臺灣博碩士論文加值系統

細菌細胞中之轉錄起始因子，σ，能否如同真核生物之轉錄因子一般，先與啟動子 DNA 結合，再招攬 RNA 聚合酶，來啟動轉錄的進行，一直是生命科學家們想解答的問題。因此，純化具有直接與啟動子 DNA 結合能力的蛋白，並在細胞中偵測到這種結合的存在，就成為解答這個問題的重點。本研究中，我發現必需採用較緩慢的體外重新摺疊，才能由不可溶的σA 蛋白聚結體(inclusion bodies)樣品中，製備具有獨立與啟動子 DNA 結合能力的σA 蛋白。不過，體外重新摺疊所獲得的σA 樣品，存在有不同構形及和啟動子 DNA 不同結合能力的σA 蛋白，必需再利用分子篩層析管柱，加以純化。此經重新摺疊、純化的σA 蛋白與啟動子 DNA 結合能力和從大腸桿菌細胞中表現、純化的自然摺疊、水溶性σA 蛋白相似，可見不需要核心酵素(core enzyme)的協助，σA 蛋白即具有與啟動子 DNA 結合之本能力。為了證明枯草桿菌細胞內之A 蛋白也具有獨立與啟動子 DNA 結合之能力 我進行了，Sequential Chromatin Immunoprecipitation (SeqChIP)的分析。結果顯示，在枯草桿菌指數生長時期中，σA 蛋白可以直接與 ezrA、sigA-P1P2、veg、spo0A、spo0H 和29噬菌體 G3b 等啟動子 DNA 結合，而且至少與 G3b 啟動子 DNA 之–10 及–35 elements 的結合具有專一性。另外，σA 蛋白也具有辨識最佳啟動子–10 及–35 elements 之區間(spacing)的能力 因子若真能在沒有核心酵素協助下即能與啟動子σDNA 結合，那麼它將帶給原核生物基因轉錄起始研究一個嶄新的方向。

It has been long a focus for scientists to answer whether the prokaryotic σ like the universal eukaryotic transcription initiation factor, the TATA box-binding protein, can be recruited to the promoter prior to association with core RNAP during transcription initiation. To answer this question, purification of σ with the promoter DNA-binding property in vitro and detection of the σ-promoter-DNA interaction in vivo are absolutely required. In my study, I found that the Bacillus subtilis σA with a promoter DNA-binding activity can be obtained if the σA was overexpressed heterologously in Escherichia coli, denatured, refolded slowly in vitro and purified with molecular sieving columns. The promoter DNA-binding activity of the in vitro refolded σA is similar to that overproduced in Escherichia coli in soluble form. These results support the idea that the observed promoter DNA-binding activity of the refolded σA is not an artifact but is an intrinsic property of σA. The interaction of σA with known promoter DNAs in B. subtilis was analyzed by Sequential Chromatin Immunoprecipitation (SeqChIP) in accompany with a Nickel-resin affinity chromatography. The results demonstrated that σA is at least able to interact with ezrA, sigA-P1P2, veg, spo0A, spo0H and σ phage G3b promoters in B. subtilis in early log phase. Both –10 and –35 elements of the G3b promoter DNA are required for the efficient promoter-specific interaction of σ A in vivo.Moreover, the promoter –10 and –35 specific interaction of σA is able to allow the σ A to discern the optimal promoter spacing in vivo. The ability of σ by itself, to interact specifically with promoter might introduce a critical new dimension of study in prokaryotic σ function and gene regulation during transcription initiation.

Contents
Introduction ............................................. 1
Materials and Methods
Overproduction and purification of σA ................... 10
Labeling of the σ phage G3b promoter DNA ................ 10
Electrophoretic mobility shift assay (EMSA) ............. 11
DNase I footprinting assay............................... 11
In vitro transcription assay ............................ 12
Sequential chromatin immunoprecipitation (SeqChIP) assay 13
Construction of B. subtilis DB430C12 .................... 14
Integration of the G3b promoter DNA into B. subtilis chromosome .............................................. 14
Construction of spacing variants of the G3b promoter DNA ......................................................... 15
Integration of each of the G3b promoter spacing variants into B. subtilis chromosome ............................. 15
Construction of temperature-sensitive mutations in the α subunit gene of B. subtilis RNA polymerase............... 16
Results Purification of the B. subtilis primary A, with a core-independent promoter DNA-binding activity ........ 18
The existence of core-independent promoter interaction of σA in B. subtilis .......................................... 19
Both –10 and –35 elements are important for core-independent promoter-specific interaction of σA in vivo ............. 19
The promoter spacing is critical for efficient formation of the σA-promoter DNA complex ............................. 20
Construction of the temperature-sensitive mutations in the α subunit gene of B. subtilis RNA polymerase .............. 21
Discussion .............................................. 22
References............................................... 39

Allison, L.A., Moyle, M., Shales, M., and Ingles, C.J. (1985). Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell 42,
599-610.

Aoyama, T., Takanami, M., Ohtsuka, E., Taniyama, Y., Marumoto, R., Sato, H., and Ikehara, M. (1983). Essential structure of E. coli promoter: effect of spacer length
between the two consensus sequences on promoter function. Nucleic Acids Res 11,5855-5864.

Arthur, T.M., and Burgess, R.R. (1998). Localization of a sigma 70 binding site on the N terminus of the Escherichia coli RNA polymerase beta'' subunit. J Biol Chem 273, 31381-31387.

Arthur, T.M., Anthony, L.C., and Burgess, R.R. (2000). Mutational analysis of beta''260-309, a sigma 70 binding site located on Escherichia coli core RNA polymerase. J Biol Chem 275, 23113-23119.

Auble, D.T., and deHaseth, P.L. (1988). Promoter recognition by Escherichia coli RNA polymerase. Influence of DNA structure in the spacer separating the –10 and –35 regions. J Mol Biol 202, 471-482.

Ayers, D.G., Auble, D.T., and deHaseth, P.L. (1989). Promoter recognition by Escherichia coli RNA polymerase. Role of the spacer DNA in functional complex formation. J Mol Biol 207, 749-756.

Barkley, M.D. (1981). Salt dependence of the kinetics of the lac repressor-operator interaction: role of nonoperator deoxyribonucleic acid in the association reaction. Biochemistry 20, 3833-3842.

Barne, K.A., Bown, J.A., Busby, S.J., and Minchin, S.D. (1997). Region 2.5 of the Escherichia coli RNA polymerase sigma 70 subunit is responsible for the recognition of
the ''extended –10'' motif at promoters. EMBO J 16, 4034-4040.

Borukhov, S., and Nudler, E. (2003). RNA polymerase holoenzyme: structure, function and biological implications. Curr Opin Microbiol 6, 93-100.

Borukhov, S., and Severinov, K. (2002). Role of the RNA polymerase sigma subunit in transcription initiation. Res Microbiol 153, 557-562.

Bowers, C.W., and Dombroski, A.J. (1999). A mutation in region 1.1 of sigma 70 affects promoter DNA binding by Escherichia coli RNA polymerase holoenzyme. EMBO J 18,
709-716.

Buck, M., Gallegos, M.T., Studholme, D. J., Guo, Y., and Gralla, J.D. (2000). The bacterial enhancer-dependent sigma(54) (sigma(N)) transcription factor. J Bacteriol 182:
4129-4136.

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

Callaci, S., Heyduk, E., and Heyduk, T. (1999). Core RNA polymerase from E. coli induces a major change in the domain arrangement of the sigma 70 subunit. Mol Cell 3, 229-238.

Camarero, J.A., Shekhtman, A., Campbell, E.A., Chlenov, M., Gruber, T.M., Bryant, D.A., Darst, S.A., Cowburn, D., and Muir, T.W. (2002). Autoregulation of a bacterial sigma factor explored by using segmental isotopic labeling and NMR. Proc Natl Acad Sci USA 99, 8536-8541.

Campbell, E.A., Muzzin, O., Chlenov, M., Sun, J.L., Olson, C.A., Weinman, O., Trester-Zedlitz, M.L., and Darst, S.A. (2002). Structure of the bacterial RNA polymerase promoter specificity sigma subunit. Mol Cell 9, 527-539.

Carpousis, A.J., and Gralla, J.D. (1980). Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter. Biochemistry 19, 3245-3253.

Cashel, M., Hsu, L.M., and Hernandez, V.J. (2003). Changes in conserved region 3 of Escherichia coli sigma 70 reduce abortive transcription and enhance promoter escape. J Biol Chem 278, 5539-5547.

Chang, B.Y., and Doi, R.H. (1990). Overproduction, purification, and characterization of Bacillus subtilis RNA polymerase sigma A factor. J Bacteriol 172, 3257-3263.

Chang, B.Y., and Doi, R.H. (1993). Conformational properties of Bacillus subtilis RNA polymerase sigma A factor during transcription initiation. Biochem J 294, 43-47.

Chang, B.Y., Shyu, Y.T., and Doi, R.H. (1992). The interaction between Bacillus subtilis sigma-A (sigma A) factor and RNA polymerase with promoters. Biochimie 74, 601-612.

Chen, Y., Ebright, Y.W., and Ebright, R.H. (1994). Identification of the target of a transcription activator protein by protein-protein photocrosslinking. Science 265, 90-92.

Chen, Y.F., and Helmann, J.D. (1995). The Bacillus subtilis flagellar regulatory protein sigma D: overproduction, domain analysis and DNA-binding properties. J Mol Biol 249,
743-753.

Conter, A., Menchon, C., and Gutierrez, C. (1997). Role of DNA supercoiling and rpoS sigma factor in the osmotic and growth phase-dependent induction of the gene osmE of
Escherichia coli K12. J Mol Biol 273, 75-83.

Cramer, P., Bushnell, D.A., and Kornberg, R.D. (2001). Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292, 1863-1876.

Das, A. (1993). Control of transcription termination by RNA-binding proteins. Annu Rev Biochem 62, 893-930.

Dombroski, A.J. (1997). Recognition of the –10 promoter sequence by a partial polypeptide of sigma 70 in vitro. J Biol Chem 272, 3487-3494.

Dombroski, A.J., Johnson, B.D., Lonetto, M., and Gross, C.A. (1996). The sigma subunit of Escherichia coli RNA polymerase senses promoter spacing. Proc Natl Acad Sci USA 93, 8858-8862.

Dombroski, A.J., Walter, W.A., and Gross, C.A. (1993).Amino-terminal amino acids modulate sigma-factor DNA-binding activity. Genes Dev 7, 2446-2455.

Dombroski, A.J., Walter, W.A., Record, M.T., Jr., Siegele, D.A., and Gross, C.A. (1992). Polypeptides containing highly conserved regions of transcription initiation factor sigma
70 exhibit specificity of binding to promoter DNA. Cell 70, 501-512.

Ebright, R.H. (2000). RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II. J Mol Biol 304, 687-698.

Ebright, R.H., and Busby, S. (1995). The Escherichia coli RNA polymerase alpha subunit: structure and function. Curr Opin Genet Dev 5, 197-203.

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

Estrem, S.T., Ross, W., Gaal, T., Chen, Z.W., Niu, W., Ebright, R.H., and Gourse, R.L.(1999). Bacterial promoter architecture: subsite structure of UP elements and
interactions with the carboxy-terminal domain of the RNA polymerase alpha subunit. Genes Dev 13, 2134-2147.

Fenton, M.S., Lee, S.J., and Gralla, J.D. (2000). Escherichia coli promoter opening and –10 recognition: mutational analysis of sigma 70. EMBO J 19, 1130-1137.

Fukushima, T., Ishikawa, S., Yamamoto, H., Ogasawara, N., and Sekiguchi, J. (2003). Transcriptional, functional and cytochemical analyses of the veg gene in Bacillus subtilis. J Biochem 133, 475-483.

Gardella, T., Moyle, H., and Susskind, M.M. (1989). A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity. J Mol Biol 206, 579-590.

Gnatt, A.L., Cramer, P., Fu, J., Bushnell, D.A., and Kornberg, R.D. (2001). Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 Å resolution.
Science 292, 1876-1882.

Gopal, V., and Chatterji, D. (1997). Mutations in the 1.1 subdomain of Escherichia coli sigma factor sigma 70 and disruption of its overall structure. Eur J Biochem 244,
613-618.

Gowrishankar, J., Yamamoto, K., Subbarayan, P.R., and Ishihama, A. (2003). In vitro properties of RpoS (sigma(S)) mutants of Escherichia coli with postulated N-terminal
subregion 1.1 or C-terminal region 4 deleted. J Bacteriol 185, 2673-2679.

Gross, C.A., Chan, C., Dombroski, A., Gruber, T., Sharp, M., Tupy, J., and Young, B.(1998). The functional and regulatory roles of sigma factors in transcription. Cold Spring Harb Symp Quant Biol 63, 141-155.

Gross, C., Lonetto, M., and Losick, R. (1992). In: Transcriptional Regulation (McKnight, S. R. & Yamamoto, K. R., Eds.), pp. 129–176. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

Gruber, T.M. and Bryant, D.A. (1997). Molecular systematic studies of eubacteria,using 70-type sigma factors of group 1 and group 2. J Bacteriol 179, 1734-1747.

Gruber, T. M., Markov, D., Sharp, M.M., Young, B.A., Lu, C.Z., Zhong, H.J., Artsimovitch, I., Geszvain, K.M., Arthur, T.M., Burgess, R.R., Landick, R., Severinov, K. and Gross, C.A. (2001). Binding of the initiation factor sigma (70) to core RNA polymerase is a multistep process. Mol Cell 8, 21-31.

Harley, C.B., and Reynolds, R.P. (1987). Analysis of E. coli promoter sequences. Nucleic Acids Res 15, 2343-2361.

Haugen, S.P., Berkmen, M.B., Ross, W., Gaal, T., Ward, C., and Gourse, R.L. (2006). rRNA promoter regulation by nonoptimal binding of sigma region 1.2: an additional
recognition element for RNA polymerase. Cell 125, 1069-1082.

Haugen, S.P., Ross, W., Manrique, M., and Gourse, R.L. (2008). Fine structure of the promoter-sigma region 1.2 interaction. Proc Natl Acad Sci USA 105, 3292-3297.

Hawley, D.K., and McClure, W.R. (1980). In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters. Proc Natl Acad Sci USA
77, 6381-6385.

Hawley, D.K., and McClure, W.R. (1983). Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res 11, 2237-2255.

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.

Helmann, J.D. (2002). The extracytoplasmic function (ECF) sigma factors. Adv Microb Physiol 46, 47-110.

Helmann, J.D., and Chamberlin, M.J. (1988). Structure and function of bacterial sigma factors. Annu Rev Biochem 57, 839-872.

Helmann, J.D., and deHaseth, P.L. (1999). Protein-nucleic acid interactions during open complex formation investigated by systematic alteration of the protein and DNA binding partners. Biochemistry 38, 5959-5967.

He, X.S., Shyu, Y.T., Nathoo, S., Wong, S.L., and Doi, R.H. (1991). Construction and use of a Bacillus subtilis mutant deficient in multiple protease genes for the expression
of eukaryotic genes. Ann N Y Acad Sci 646, 69-77.

Hernandez, V.J., and Cashel, M. (1995). Changes in conserved region 3 of Escherichia coli sigma 70 mediate ppGpp dependent functions in vivo. J Mol Biol 252, 536-549.

Hernandez, V.J., Hsu, L.M., and Cashel, M. (1996). Conserved region 3 of Escherichia coli final sigma70 is implicated in the process of abortive transcription. J Biol Chem 271, 18775-18779.

Hidalgo, E., and Demple, B. (1997). Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor. EMBO J 16, 1056-1065.

Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. (1989). Site-directed mutagenesis by overlapping extension using the polymerase chain reaction. Gene 77,
51-59.

Horton, R.M., Cai, Z., Ho, S.N., and Pease, L.R. (1990). Gene splicing by overlapping extension: tailor-made genes using the polymerase chain reaction. BioTechniques 8,
528-535.

Hsu, L.M. (2002). Promoter clearance and escape in prokaryotes. Biochim Biophys Acta 1577, 191-207.

Huang, X., Lopez de Saro, F.J., and Helmann, J.D. (1997). sigma factor mutations affecting the sequence-selective interaction of RNA polymerase with –10 region single-stranded DNA. Nucleic Acids Res 25, 2603-2609.

Igarashi, K., and Ishihama, A. (1991). Bipartite functional map of the E. coli RNA polymerase alpha subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell 65, 1015-1022.

Ishihama, A. (1981). Subunit of assembly of Escherichia coli RNA polymerase. Adv Biophys 14, 1-35.

Jaurin, B., Grundstrom, T., Edlund, T., and Normark, S. (1981). The E. coli beta-lactamase attenuator mediates growth rate-dependent regulation. Nature 290, 221-225.

Jeon, Y.H., Negishi, T., Shirakawa, M., Yamazaki, T., Fujita, N., Ishihama, A., and Kyogoku, Y. (1995). Solution structure of the activator contact domain of the RNA
polymerase alpha subunit. Science 270, 1495-1497.

Jyothirmai, G., and Mishra, R.K. (1994). Differential influence of DNA supercoiling on in vivo strength of promoters varying in structure and organisation in E. coli. FEBS Lett 340, 189-192.

Juang, Y.L., and 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.

Kammerer, W., Deuschle, U., Gentz, R., and Bujard, H. (1986). Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late
steps of the overall process. EMBO J 5, 2995-3000.

Keilty, S., and Rosenberg, M. (1987). Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J Biol Chem 262, 6389-6395.

Korzheva, N., and Mustaev, A. (2001). Transcription elongation complex: structure and function. Curr Opin Microbiol 4, 119-125.

Kovacic, R.T. (1987). The 0 degree C closed complexes between Escherichia coli RNA polymerase and two promoters, T7-A3 and lacUV5. J Biol Chem 262:13654-13661.

Kramer, H., Amouyal, M., Nordheim, A., and Muller-Hill, B. (1988). DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor. EMBO J 7, 547-556.

Kudo, T., and Doi, R.H. (1981). Free sigma factor of Escherichia coli RNA polymerase can bind to DNA. J Biol Chem 256, 9778-9781.

Kudo, T., Jaffe, D., and Doi, R.H. (1981). Free sigma subunit of Bacillus subtilis RNA polymerase binds to DNA. Mol Gen Genet 181, 63-68.

Kulbachinskiy, A., Mustaev, A., Goldfarb, A., and Nikiforov, V. (1999). Interaction with free beta'' subunit unmasks DNA-binding domain of RNA polymerase sigma subunit. FEBS Lett 454, 71-74.

Kuldell, N., and Hochschild, A. (1994). Amino acid substitutions in the –35 recognition motif of sigma 70 that result in defects in phage lambda repressor-stimulated
transcription. J Bacteriol 176, 2991-2998.

Kumar, A., Malloch, R.A., Fujita, N., Smillie, D.A., Ishihama, A., and Hayward, R.S. (1993). The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an "extended minus 10" promoter. J Mol Biol 232, 406-418.

Kuznedelov, K., Minakhin, L., Niedziela-Majka, A., Dove, S.L., Rogulja, D., Nickels, B.E., Hochschild, A., Heyduk, T., and Severinov, K. (2002). A role for interaction of the
RNA polymerase flap domain with the sigma subunit in promoter recognition. Science 295, 855-857.

Landini, P., and Busby, S.J. (1999). The Escherichia coli Ada protein can interact with two distinct determinants in the sigma70 subunit of RNA polymerase according to
promoter architecture: identification of the target of Ada activation at the alkA promoter. J Bacteriol 181, 1524-1529.

Li, M. Moyle, H. and Susskind, M.M. (1994). Target of the transcriptional activation function of phage lambda cI protein. Science 263, 75-77.

Liao, C.T., Wen, Y.D., Wang., W.H., and Chang, B.Y. (1999). Idetification and characterization of a stress-responsive promoter in the macromolecular synthesis operon of Bacillus subtilis. Mol. Microbiol 33, 377-388.

Lin, T.F. (2014). Study on the core-independent promoter-specific interaction of primary sigma factor. Master Thesis.
Lonetto, M., Gribskov, M., and Gross, C.A. (1992). The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol 174, 3843-3849.

Lonetto, M.A., Brown, K.L., Rudd, K.E., and Buttner, M.J. (1994). Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial
RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions.Proc Natl Acad Sci USA 91, 7573-7577.

Lord, M., Barilla, D. and Yudkin, M.D. (1999). Replacement of vegetative sigmaA by sporulation-specific sigmaF as a component of the RNA polymerase holoenzyme in sporulating Bacillus subtilis. J Bacteriol 181, 2346-2350.

Malhotra, A., Severinova, E., and Darst, S.A. (1996). Crystal structure of a sigma 70 subunit fragment from E. coli RNA polymerase. Cell 87, 127-136.

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

McClure, W.R., Cech, C.L., and Johnston, D.E. (1978). A steady state assay for the RNA polymerase initiation reaction. J Biol Chem 253, 8941-8948.

McKane, M., and Gussin, G.N. (2000). Changes in the 17 bp spacer in the PR promoter of bacteriophage lambda affect steps in open complex formation that precede DNA
strand separation. J Mol Biol 299, 337-349.

Merrick, M. J., Gibbin, J., and Toukdarian, A. (1987). The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins. Mol Gen Genet. 210, 323-330.

Merrick, M.J. (1993). In a class of its own--the RNA polymerase sigma factor sigma 54(sigma N). Mol Microbiol 10, 903-909.

Minakhin, L., Bhagat, S., Brunning, A., Campbell, E.A., Darst, S.A., Ebright, R.H., and Severinov, K. (2001). Bacterial RNA polymerase subunit omega and eukaryotic RNA
polymerase subunit RPB6 are sequence, structure, and functional homologs and promote RNA polymerase assembly. Proc Natl Acad Sci USA 98, 892-897.

Moran, C.P. Jr., Lang, N., LeGrice, S.F., Lee,G., Stephens, M., Sonenshein, A.L., Pero, J. and Losick, R. (1982). Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet 186, 339-346.

Mukherjee, K., and Chatterji, D. (1997). Studies on the omega subunit of Escherichia coli RNA polymerase--its role in the recovery of denatured enzyme activity. Eur J
Biochem 247, 884-889.

Mukherjee, K., Nagai, H., Shimamoto, N., and Chatterji, D. (1999). GroEL is involved in activation of Escherichia coli RNA polymerase devoid of the omega subunit in vivo.
Eur J Biochem 266, 228-235.

Mulligan, M.E., Brosius, J., and McClure, W.R. (1985). Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC
promoter. J Biol Chem 260, 3529-3538.

Murakami, K.S., Masuda, S., Campbell, E.A., Muzzin, O., and Darst, S.A. (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science 296, 1285-1290

Murakami, K.S., Masuda, S., and Darst, S.A. (2002b). Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution. Science 296, 1280-1284.

Niedziela-Majka, A., and Heyduk, T. (2005). Escherichia coli RNA polymerase contacts outside the –10 promoter element are not essential for promoter melting. J Biol Chem 280, 38219-38227.

Panaghie, G., Aiyar, S.E., Bobb, K.L., Hayward, R.S., and de Haseth, P.L. (2000). Aromatic amino acids in region 2.3 of Escherichia coli sigma 70 participate collectively
in the formation of an RNA polymerase-promoter open complex. J Mol Biol 299, 1217-1230.

Parkhill, J., and Brown, N.L. (1990). Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR. Nucleic Acids Res 18, 5157-5162.

Perez-Martin, J., Rojo, F., and de Lorenzo, V. (1994). Promoters responsive to DNA bending: a common theme in prokaryotic gene expression. Microbiol Rev 58, 268-290.
Plaskon, R.R., and Wartell, R.M. (1987). Sequence distributions associated with DNA curvature are found upstream of strong E. coli promoters. Nucleic Acids Res 15,
785-796.

Ptashne, M., and Gann, A. (1997). Transcriptional activation by recruitment. Nature 386, 569-577.

Qi, F.X., He, X.S., and Doi, R.H. (1991). Localization of a new promoter, P5, in the sigA operon of Bacillus subtilis and its regulation in some spo mutant strains. J Bacteriol 173, 7050-7054.

Ramesh, U., and Meares, C.F. (1989). Footprint of the sigma protein. Biochem Biophys Res Commun 160, 121-125.

Rhodius, V.A., and Busby, S.J. (2000). Transcription activation by the Escherichia coli cyclic AMP receptor protein: determinants within activating region 3. J Mol Biol 299,295-310.

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

Romero, Obradovic, and Dunker, K. (1997). Sequence Data Analysis for Long Disordered Regions Prediction in the Calcineurin Family. Genome Inform Ser Workshop Genome Inform 8, 110-124.

Rong, J.C., and Helmann, J.D. (1994). Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting. J Bacteriol 176, 5218-5224.

Ross, W., Ernst, A., and Gourse, R.L. (2001). Fine structure of E. coli RNA polymerase-promoter interactions: alpha subunit binding to the UP element minor groove. Genes Dev 15, 491-506.

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

Russell, D.R., and Bennett, G.N. (1982). Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the –35 to –10 spacing. Gene
20, 231-243.

Sanderson, A., Mitchell, J.E., Minchin, S.D., and Busby, S.J. (2003). Substitutions in the Escherichia coli RNA polymerase sigma 70 factor that affect recognition of extended – 10 elements at promoters. FEBS Lett 544, 199-205.
Sasse, D. S., and Gralla, J. D. (1990) Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor 54. Cell 62, 945-954.

Schwartz, E.C., Shekhtman, A., Dutta, K., Pratt, M.R., Cowburn, D., Darst, S., and Muir,T.W. (2008). A full-length group 1 bacterial sigma factor adopts a compact structure
incompatible with DNA binding. Chem Biol 15, 1091-1103.
Sen, R., Nagai, H., Hernandez, V.J., and Shimamoto, N. (1998). Reduction in abortive transcription from the lambda PR promoter by mutations in region 3 of the sigma70
subunit of Escherichia coli RNA polymerase. J Biol Chem 273, 9872-9877.

Severinova, E., Severinov, K., Fenyo, D., Marr, M., Brody, E.N., Roberts, J.W., Chait, B.T., and Darst, S.A. (1996). Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit. J Mol Biol 263, 637-647.

Sevostyanova, A., Feklistov, A., Barinova, N., Heyduk, E., Bass, I., Klimasauskas, S., Sharp, M.M., Chan, C.L., Lu, C.Z., Marr, M.T., Nechaev, S., Merritt, E.W., Severinov,
K., Roberts, J.W., and Gross, C.A. (1999). The interface of sigma with core RNA polymerase is extensive, conserved, and functionally specialized. Genes Dev 13, 3015-3026.

Shorenstein, R.G., and Losick, R. (1973). Comparative size and properties of the sigma subunits of ribonucleic acid polymerase from Bacillus subtilis and Escherichia coli. J
Biol Chem 248, 6170-6173.

Siegele, D.A., Hu, J.C., Walter, W.A., and 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.

Sorenson, M.K., and Darst, S.A. (2006). Disulfide cross-linking indicates that FlgM-bound and free sigma 28 adopt similar conformations. Proc Natl Acad Sci USA 103, 16722-16727.

Sorenson, M.K., Ray, S.S., and Darst, S.A. (2004). Crystal structure of the flagellar sigma/anti-sigma complex sigma(28)/FlgM reveals an intact sigma factor in an inactive
conformation. Mol Cell 14, 127-138.

Stefano, J.E., and Gralla, J.D. (1982). Spacer mutations in the lac ps promoter. Proc Natl Acad Sci USA 79, 1069-1072.
Strainic, M.G., Jr., Sullivan, J.J., Velevis, A., and deHaseth, P.L. (1998). Promoter recognition by Escherichia coli RNA polymerase: effects of the UP element on open
complex formation and promoter clearance. Biochemistry 37, 18074-18080.

Summers, A.O. (1992). Untwist and shout: a heavy metal-responsive transcriptional regulator. J Bacteriol 174, 3097-3101.

Sweetser, D., Nonet, M., and Young, R.A. (1987). Prokaryotic and eukaryotic RNA polymerases have homologous core subunits. Proc Natl Acad Sci USA 84, 1192-1196.

Tomsic, M., Tsujikawa, L., Panaghie, G., Wang, Y., Azok, J., and deHaseth, P.L. (2001). Different roles for basic and aromatic amino acids in conserved region 2 of Escherichia
coli sigma(70) in the nucleation and maintenance of the single-stranded DNA bubble in open RNA polymerase-promoter complexes. J Biol Chem 276, 31891-31896.

Travers, A.A. (1987). Structure and function of E. coli promoter DNA. CRC Crit Rev Biochem 22, 181-219.

Vassylyev, D.G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M.N., Borukhov, S., and Yokoyama, S. (2002). Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 Å resolution. Nature 417, 712-719.

von Hippel, P.H. (1998). An integrated model of the transcription complex in elongation, termination, and editing. Science 281, 660-665.

Voskuil, M.I., and Chambliss, G.H. (2002). The TRTGn motif stabilizes the transcription initiation open complex. J Mol Biol 322, 521-532.

Voskuil, M.I., Voepel, K., and Chambliss, G.H. (1995). The –16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli. Mol Microbiol 17, 271-279.

Waldburger, C., Gardella, T., Wong, R., and Susskind, M.M. (1990). Changes in conserved region 2 of Escherichia coli sigma 70 affecting promoter recognition. J Mol Biol 215, 267-276.

Walter, G., Zillig, W., Palm, P., and Fuchs, E. (1967). Initiation of DNA-dependent RNA synthesis and the effect of heparin on RNA polymerase. Eur J Biochem 3, 194-201.

Warne, S.E., and deHaseth, P.L. (1993). Promoter recognition by Escherichia coli RNA polymerase. Effects of single base pair deletions and insertions in the spacer DNA separating the –10 and –35 regions are dependent on spacer DNA sequence. Biochemistry 32, 6134-6140.

Wellman, A., and Meares, C.F. (1991). Footprint of the sigma protein: a re-examination. Biochem Biophys Res Commun 177, 140-144.

Wiggs, J.L., Bush, J.W., and Chamberlin, M.J. (1979). Utilization of promoter and terminator sites on bacteriophage T7 DNA by RNA polymerases from a variety of
bacterial orders. Cell 16, 97-109.

Wilson, C. and Dombroski, A.J. (1997). Regoion 1of 70 is required for efficient isomerization and initiation of transcription by Escherichia coli RNA polymerase. J Mol Biol 267, 60-74.

Winter, R.B., Berg, O.G., and von Hippel, P.H. (1981). Diffusion-driven mechanisms of protein translocation on nucleic acids. 3. The Escherichia coli lac repressor--operator interaction: kinetic measurements and conclusions. Biochemistry 20, 6961-6977.

Yeh, H.Y., Chen, T.C., Liou, K.M., Hsu, H.T., Chung, K.M., Hsu, L.L., and Chang, B.Y. (2011). The core-independent promoter-specific interaction of primary sigma factor. Nucleic Acids Res 39, 913-925.

Young, B.A., Anthony, L.C., Gruber, T.M., Arthur, T.M., Heyduk, E., Lu, C.Z., Sharp, M.M., Heyduk, T., Burgess, R.R., and Gross, C.A. (2001). A coiled-coil from the RNA polymerase beta'' subunit allosterically induces selective nontemplate strand binding by sigma(70). Cell 105, 935-944.

Young, B.A., Gruber, T.M., and Gross, C.A. (2004). Minimal machinery of RNA olymerase holoenzyme sufficient for promoter melting. Science 303, 1382-1384.

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

Zuber, P., Healy, J., Carter, H.L., 3rd, Cutting, S., Moran, C.P., Jr., and Losick, R. (1989). Mutation changing the specificity of an RNA polymerase sigma factor. J Mol Biol 206,605-614.