Изучение взаимодействий РНК-полимеразы с однонитевыми и двунитевыми ДНК-аптамерами тема диссертации и автореферата по ВАК РФ 03.00.03, кандидат биологических наук Феклистов, Андрей Владимирович

• Феклистов, Андрей Владимирович
• кандидат биологических наук
• 2005

Заключение диссертации по теме «Молекулярная биология», Феклистов, Андрей Владимирович

Выводы

1. Получены и охарактеризованы высоаффинные ДНК-аптамеры к кор-РНК-полимеразам Е. coli и Т. aquaticus, которые являются эффективными ингибиторами транскрипции. При помощи аптамеров показано, что фактор GreB вызывает конформационные перестройки внутри главного канала РНКП.

2. Осуществлён поиск ДНК-аптамеров к изолированной а-субъединице РНК-полимеразы Т. aquaticus. Отобранные аптамеры содержат последовательность нематричной нити -10 области бактериальных промоторов. Таким образом, свободная а-субъединица способна узнавать промоторную последовательность в отсутствие кор-фермента РНК-полимеразы. Аптамеры взаимодействуют с РНК-полимеразой с высокой аффинностью и являются эффективными ингибиторами синтеза РНК in vitro.

3. Двунитевой фрагмент ДНК, содержащий последовательность аптамера к ст-субъединице Т. aquaticus, является промотором нового типа, не содержащим -35 области и TG-элемента и специфичным для холофермента РНК-полимеразы Т. aquaticus. Для узнавания аптамерного промотора необходимо наличие расширенного мотива -10 области TATAATGGGA, содержащего ранее не известный GGGA-элемент.

4. Однонитевой ДНК-аптамер к изолированной <т-субъединице Т. aquaticus служит матрицей для специфической ст-зависимой инициации транскрипции РНК-полимеразой Т. aquaticus.

Список работ, опубликованных по теме диссертации

Kulbachinskiy A., Feklistov A., Krasheninnikov I., Goldfarb A., Nikiforov V. 2004. Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, o-subunit and GreB. Eur. J. Biochem. 271: 4921-4931.

Feklistov A., Kulbachinsky A., Nikiforov V. DNA aptamers unveil sequence specificity of free sigma factor. FASEB Summer Research Conference "Transcription initiation in Prokaryotes". June 12-17, 2004, Vermont Academy in Saxtons River, Vermont, USA.

Феклистов А. В., Кульбачинский А. В., Никифоров В. Г. ДПК-связывающая специфичность ст-субъединицы РНК-полимеразы Т. aquaticus, изученная методом SELEX. Конференция молодых учёных, аспирантов и студентов по молекулярной биологии и генетике, посвященная 50-летию открытия двойной спирали ДНК, а также 30-ой годовщине Института молекулярной биологии и генетики академии наук Украины. 25-27 сентября 2003 г., Украина, Киев.

Благодарности

Автор благодарит А. В. Кульбачинского и В. Г. Никифорова за организацию работы, руководство, советы и критику, А. Гольдфарба и И.А. Крашенинникова за создание условий для работы.

Автор благодарен сотрудникам ЛМГМ, ОМГЖ ИМГ РАН и лаборатории молекулярной биофизики Рокфеллеровского университета, а также коллективу Кафедры молекулярной биологии Биологического факультета МГУ за внимание, советы и помощь в работе.

Автор очень признателен Н. Наумовой, С. Колбу, А. Кульбачинскому, Н. Севостьяновой, В. Вагину, А. и П. Моториным, М. и М. Фрих-Харам, А. Мустаеву, И. Аджубею, И. Абрамсон, А. Машину, О. Глебову, А. Ленцу, В. Эпштейну, Д. Альперну, Л. Вестблэйд и Д. Мюррей за неоценимую дружескую помощь и поддержку в процессе выполнения работы.

Список литературы диссертационного исследования кандидат биологических наук Феклистов, Андрей Владимирович, 2005 год

1. Савинкова Л.К., Кнорре В.Л., Салганик Р.И. 1983. Избирательное связывание определенных нуклеотидных последовательностей промоторов генов Escherichia coli и фага Т7 с соответствующими РНК-полимеразами. Докл. АН СССР 270: 1501-1504.

2. Тулохонов И. И., Савинкова Л. К., Pay В. А., Pap В. А. 1999. Взаимодействие изолированной субъединицы <т-70 с олигодезоксинуклеотидами, идентичными участкам -10 и —35 промотора spc. Молекулярная Биология, 33: 169 — 173.

3. Bar-Nahum G., Nudler Е. 2001. Isolation and characterization of sigma(70)-retaining transcription elongation complexes from Escherichia coli. Cell, 2001,106: 443-451.

4. Barne K. A., Bown J. A., Busby S. J., Minchin S. D. 1997. Region 2.5 of the Escherichia coli RNA polymerase sigma70 subunit is responsible for the recognition of the 'extended-10' motif at promoters. EMBOJ., 16: 4034-4040.

5. Blackburn E. H. 1975. Transcription by Escherichia coli RNA polymerase of a single-stranded fragment by 0X174 bacteriophage DNA 48 residues in length. J. Mol. Biol. 93: 367-370.

6. Bock L. C., Griffin L. C., Latham J. A., Vermaas E. H., Toole J. J. 1992. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature, 355: 564-566.

7. Breaker R. R., Banerji A., Joyce G. F. 1994. Continuous in vitro evolution of bacteriophage RNA polymerase promoters. Biochemistry, 33: 11980-11986.

8. Burden D.A., OsherofTN. 1999. In vitro evolution of preferred topoisomerase II DNA cleavage sites. J. Biol. Chem., 274: 5227-5235.

9. Burgess R. R., Travers A. A., Dunn J. J., Bautz E. K. 1969. Factor stimulating transcription by RNA polymerase. Nature, 221: 43-46.

10. Callaci S., Heyduk T. 1998. Conformation and DNA binding properties of a single-stranded DNA binding region of sigma 70 subunit from Escherichia coli RNA polymerase are modulated by an interaction with the core enzyme. Biochemistry, 37: 3312-3320.

11. Campbell E. A., Korzheva N., Mustaev A., Murakami K., Nair S., Goldfarb A., Darst S. A. 2001. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell, 104: 901912.

12. Campbell E. A., Muzzin O., Chlenov M., Sun J. L., Olson C. A., Weinman O., Trester-Zedlitz M. L., Darst S. A. 2002. Structure of the Bacterial RNA Polymerase Promoter Specificity sigma Subunit. Mol. Cell, 9: 527-539.

13. Chan В., Spassky A., Busby S. 1990. The organization of open complexes between Escherichia coli RNA polymerase and DNA fragments carrying promoters either with or without consensus -35 region sequences. Biochem. J., I: 141-148.

14. Chan C. L., Gross C. A. 2001. The anti-initial transcribed sequence, a portable sequence that impedes promoter escape, requires sigma70 for function. J. Biol. Chem., 276: 38201-38209.

15. Colland F., Orsini G., Brody E.N., Buc H., Kolb A. 1998. The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters. Mol. Microbiol., 27: 819-829.

16. Convery, M.A., Rowsell, S., Stonehouse, N.J., Ellington, A.D., Hirao, I., Murray, J.B., Peabody, D.S., Phillips, S.E., Stockley, P.G. 1998. Crystal structure of an RNA aptamer-protein complex at 2.8 A resolution. Nat. Struct. Biol., 5: 133-139.

17. Daube S. S and von Hippel P. H. 1992. Functional transcription elongation complexes from synthetic RNA-DNA bubble duplexes. Science, 258: 1320-4.

18. Daube S. S and von Hippel P. H. 1994. RNA displacement pathways during transcription from synthetic RNA-DNA bubble duplexes. Biochemistry 33: 340-7

19. Dedrick R. L and Chamberlin M. J. 1985. Studies on transcription of 3'-extended templates by mammalian RNA polymerase II. Parameters that affect the initiation and elongation reactions. Biochemistry. 24: 2245-53.

20. Dombroski, A. J., Walter W. A., Record M. T., Jr., Siegele D. A., Gross C. A. 1992. Polypeptides, containing higly conserved regions of transcription initiation factor cr70 exhibit speficity of binding to promoter DNA. Cell, 70: 501-512.

21. Dombroski A. J. 1997. Recognition of the -10 promoter sequence by a partial polypeptide of sigma70 in vitro. J. Biol. Chem., 111-. 3487-3494.

22. Eaton B. E., Gold L., Zichi D. A. 1995. Let's get specific: the relationship between specificity and affinity. Chemistry and Biology, 2: 633 — 638.

23. Eichenberger P., Dethiollaz S., Buc H., Geiselmann J. 1997. Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting. Proc. Natl. Acad. Sei. USA, 94: 9022-9027.

24. Ellington A. D. and Szostak J. W. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature, 346: 818-22

25. Estrem, S.T., Gaal, T., Ross, W., Gourse, R.L. 1998. Identification of an UP element consensus sequence for bacterial promoters. Proc. Natl. Acad. Sei., USA 95: 9761-9766.

26. Falashi A., Adler J., Khorana H. G. 1963. Chemical synthesized deoxypolynucleotides as templates for ribonucleic acid polymerase. J. Biol. Chem. 238: 3080-3085.

27. Fedoriw A. M., Liu H., Anderson V. E., DeHaseth P. L. 1998. Equilibrium and kinetic parameters of the sequence-specific interaction of Escherichia coli RNA polymerase with nontemplate strand oligodeoxyribonucleotides. Biochemistry 37: 11971-11979.

28. Fenton M. S., Gralla J. D. 2001. Function of the bacterial TATAAT -10 element as single-stranded DNA during RNA polymerase isomerization. Proc. Natl. Acad. Sei. USA, 98: 90209025.

29. Fenton M. S., Lee S. J., Gralla J. D. 2000. Escherichia coli promoter opening and -10 recognition: mutational analysis of sigma70. EMBOJ., 19: 1130-1137.

30. Gold L., Brown D., He Y., Shtatland T., Singer B. S., Wu Y. 1997. From oligonucleotide shapes to genomic SELEX: Novel biological regulatory loops. Proc. Natl. Acad. Sei., 94: 59-64.

31. Gold L., Polisky B., Uhlenbeck O., Yarus M. 1995. Diversity of oligonucleotide functions. Annu. Rev. Biochem., 64: 763-797.

32. Gribskov M., Burgess R.R. 1983. Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene 26: 109-118.

33. Guo Y., Gralla J. D. 1998. Promoter opening via a DNA fork junction binding activity. Proc. Natl. Acad. Sei. USA, 95: 11655-11660.

34. Hale S. P., Schimmel P. 1996. Protein synthesis editing by a DNA aptamer. Proc. Natl. Acad. Sei. USA., 93: 2755-2758.

35. He, Y.Y., Stockley, P.G., Gold, L. 1996. In vitro evolution of the DNA binding sites of Escherichia coli methionine repressor, MetJ. J. Mol. Biol., 255: 55-66.

36. Helmann J. D. 1995. Compilation and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res., 23: 2351-2360.

37. Hermann T., Patel D. J. 2000. Adaptive recognition by nucleic acid aptamers. Science, 287: 820825.

38. Hesselberth J. R., Miller D., Robertus J., Ellington A. D. 2000. In vitro selection of RNA molecules that inhibit the activity of ricin A-chain. J. Biol. Chem., 275: 4937-4942.

39. Higashitani A, Higashitani N, Horiuchi K. 1997. Minus-strand origin of filamentous phage versus transcriptional promoters in recognition of RNA polymerase. Proc. Natl. Acad. Sei. USA. 94: 2909-14

40. Jaeger J., Restle T., Steitz T. A. 1998. The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor EMBOJ., 17: 4535-4542.

41. Jenison R. D., Gill S. C., Pardi A., Polisky B. 1994. High-resolution molecular discrimination by RNA. Science, 263: 1425-1429.

42. Jeon C., Agarwal K. 1996. Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS. Proc. Natl. Acad. Sei. USA. 93: 13677-82

43. Jing N., Hogan M. E. 1998. Structure-activity of tetrad-forming oligonucleotides as a potent anti-HIV therapeutic drug. J. Biol. Chem., 273: 34992-34999.

44. Jing N., Marchand С., Liu J., Mitra R., Hogan M. E., Pommier Y. 2000. Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in vitro. J. Biol. Chem., 275: 2146021467.

45. Juang Y. L., Helmann J. D. 1994. A promoter melting region in the primary sigma factor of Bacillus subtilis. Identification of functionally important aromatic amino acids. J. Mol. Biol, 235: 1470-1488.

46. Kaguni J. M and Kornberg A. 1982. The rho subunit of RNA polymerase holoenzyme confers specificity in priming M13 viral DNA replication. J. Biol. Chem. 257: 5437-43.

47. Kimoto M., Sakamoto K., Shirouzu M., Hirao I., Yokoyama S. 1998. RNA aptamers that specifically bind to the Ras-binding domain of Raf-1. FEBS Lett., 441: 322-326.

48. Korzheva N., Mustaev A., Nudler E., Nikiforov V., Goldfarb A. 1998. Mechanistic model of the elongation complex of Escherichia coli RNA polymerase. Cold Spring Harb. Symp. Quant. Biol. 63: 337-345.

49. Korzheva N., Mustaev A., Kozlov M., Malhotra A., Nikiforov V., Goldfarb A., Darst S.A. 2000. A structural model of transcription elongation. Science. 289: 619-625.

50. Korzheva N., Mustaev A. 2001. Transcription elongation complex: structure and function. Current Opinion in Microbiology, 4: 119-125.

51. Kubik M. F, Stephens A. W., Schneider D., Marlar R. A., Tasset D. 1994. High-affinity RNA ligands to human alpha-thrombin. Nucleic Acids Res., 22: 2619-2626.

52. Kudo Т., Doi R. H. 1981. Free sigma factor of Escherichia coli RNA polymerase can bind to DNA. J. Biol. Chem., 256: 9778-9781.

53. Kudo Т., Jaffe D., Doi R. H. 1981. Free sigma subunit of Bacillus subtilis RNA polymerase binds to DNA. Mol. Gen. Genet., 181: 63-68.

54. Kulbachinskiy A., Mustaev A., Goldfarb A., Nikiforov V. 1999. Interaction with free beta' subunit unmasks DNA-binding domain of RNA polymerase sigma subunit. FEBS Lett., 454: 7174.

55. Kulbachinskiy A., Feklistov A., Krasheninnikov I., Goldfarb A., Nikiforov V. 2004. Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, o-subunit and GreB. Eur. J. Biochem. Ill: 4921-4931.

56. Marr M. T., Roberts J. W. 1997. Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide. Science., 276: 1258-1260.

57. Masukata H. and Tomizawa J. 1990. A mechanism of formation of a persistent hybrid between elongating RNA and template DNA. Cell. 62: 331-8.

58. McClure W. R. 1985. Mechanizm and control of transcription initiation in prokariotes. Ann. Rev. Biochem., 54: 171-204.

59. Mecsas J., Cowing D. W., Gross C. A. 1991. Development of RNA polymerase-promoter contacts during open complex formation. J. Mol. Biol., 220: 585-597.

60. Meima R., Rothfuss H. M., Gewin L., Lidstrom M. E. 2001. Promoter cloning in the radioresistant bacterium Deinococcus radiodurans. J. Bacteriol., 183: 3169-3175

61. Michaud M., Jourdan E., Ravelet C., Villet A., Ravel A., Grosset C., Peyrin E. 2004. Immobilized DNA aptamers as target-specific chiral stationary phases for resolution of nucleoside and amino acid derivative enantiomers. Anal. Chem. 76: 1015-20.

62. Minakhin L., Nechaev S., Campbell E. A., Severinov K. 2001. Recombinant Thermus aquaticus RNA polymerase, a new tool for structure-based analysis of transcription. J. Bacteriol., 183: 7176.

63. Morris K.N., Jensen K.B., Jul in C.M., Weil M., Gold, L. 1998. High affinity ligands from in vitro selection: complex targets. Proc. Natl. Acad. Sei. USA, 95: 2902-2907.

64. Murakami K. S., Masuda S., Darst S. A. 2002a. Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Ä resolution. Science, 296: 1280-1284.

65. Murakami K. S., Masuda S., Campbell E. A., Muzzin O., Darst S. A. 2002b. Structural Basis of Transcription Initiation: An RNA Polymerase Holoenzyme-DNA Complex. Science, 296: 12851290.

66. Naryshkin N., Revyakin A., Kim Y., Mekler V., Ebright R. H. 2000. Structural organization of the RNA polymerase-promoter open complex. Cell, 101: 601-611.

67. Pan, W., Craven, R.C., Qiu, Q., Wilson, C.B., Wills, J.W., Golovine, S., Wang, J.F. 1995. Isolation of virus-neutralizing RNAs from a large pool of random sequences. Proc. Natl. Acad. Sei. USA, 92: 11509-11513.

68. Patel, D.J., Suri, A.K., Jiang, F., Jiang, L., Fan, P., Kumar, R.A., Nonin, S. 1997. Structure, recognition and adaptive binding in RNA aptamer complexes. J. Mol. Biol. 272: 645-664.

69. Peterson E. T., Pan T., Coleman J., Uhlenbeck O. C. 1994. In vitro selection of small RNAs that bind to Escherichia coli phenylalanyl-tRNA synthetase. J. Mol. Biol., 242: 186-192.

70. Pleij C. 1994. RNA pseudoknots. Current Opinion in Structural Biology, 4: 337-344.

71. Pribnow D. 1975. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc. Natl. Acad. Sei. USA, 72: 784-788.

72. Qiu J., Helmann J. D. 1999. Adenines at -11, -9 and -8 play a key role in the binding of Bacillus subtilis Esigma(A) RNA polymerase to -10 region single-stranded DNA. Nucleic Acids Res., 27: 4541-4546.

73. Ring B. Z., Yarnell W. S., Roberts J. W. 1996. Function of E. coli RNA polymerase sigma factor sigma 70 in promoter-proximal pausing. Cell, 86: 485-493.

74. Ringquist, S., Jones, T., Snyder, E.E., Gibson, T., Boni, I., Gold, L. 1995. High-affinity RNA ligands to Escherichia coli ribosomes and ribosomal protein SI: comparison of natural and unnatural binding sites. Biochemistry, 34: 3640-3648.

75. Roberts C. W., Roberts J. W. 1996. Base-Specific Recognition of the Nontemplate strand of Promoter DNA by E. coli RNA Polymerase. Cell, 86: 495-501.

76. Ross W., Gosink K. K., Salomon J., Igarashi K., Zhou C., Ishihama A., Severinov K., Gourse R. L. 1993. A third recognition element in bacterial promoters: DNA binding by the a-subunit of RNA polymerase. Science, 262: 1407-1413.

77. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular cloning. Cold spring harbor press.

78. Santoro, S. W., Joyce, G. F. 1997. A general purpose RNA-cIeaving DNA enzyme. Proc. Natl. Acad. Sei. USA, 94: 4262-4266.

79. Schickor P., Metzger W., Werel W., Lederer H., Heumann H. 1990. Topography of intermediates in transcription initiation of E.coli. EMBOJ. 9: 2215-2220.

80. Schneider, D., Feigon, J., Hostomsky, Z., Gold, L. 1995. High-affinity ssDNA inhibitors of the reverse transcriptase of type 1 human immunodeficiency virus. Biochemistry, 34: 9599-9610.

81. Siebenlist U., Simpson R. B., Gilbert W. 1980. Escherichia coli RNA polymerase interacts gomologously with two different promoters. Cell, 20: 269-281.

82. Siegele D. A., Hu J. C., Walter W. A., Gross C. A. 1989. Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase. J Mol Biol 206: 591-603.

83. Simpson R. B. 1979. The molecular topography of RNA polymerase-promoter interaction. Cell, 18: 277-285.

84. Szkaradkiewicz K., Nanninga M., Nesper-Brock M., Gerrits M., Erdmann V. A., Sprinzl M. 2002. RNA aptamers directed against release factor 1 from Thermus thermophilics. FEBS Lett., 514: 90-95.

85. Tasset D. M., Kubik M. F., Steiner W. 1997. Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J. Mol. Biol., 272: 688-698.

86. Terao T., Dahlberg J. E., Khorana H. G. 1972. Studies on polynucleotides. CXX. On the transcription of a synthetic 29-unit long deoxyribopolynucleotide. J. Biol. Chem. 247: 61576166.

87. Tombelli S., Minunni M., Mascini M. 2005. Analytical applications of aptamers. Biosens. Bioelectron. 20: 2424-34.

88. Triqueneaux G., Velten M., Franzon P., Dautry F., Jacquemin-Sablon H. 1999. RNA binding specificity of Unr, a protein with five cold shock domains. Nucleic Acids Res., 27: 1926-1934.

89. Tsai, R.Y., Reed, R.R. 1998. Identification of DNA recognition sequences and protein interaction domains of the multiple-Zn-finger protein Roaz. Mol. Cell. Biol., 18: 6447-6456.

90. Tsiang M., Gibbs C. S., Griffin L. C., Dunn K. E., Leung L. L. 1995. Selection of a suppressor mutation that restores affinity of an oligonucleotide inhibitor for thrombin using in vitro genetics. J. Biol. Chem., 270: 19370-19376.

91. Tuerk C., MacDougal S., Gold L. 1992. RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. Proc. Natl. Acad. Sei. USA., 89: 6988-6992.

92. Tuerk, C., Gold, L. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science, 249: 505-510.

93. Vassylyev D. G., Sekine S., Laptenko O., Lee J., Vassylyeva M. N., Borukhov S., Yokoyama S. 2002. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution. Nature. 417: 712-9.

94. Venables J. P., Ruggiu M., Cooke H. J. 2001. The RNA-binding specificity of the mouse Dazl protein. Nucleic Acids Res., 29: 2479-2483.

95. Wang K. Y., McCurdy S., Shea R. G., Swaminathan S., Bolton P. H. 1993a. A DNA aptamer which binds to and inhibits thrombin exhibits a new structural motif for DNA. Biochemistry, 32: 1899-1904.

96. Wang K. Y., Krawczyk S. H., Bischofberger N., Swaminathan S„ Bolton P. H. 1993b. The tertiary structure of a DNA aptamer which binds to and inhibits thrombin determines activity. Biochemistry, 32: 11285-11292.

97. Wilson D. S., Szostak J. W. 1999. In vitro selection of functional nucleic acids. Annu. Rev. Biochem., 68: 611-647.

98. Xu J., McCabe B. C., Koudelka G. B. 2001. Function-based selection and characterization of base-pair polymorphisms in a promoter of Escherichia coli RNA polymerase-sigma(70). J. Bacteriol., 183:2866-2873.

99. Ye X., Gorin A., Frederick R., Hu W., Majumdar A., Xu W., McLendon G., Ellington A., Patel D. J. 1999. RNA architecture dictates the conformations of a bound peptide. Chem. Biol., 6: 657669.

100. Zalenskaya K., Lee J., Gujulova C.N., Shin Y.K., Slutsky M., Goldfarb A. 1990. Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products. Gene. 89: 7-12.

101. Zenkin N, Severinov K. 2004. The role of RNA polymerase sigma subunit in promoter-independent initiation of transcription. Proc. Natl. Acad. Sei. USA. 101: 4396-400.

102. Zenkin N., Naryshkina T., Kuznedelov K. and Severinov K. 2005. The mechanism of DNA replication primer synthesis by RNA polymerase. Nature, in press.

103. Zhang G., Campbell E. A., Minakhin L., Richter C., Severinov K., Darst S. A. 1999. Crystal structure of Thermus aquaticus core RNA polymerase at 3.3 Ä resolution. Cell, 98: 811-824.