8.10.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marzouki N., Camier S., Ruet A., Moenne A., Sentenac A. Selective proteolysis defines two DNA binding domains in yeast transcription factor tau. Nature. 1986 Sep 11;323(6084):176–178. doi: 10.1038/323176a0. [DOI] [PubMed] [Google Scholar]
  22. McCall M., Brown T., Hunter W. N., Kennard O. The crystal structure of d(GGATGGGAG): an essential part of the binding site for transcription factor IIIA. Nature. 1986 Aug 14;322(6080):661–664. doi: 10.1038/322661a0. [DOI] [PubMed] [Google Scholar]
  23. McClarin J. A., Frederick C. A., Wang B. C., Greene P., Boyer H. W., Grable J., Rosenberg J. M. Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution. Science. 1986 Dec 19;234(4783):1526–1541. doi: 10.1126/science.3024321. [DOI] [PubMed] [Google Scholar]
  24. Miyada C. G., Wallace R. B. Oligonucleotide hybridization techniques. Methods Enzymol. 1987;154:94–107. doi: 10.1016/0076-6879(87)54072-1. [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. Otwinowski Z., Schevitz R. W., Zhang R. G., Lawson C. L., Joachimiak A., Marmorstein R. Q., Luisi B. F., Sigler P. B. Crystal structure of trp repressor/operator complex at atomic resolution. Nature. 1988 Sep 22;335(6188):321–329. doi: 10.1038/335321a0. [DOI] [PubMed] [Google Scholar]
  27. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  28. Rhodes D., Klug A. An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA. Cell. 1986 Jul 4;46(1):123–132. doi: 10.1016/0092-8674(86)90866-4. [DOI] [PubMed] [Google Scholar]
  29. Rich A., RajBhandary U. L. Transfer RNA: molecular structure, sequence, and properties. Annu Rev Biochem. 1976;45:805–860. doi: 10.1146/annurev.bi.45.070176.004105. [DOI] [PubMed] [Google Scholar]
  30. Richmond T. J. Protein--DNA interaction. Another folding problem? Nature. 1987 Mar 5;326(6108):18–19. doi: 10.1038/326018a0. [DOI] [PubMed] [Google Scholar]
  31. Schaack J., Sharp S., Dingermann T., Burke D. J., Cooley L., Söll D. The extent of a eukaryotic tRNA gene. 5'- and 3'-flanking sequence dependence for transcription and stable complex formation. J Biol Chem. 1984 Feb 10;259(3):1461–1467. [PubMed] [Google Scholar]
  32. 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]
  33. Seeman N. C., Rosenberg J. M., Rich A. Sequence-specific recognition of double helical nucleic acids by proteins. Proc Natl Acad Sci U S A. 1976 Mar;73(3):804–808. doi: 10.1073/pnas.73.3.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Sharp S. J., Schaack J., Cooley L., Burke D. J., Söll D. Structure and transcription of eukaryotic tRNA genes. CRC Crit Rev Biochem. 1985;19(2):107–144. doi: 10.3109/10409238509082541. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Sprinzl M., Hartmann T., Weber J., Blank J., Zeidler R. Compilation of tRNA sequences and sequences of tRNA genes. Nucleic Acids Res. 1989;17 (Suppl):r1–172. doi: 10.1093/nar/17.suppl.r1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stewart T. S., Söll D., Sharp S. Point mutations in the 5' ICR and anticodon region of a Drosophila tRNAArg gene decrease in vitro transcription. Nucleic Acids Res. 1985 Jan 25;13(2):435–447. doi: 10.1093/nar/13.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stillman D. J., Geiduschek E. P. Differential binding of a S. cerevisiae RNA polymerase III transcription factor to two promoter segments of a tRNA gene. EMBO J. 1984 Apr;3(4):847–853. doi: 10.1002/j.1460-2075.1984.tb01895.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Traboni C., Ciliberto G., Cortese R. Mutations in Box B of the promoter of a eucaryotic tRNAPro gene affect rate of transcription, processing, and stability of the transcripts. Cell. 1984 Jan;36(1):179–187. doi: 10.1016/0092-8674(84)90087-4. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. von Hippel P. H., Berg O. G. On the specificity of DNA-protein interactions. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1608–1612. doi: 10.1073/pnas.83.6.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]

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