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. 1996 May 15;15(10):2593–2603.

Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and catalytic domain, homologous to the family X polymerases, and to other nucleotidyltransferases.

G Martin 1, W Keller 1
PMCID: PMC450192  PMID: 8665867

Abstract

We have tested deletion and substitution mutants of bovine poly(A) polymerase, and have identified a small region that overlaps with a nuclear localization signal and binds to the RNA primer. Systematic mutagenesis of carboxylic amino acids led to the identification of three aspartates that are essential for catalysis. Sequence and secondary structure comparisons of regions surrounding these aspartates with sequences of other polymerases revealed a significant homology to the palm structure of DNA polymerase beta, terminal deoxynucleotidyltransferase and DNA polymerase IV of Saccharomyces cerevisiae, all members of the family X of polymerases. This homology extends as far as cca: tRNA nucleotidyltransferase and streptomycin adenylyltransferase, an antibiotic resistance factor.

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Selected References

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

  1. Aebi M., Kirchner G., Chen J. Y., Vijayraghavan U., Jacobson A., Martin N. C., Abelson J. Isolation of a temperature-sensitive mutant with an altered tRNA nucleotidyltransferase and cloning of the gene encoding tRNA nucleotidyltransferase in the yeast Saccharomyces cerevisiae. J Biol Chem. 1990 Sep 25;265(27):16216–16220. [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Argos P. A sequence motif in many polymerases. Nucleic Acids Res. 1988 Nov 11;16(21):9909–9916. doi: 10.1093/nar/16.21.9909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Astatke M., Grindley N. D., Joyce C. M. Deoxynucleoside triphosphate and pyrophosphate binding sites in the catalytically competent ternary complex for the polymerase reaction catalyzed by DNA polymerase I (Klenow fragment). J Biol Chem. 1995 Jan 27;270(4):1945–1954. doi: 10.1074/jbc.270.4.1945. [DOI] [PubMed] [Google Scholar]
  5. Bairoch A., Boeckmann B. The SWISS-PROT protein sequence data bank, recent developments. Nucleic Acids Res. 1993 Jul 1;21(13):3093–3096. doi: 10.1093/nar/21.13.3093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ballantyne S., Bilger A., Astrom J., Virtanen A., Wickens M. Poly (A) polymerases in the nucleus and cytoplasm of frog oocytes: dynamic changes during oocyte maturation and early development. RNA. 1995 Mar;1(1):64–78. [PMC free article] [PubMed] [Google Scholar]
  7. Beese L. S., Friedman J. M., Steitz T. A. Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. Biochemistry. 1993 Dec 28;32(51):14095–14101. doi: 10.1021/bi00214a004. [DOI] [PubMed] [Google Scholar]
  8. Bienroth S., Keller W., Wahle E. Assembly of a processive messenger RNA polyadenylation complex. EMBO J. 1993 Feb;12(2):585–594. doi: 10.1002/j.1460-2075.1993.tb05690.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bienroth S., Wahle E., Suter-Crazzolara C., Keller W. Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors. J Biol Chem. 1991 Oct 15;266(29):19768–19776. [PubMed] [Google Scholar]
  10. Birney E., Kumar S., Krainer A. R. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 1993 Dec 25;21(25):5803–5816. doi: 10.1093/nar/21.25.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bonner G., Patra D., Lafer E. M., Sousa R. Mutations in T7 RNA polymerase that support the proposal for a common polymerase active site structure. EMBO J. 1992 Oct;11(10):3767–3775. doi: 10.1002/j.1460-2075.1992.tb05462.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  13. Carey J. Gel retardation. Methods Enzymol. 1991;208:103–117. doi: 10.1016/0076-6879(91)08010-f. [DOI] [PubMed] [Google Scholar]
  14. Christofori G., Keller W. 3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP. Cell. 1988 Sep 9;54(6):875–889. doi: 10.1016/s0092-8674(88)91263-9. [DOI] [PubMed] [Google Scholar]
  15. Clos J., Westwood J. T., Becker P. B., Wilson S., Lambert K., Wu C. Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation. Cell. 1990 Nov 30;63(5):1085–1097. doi: 10.1016/0092-8674(90)90511-c. [DOI] [PubMed] [Google Scholar]
  16. Cudny H., Lupski J. R., Godson G. N., Deutscher M. P. Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase. J Biol Chem. 1986 May 15;261(14):6444–6449. [PubMed] [Google Scholar]
  17. Date T., Yamamoto S., Tanihara K., Nishimoto Y., Matsukage A. Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding. Biochemistry. 1991 May 28;30(21):5286–5292. doi: 10.1021/bi00235a023. [DOI] [PubMed] [Google Scholar]
  18. Davies J. F., 2nd, Almassy R. J., Hostomska Z., Ferre R. A., Hostomsky Z. 2.3 A crystal structure of the catalytic domain of DNA polymerase beta. Cell. 1994 Mar 25;76(6):1123–1133. doi: 10.1016/0092-8674(94)90388-3. [DOI] [PubMed] [Google Scholar]
  19. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  20. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  22. Dong Q., Wang T. S. Mutational studies of human DNA polymerase alpha. Lysine 950 in the third most conserved region of alpha-like DNA polymerases is involved in binding the deoxynucleoside triphosphate. J Biol Chem. 1995 Sep 15;270(37):21563–21570. doi: 10.1074/jbc.270.37.21563. [DOI] [PubMed] [Google Scholar]
  23. Gibson T. J., Rice P. M., Thompson J. D., Heringa J. KH domains within the FMR1 sequence suggest that fragile X syndrome stems from a defect in RNA metabolism. Trends Biochem Sci. 1993 Sep;18(9):331–333. doi: 10.1016/0968-0004(93)90068-x. [DOI] [PubMed] [Google Scholar]
  24. Gibson T. J., Thompson J. D., Heringa J. The KH domain occurs in a diverse set of RNA-binding proteins that include the antiterminator NusA and is probably involved in binding to nucleic acid. FEBS Lett. 1993 Jun 21;324(3):361–366. doi: 10.1016/0014-5793(93)80152-k. [DOI] [PubMed] [Google Scholar]
  25. Gribskov M., McLachlan A. D., Eisenberg D. Profile analysis: detection of distantly related proteins. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4355–4358. doi: 10.1073/pnas.84.13.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gunderson S. I., Beyer K., Martin G., Keller W., Boelens W. C., Mattaj L. W. The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymerase. Cell. 1994 Feb 11;76(3):531–541. doi: 10.1016/0092-8674(94)90116-3. [DOI] [PubMed] [Google Scholar]
  27. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  28. Haynes S. R. The RNP motif protein family. New Biol. 1992 May;4(5):421–429. [PubMed] [Google Scholar]
  29. Higgins D. G., Fuchs R., Stoehr P. J., Cameron G. N. The EMBL Data Library. Nucleic Acids Res. 1992 May 11;20 (Suppl):2071–2074. doi: 10.1093/nar/20.suppl.2071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Holm L., Sander C. DNA polymerase beta belongs to an ancient nucleotidyltransferase superfamily. Trends Biochem Sci. 1995 Sep;20(9):345–347. doi: 10.1016/s0968-0004(00)89071-4. [DOI] [PubMed] [Google Scholar]
  31. Ito J., Braithwaite D. K. Compilation and alignment of DNA polymerase sequences. Nucleic Acids Res. 1991 Aug 11;19(15):4045–4057. doi: 10.1093/nar/19.15.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Joyce C. M., Steitz T. A. Function and structure relationships in DNA polymerases. Annu Rev Biochem. 1994;63:777–822. doi: 10.1146/annurev.bi.63.070194.004021. [DOI] [PubMed] [Google Scholar]
  33. Keller W., Bienroth S., Lang K. M., Christofori G. Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3' processing signal AAUAAA. EMBO J. 1991 Dec;10(13):4241–4249. doi: 10.1002/j.1460-2075.1991.tb05002.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Keller W. No end yet to messenger RNA 3' processing! Cell. 1995 Jun 16;81(6):829–832. doi: 10.1016/0092-8674(95)90001-2. [DOI] [PubMed] [Google Scholar]
  35. Kenan D. J., Query C. C., Keene J. D. RNA recognition: towards identifying determinants of specificity. Trends Biochem Sci. 1991 Jun;16(6):214–220. doi: 10.1016/0968-0004(91)90088-d. [DOI] [PubMed] [Google Scholar]
  36. Kim Y., Eom S. H., Wang J., Lee D. S., Suh S. W., Steitz T. A. Crystal structure of Thermus aquaticus DNA polymerase. Nature. 1995 Aug 17;376(6541):612–616. doi: 10.1038/376612a0. [DOI] [PubMed] [Google Scholar]
  37. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  38. Korolev S., Nayal M., Barnes W. M., Di Cera E., Waksman G. Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9264–9268. doi: 10.1073/pnas.92.20.9264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. LaCasse E. C., Lefebvre Y. A. Nuclear localization signals overlap DNA- or RNA-binding domains in nucleic acid-binding proteins. Nucleic Acids Res. 1995 May 25;23(10):1647–1656. doi: 10.1093/nar/23.10.1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lingner J., Kellermann J., Keller W. Cloning and expression of the essential gene for poly(A) polymerase from S. cerevisiae. Nature. 1991 Dec 12;354(6353):496–498. doi: 10.1038/354496a0. [DOI] [PubMed] [Google Scholar]
  41. Manley J. L. A complex protein assembly catalyzes polyadenylation of mRNA precursors. Curr Opin Genet Dev. 1995 Apr;5(2):222–228. doi: 10.1016/0959-437x(95)80012-3. [DOI] [PubMed] [Google Scholar]
  42. Mikaelian I., Sergeant A. A general and fast method to generate multiple site directed mutations. Nucleic Acids Res. 1992 Jan 25;20(2):376–376. doi: 10.1093/nar/20.2.376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nagai K., Oubridge C., Ito N., Avis J., Evans P. The RNP domain: a sequence-specific RNA-binding domain involved in processing and transport of RNA. Trends Biochem Sci. 1995 Jun;20(6):235–240. doi: 10.1016/s0968-0004(00)89024-6. [DOI] [PubMed] [Google Scholar]
  44. Ollis D. L., Kline C., Steitz T. A. Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase. 1985 Feb 28-Mar 6Nature. 313(6005):818–819. doi: 10.1038/313818a0. [DOI] [PubMed] [Google Scholar]
  45. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pedersen L. C., Benning M. M., Holden H. M. Structural investigation of the antibiotic and ATP-binding sites in kanamycin nucleotidyltransferase. Biochemistry. 1995 Oct 17;34(41):13305–13311. doi: 10.1021/bi00041a005. [DOI] [PubMed] [Google Scholar]
  47. Pelletier H., Sawaya M. R., Kumar A., Wilson S. H., Kraut J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science. 1994 Jun 24;264(5167):1891–1903. [PubMed] [Google Scholar]
  48. Poch O., Sauvaget I., Delarue M., Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989 Dec 1;8(12):3867–3874. doi: 10.1002/j.1460-2075.1989.tb08565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Polesky A. H., Steitz T. A., Grindley N. D., Joyce C. M. Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli. J Biol Chem. 1990 Aug 25;265(24):14579–14591. [PubMed] [Google Scholar]
  50. Prasad R., Widen S. G., Singhal R. K., Watkins J., Prakash L., Wilson S. H. Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme. Nucleic Acids Res. 1993 Nov 25;21(23):5301–5307. doi: 10.1093/nar/21.23.5301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Raabe T., Bollum F. J., Manley J. L. Primary structure and expression of bovine poly(A) polymerase. Nature. 1991 Sep 19;353(6341):229–234. doi: 10.1038/353229a0. [DOI] [PubMed] [Google Scholar]
  52. Raabe T., Murthy K. G., Manley J. L. Poly(A) polymerase contains multiple functional domains. Mol Cell Biol. 1994 May;14(5):2946–2957. doi: 10.1128/mcb.14.5.2946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  54. Rost B., Sander C. Combining evolutionary information and neural networks to predict protein secondary structure. Proteins. 1994 May;19(1):55–72. doi: 10.1002/prot.340190108. [DOI] [PubMed] [Google Scholar]
  55. Rost B., Sander C. Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol. 1993 Jul 20;232(2):584–599. doi: 10.1006/jmbi.1993.1413. [DOI] [PubMed] [Google Scholar]
  56. Sakon J., Liao H. H., Kanikula A. M., Benning M. M., Rayment I., Holden H. M. Molecular structure of kanamycin nucleotidyltransferase determined to 3.0-A resolution. Biochemistry. 1993 Nov 16;32(45):11977–11984. doi: 10.1021/bi00096a006. [DOI] [PubMed] [Google Scholar]
  57. Sawaya M. R., Pelletier H., Kumar A., Wilson S. H., Kraut J. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. Science. 1994 Jun 24;264(5167):1930–1935. doi: 10.1126/science.7516581. [DOI] [PubMed] [Google Scholar]
  58. Shaw K. J., Rather P. N., Hare R. S., Miller G. H. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev. 1993 Mar;57(1):138–163. doi: 10.1128/mr.57.1.138-163.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sousa R., Chung Y. J., Rose J. P., Wang B. C. Crystal structure of bacteriophage T7 RNA polymerase at 3.3 A resolution. Nature. 1993 Aug 12;364(6438):593–599. doi: 10.1038/364593a0. [DOI] [PubMed] [Google Scholar]
  60. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  61. Thuresson A. C., Aström J., Aström A., Grönvik K. O., Virtanen A. Multiple forms of poly(A) polymerases in human cells. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):979–983. doi: 10.1073/pnas.91.3.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wahle E. 3'-end cleavage and polyadenylation of mRNA precursors. Biochim Biophys Acta. 1995 Apr 4;1261(2):183–194. doi: 10.1016/0167-4781(94)00248-2. [DOI] [PubMed] [Google Scholar]
  63. Wahle E. A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation. Cell. 1991 Aug 23;66(4):759–768. doi: 10.1016/0092-8674(91)90119-j. [DOI] [PubMed] [Google Scholar]
  64. Wahle E., Lustig A., Jenö P., Maurer P. Mammalian poly(A)-binding protein II. Physical properties and binding to polynucleotides. J Biol Chem. 1993 Feb 5;268(4):2937–2945. [PubMed] [Google Scholar]
  65. Wahle E., Martin G., Schiltz E., Keller W. Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase. EMBO J. 1991 Dec;10(13):4251–4257. doi: 10.1002/j.1460-2075.1991.tb05003.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wahle E. Purification and characterization of a mammalian polyadenylate polymerase involved in the 3' end processing of messenger RNA precursors. J Biol Chem. 1991 Feb 15;266(5):3131–3139. [PubMed] [Google Scholar]
  67. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  68. Yang W., Steitz T. A. Recombining the structures of HIV integrase, RuvC and RNase H. Structure. 1995 Feb 15;3(2):131–134. doi: 10.1016/s0969-2126(01)00142-3. [DOI] [PubMed] [Google Scholar]
  69. Zhelkovsky A. M., Kessler M. M., Moore C. L. Structure-function relationships in the Saccharomyces cerevisiae poly(A) polymerase. Identification of a novel RNA binding site and a domain that interacts with specificity factor(s). J Biol Chem. 1995 Nov 3;270(44):26715–26720. doi: 10.1074/jbc.270.44.26715. [DOI] [PubMed] [Google Scholar]

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