title "version = 2.31 of inst.dnaa 2000 Sep 20"; (* DnaA binding sites. General review: Skarstad.Boye1994 Only sites that have been rigorously shown by footprinting or direct mutation are accepted here. *) default numbering piece; default numbering 1; default out-of-range reduce-range; (******************************************************************) organism E.coli; chromosome E.coli; (******************************************************************) (* dnaA sites in the origin of E. coli *) name "oriC"; piece K01789; (* reference: Fuller1983, Fuller1984 *) name "oriC R1"; (* Fuller1984 R1, Matsui1985 a, Samitt1989 *) get from 80 -100 to same +100 direction +; name "oriC R*"; (* R*, Matsui1985 X1 *) get from 135 -100 to same +100 direction +; (* REMOVE *) name "oriC R2"; (* Fuller1984 R2, Matsui1985 b, Samitt1989 *) get from 194 +100 to same -100 direction -; (* REMOVE *) name "oriC R3"; (* Fuller1984 R3, Matsui1985 X2 *) get from 221 -100 to same +100 direction +; (* REMOVE *) name "oriC R4"; (* Fuller1984 R4, Matsui1985 c, Samitt1989 *) get from 268 +100 to same -100 direction -; (* REMOVE *) (******************************************************************) (* nrd promoter *) piece K02672; (* binding data: Cu-phenanthroline footprinting Augustin1994 Fig. 4, location: fig 1 *) name "nrd -52"; (* from promoter *) get from 3351 +100 to same -100 direction -; name "nrd -40"; (* from promoter *) get from 3363 +100 to same -100 direction -; (******************************************************************) (* dnaA site controlling expression of dnaA itself Only one site is confirmed, the rest are a mess. This paper contributed nothing solid: @article{Polaczek.Wright1990, author = "P. Polaczek and A. Wright", title = "Regulation of Expression of the {{\em dnaA}} Gene in {{\em Escherichia coli\/}}: Role of the Two Promoters and the {DnaA} Box", journal = "The New Biologist", volume = "2", pages = "574-582", year = "1990"} Fuller1984 claims that there is a site in the middle of the gene, but give only a reference to a sequencing paper tha does NOT cover the relevant sequence! Schaefer1989 mutated the site and claim it has effects but Perez-Roger1995 disagree, and demonstrate that the site is not functional. get from 1729 -100 to same +100 direction +; Braun1985 reports two sites that fall to 867-847 and 647-677, just around the two dnaA promoters, these do not show anything above Ri zero, but do each contain a "ttat". This is too vague to include and others don't mention them. *) piece J01602; name "DnaA 1"; (* footprint: Fuller1984, Braun1985 *) get from 763 -100 to same +100 direction +; (* footprint: Speck.Messer1999 *) get from 784 -100 to same +100 direction +; name "DnaA 1"; (******************************************************************) (* rpoH dnaA protein regulates transcriptions of the rpoH gene of Escherichia coli. Wang QP; Kaguni JM J Biol Chem 264: 7338-44 (1989) The rpoH (htpR) gene of Escherichia coli encodes a sigma factor which confers upon RNA polymerase the ability to recognize the promoters for genes responsive to the phenomenon termed the heat shock response. dnaA protein, a sequence-specific DNA binding protein, is required for initiation of chromosomal replication by binding to sites within the chromosomal origin. dnaA protein also autoregulates its expression by binding to a site in the dnaA promoter region. Two copies of the dnaA protein recognition sequence are present within the rpoH promoter region. Using filter binding assays, dnaA protein was observed to bind specifically to DNA fragments containing the rpoH promoter region with greater affinity than its binding to the dnaA promoter region. By contrast, reduced binding to a DNA fragment containing the lacUV5 promoter was observed. DNase I footprint analysis indicated that dnaA protein protected specific sites within the rpoH promoter region. The binding of dnaA protein to the rpoH promoter region resulted in transcriptional repression from two of the three promoters of the rpoH gene in vitro. Elevated levels of dnaA protein repressed transcription from these two rpoH promoters in vivo. These results indicate that dnaA protein regulates rpoH transcription to influence the expression of genes under rpoH control. footprint data! A novel sigma factor is involved in expression of the rpoH gene of Escherichia coli. Wang QP; Kaguni JM J Bacteriol 171: 4248-53 (1989) The Escherichia coli rpoH gene encoding sigma 32, which is involved in the heat shock response, is transcribed from as many as four promoters. We have isolated a novel sigma factor of about 24 kilodaltons that allows core RNA polymerase to transcribe preferentially from one of these promoters, rpoH3p. This promoter is known to be regulated by DnaA protein. The sigma 24 factor was isolated from a preparation of RNA polymerase by electroelution from sodium dodecyl sulfate-polyacrylamide gels followed by renaturation. Expression of heat shock proteins is induced by treatments which include those that induce the stringent response. Under such conditions, decreased transcription from rpoH3p and no increase in transcription from other rpoH promoters were observed. This result suggests that induction of heat shock proteins by the stringent response is not mediated by increased transcription of the rpoH gene. confirms under control of dnaA RNA polymerase; rpoH gene. This is correct: Wang.Kaguni1989 to right of HindIII piece M20668; name "rpoH M20668"; get from 25 -100 to same +100 direction +; But it is out of date, the other piece is better to use. (coordinate 15152 gives identical sequence) *) (* Wang.Kaguni1989 fig 1 gives a fragment with ftsX: search for ftsX: U00039 Fig 1B is: organism E.coli; chromosome E.coli; piece U00039; get from 15322 to same; *) piece U00039; name "rpoH 1"; (* Wang.Kaguni1989 footprinting, at HindIII *) get from 15160 +100 to same -100 direction -; (* Wang.Kaguni1989 footprinting, Schaper.Messer1995 binding constant (with badly messed up flanks!) *) name "rpoH 2"; get from 15176 -100 to same +100 direction +; (* REMOVE *) (******************************************************************) (* 94.7 region: Kitagawa1996 page 1138 says the 5 sites in fig 1 were DNase I footprinted. h44851a@nucc.cc.nagoya-u.ac.jp Tohru Ogawa *) piece U14003; name "94.7-1"; get from 83513 +100 to same -100 direction -; name "94.7-2"; get from 83580 +100 to same -100 direction -; name "94.7-3"; get from 83742 +100 to same -100 direction -; name "94.7-4"; get from 83746 -100 to same +100 direction +; (* name "94.7-5"; get from 84403 -100 to same +100 direction +;*) (******************************************************************) (* mioC-asnC interval | MUST CHECK THESE REFERENCES: Cell cycle-dependent transcription from the gid and mioC promoters of Escherichia coli. Ogawa T; Okazaki T Department of Molecular Biology, School of Science, Nagoya University, Japan. J Bacteriol 176: 1609-15 (1994) Transcription from the gid and mioC promoters, which neighbor the origin of replication of the Escherichia coli chromosome (oriC), has been implicated in the control of initiation of replication of minichromosomes. The amounts of transcripts from these two promoters on the chromosome were quantified at various times in a synchronized culture of a temperature-sensitive dnaC mutant strain. Transcription from the gid promoter was most active before the initiation of replication and was inhibited after initiation, during the time corresponding to the period of sequestration of the oriC region from the dam methyltransferase. On the other hand, transcription from the mioC promoter was inhibited before initiation and the inhibition was relieved after initiation prior to the recovery of gid transcription. The strict regulation of transcription from the gid and mioC promoters may be involved in positive and negative control of chromosomal replication, respectively, as has been suggested for minichromosome replication. The DnaA protein was involved in repression of mioC transcription, indicating that the activity of the DnaA protein changes during the cell cycle. | Participation of the histone-like protein HU and of IHF in minichromosomal maintenance in Escherichia coli. Kano Y; Ogawa T; Ogura T; Hiraga S; Okazaki T; Imamoto F Department of Molecular Genetics, Kyoto Pharmaceutical University, Japan. Gene 103: 25-30 (1991) The closely related Escherichia coli genes, hupA, hupB, himA and himD (hip), encode the bacterial histone-like protein subunits, HU-2, HU-1, IHF chi and IHF beta, respectively. We report here that E. coli minichromosomes [plasmids (2.7-12.2 kb) with oriC] carrying the intact mioC region were unable to transform mutants deficient in both HU and integration host factor (IHF), whereas they could transform mutants deficient in either HU or IHF as efficiently as the wild-type strain. Minichromosomes carrying a deletion of the proximal part of mioC or a DnaA box just upstream from mioC could not transform cells deficient in IHF, but could transform cells deficient in HU. These results suggested that HU and IHF participate in minichromosomal replication from oriC in E. coli. | L10328 | rep_origin 115133..115364 /gene="oriC" /note="origin of replication of chromosome" misc_difference 115147 /note="GG in K01789; G in X01631, K00826, J01657, V00308, X02820, M10566, M10679 and here" CDS complement(115401..115844) /gene="mioC (CG Site No. 18154)" /note="involved in modulation of initiation at oriC; NCBI gi: 290591" /codon_start=1 /function="unknown" /translation="MADITLISGSTLGGAEYVAEHLAEKLEEAGFTTETLHGPLLEDL PASGIWLVISSTHGAGDIPDNLSPFYEALQEQKPDLSAVRFGAIGIGSREYDTFCGAI DKLEAELKNSGAKQTGETLKINILDHDIPEDPAEEWLGSWVNLLK" misc_feature 115511..117681 /note="V00263; ECOASNA(1..2170)" misc_difference 115544..115547 /note="CCCCC in J01657, V00308, M10679 and V00263; CCCC in K00826 and here" misc_difference 115680..115681 /note="G in J01657, M10679 and V00263; GG in K00826 and here" promoter complement(115873..115901) /note="promoter-like sequence; promoter matrix score of 54" CDS complement(115934..116392) /gene="asnC (CG Site No. 18520)" | 115844 to 115934 Scan shows: 1 91 L10328 115912 -1 6.825711 -2.081383 0.018699 *) organism E.coli; chromosome E.coli; piece L10328; (* mioC-asnC interval *) name "mioC"; (* Schaper.Messer1995: binding constant to oligo *) get from 115904 -100 to same +100 direction +; (******************************************************************) (* Resnikoff1993, Fuller.Kornberg1983, Yin1987 Fuller1984: Fig 9 footprint, table 1 sequence. *) piece U00004; name "Tn5 IRL"; get from 16 +100 to same -100 direction -; (******************************************************************) (* P1 plasmid replicon footprinting: 5 sites! ACCESSION K02380 M24626 X02954 AUTHORS Mukhopadhyay,G., Carr,K.M., Kaguni,J.M. and Chattoraj,D.K. TITLE Open-complex formation by the host initiator, DnaA, at the origin of P1 plasmid replication JOURNAL EMBO J. 12 (12), 4547-4554 (1993) MEDLINE 94038938 Footprints: @article{Mukhopadhyay.Chattoraj.emboj1993, author = "G. Mukhopadhyay and K. M. Carr and J. M. Kaguni and D. K. Chattoraj", title = "Open-complex formation by the host initiator, {DnaA} at the origin of {P1} plasmid replication", journal = "EMBO J.", volume = "12", pages = "4547-4554", comment = "was Mukhopadhyay1993", year = "1993"} *) organism B.P1; chromosome B.P1; piece K02380; name "P1-1"; get from 737 +100 to same -100 direction -; name "P1-2"; get from 746 +100 to same -100 direction -; (* REMOVE *) name "P1-3"; get from 968 +100 to same -100 direction -; (* REMOVE *) name "P1-4"; get from 978 +100 to same -100 direction -; name "P1-5"; get from 987 +100 to same -100 direction -; (* REMOVE*) (******************************************************************) (* pSC101 *) organism S.typhimurium; chromosome S.typhimurium; piece X01654; (* complete sequence of pSC101 *) (* Fuller.Kornberg1983: Fuller1984: fig 3, fragment retention. Stenzel1991: footprint data shows strong and weak sites. I don't believe the weak sites since they require IHF or RepA binding. Also, the paper reports only one IHF site, but a scan clearly reveals two back to back in opposite directions. Sugiura1993: deletions clearly show that the site upstream of IHF on side opposite from repeats is required. Ohkubo.Yamaguchi1995: collaborates the binding site. The sequence order is: DnaA-IHF-repA repeats-repA gene. *) name "pSC101 s"; get from 5014 +100 to same -100 direction -; (******************************************************************) (* RK2 Gaylo1987 says the "second site" is gcttttaaaccaatatttata This is at 910 of L36668. Gaylo is garbage, scan shows no site there!!! Shah created del114; it deletes one of two sites. It is not clear what this did, because it makes the remaining sites stronger. DO NOT USE THESE SITES: there are no solid data *) (* organism P.pTJS75; chromosome P.pTJS75; piece L36668; name "TODO RK2"; (@ Gaylo1987: DNA binding, mutation @) get from 704 -100 to same +100 direction +; better: organism B.IncP-alpha; chromosome B.IncP-alpha; piece L22758; name "TODO RK2"; (@ Shah(1995) JMB 254:608: mutation @) get from 12508 -100 to same +100 direction +; (@ deleted by del114 @) get from 12496 -100 to same +100 direction +; *) (* New data in Doran.Konieczny1999 support 4 sites. "box 4" 12496 is 12 bits, "box 3" 12508 is 11 bits, "box 2" 12514 is 7.3 bits "box 1" ? is <0.0 bits If box 4 is deleted the others go away. So Others depend on box 4 cooperatively. Therefore use only box 4. *) organism B.IncP-alpha; chromosome B.IncP-alpha; piece L27758; name "RK2"; get from 12496 +100 to same -100 direction -; (******************************************************************) (* ori-2-dependent mini-F *) (* From fig 3 of Murakami1987 ttgggg#ttatccacttatccacggggatatttt *) (* I don't believe the deletion data. The mutations do not fit to the observed Origin activity. In particular, the second DnaA site is completely zapped in the last 5 mutations, yet there is origin activity. This may mean that something else is going on. It means that we really don't know where the site(s) are precisely. *) (* organism P.F; chromosome P.F; piece M12987; name "TODO ori2-F-1"; get from 4869 +100 to same -100 direction -; name "TODO ori2-F-2"; get from 4877 +100 to same -100 direction -; *) (* TODO: still need to finish work in f directory to see effect of mutations. Then decide which sites are legit. *) (******************************************************************) (* Rts1 Masai H; Asai T; Kubota Y; Arai K; Kogoma T EMBO J 13: 5338-45 (1994) DnaA-dependent plasmid replicons such as F, R6K, Rts1 and RK2... Function of the N-terminal half of RepA in activation of Rts1 ori. Terawaki Y; Itoh Y; Zeng H; Hayashi T; Tabuchi A Department of Bacteriology, Shinshu University School of Medicine, Matsumoto, Japan. J Bacteriol 174: 6904-10 (1992) The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37 degrees C. At 42 degrees C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin. 3 1855 K00053 1403 -1 13.615355 -0.092534 0.463137 3 1855 K00053 1175 -1 8.278178 -1.527327 0.063340 3 1855 K00053 78 -1 6.436748 -2.022358 0.021570 3 1855 K00053 1428 -1 5.124413 -2.375153 0.008771 3 1855 K00053 308 -1 4.548592 -2.529951 0.005704 3 1855 K00053 1395 -1 4.501164 -2.542701 0.005500 Essential DNA sequence for the replication of Rts1. Itoh Y; Kamio Y; Terawaki Y J Bacteriol 169: 1153-60 (1987) The promoter sequence of the mini-Rts1 repA gene encoding the 33,000-dalton RepA protein that is essential for replication was defined by RNA polymerase protection experiments and by analyzing RepA protein synthesized in maxicells harboring mini-Rts1 derivatives deleted upstream of or within the presumptive promoter region. The -10 region of the promoter which shows homology to the incII repeat sequences overlaps two inverted repeats. One of the repeats forms a pair with a sequence in the -35 region, and the other forms a pair with the translation initiation region. The replication origin region, ori(Rts1), which was determined by supplying RepA protein in trans, was localized within 188 base pairs in a region containing three incII repeats and four GATC sequences. Dyad dnaA boxes that exist upstream from the GATC sequences appeared to be dispensable for the origin function, but deletion of both dnaA boxes from ori(Rts1) resulted in reduced replication frequency, suggesting that host-encoded DnaA protein is involved in the replication of Rts1 as a stimulatory element. Combination of the minimal repA and ori(Rts1) segments, even in the reverse orientation compared with the natural sequence, resulted in reconstitution of an autonomously replicating molecule. This referece IS in K00053. *) (******************************************************************) (* Scan of Xer sequences for Ri > 3 CloDF13 3 9957 X04466 8745 -1 4.191581 -3.278879 0.000521 AUTHORS Stuitje,A.R., Veltkamp,E., Maat,J. and Heyneker,H.L. TITLE The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacteriocinogenic plasmid Clo DF13 JOURNAL Nucleic Acids Res. 8 (7), 1459-1473 (1980) MEDLINE 81053824 Scan of Xer sequences for Ri > 3 4 374 M29821 192 1 10.747398 -1.441155 0.074770 DEFINITION Plasmid Colicin K stability (ckr) function DNA. Scan of Xer sequences for Ri > 3 6 2235 M61242 2040 1 4.846934 -3.095170 0.000983 just outside dif, near E. coli terminus *) (******************************************************************) (* lambda o Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda. Kur J; Gorska I; Taylor K Department of Microbiology, University of Gdansk, Poland. J Mol Biol 198: 203-10 (1987) The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda. The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either. However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent. After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication. Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase. The rifampicin resistance of the normal replication of lambda plasmids in E. coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process. We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda. The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed. scan with 19 sites: 1 48502 J02459 21692 1 12.799319 -0.194192 0.423013 1 48502 J02459 47961 1 9.918903 -1.104177 0.134758 1 48502 J02459 27149 -1 9.588753 -1.208479 0.113432 1 48502 J02459 47546 -1 8.819367 -1.451544 0.073314 1 48502 J02459 34249 1 8.798705 -1.458072 0.072410 REFERENCE 20 (bases 39062 to 39170) AUTHORS Denniston-Thompson,K., Moore,D.D., Kruger,K.E., Furth,M.E. and Blattner,F.R. TITLE Physical structure of the replication origin of bacteriophage lambda JOURNAL Science 198 (4321), 1051-1056 (1977) MEDLINE 78054731 REFERENCE 54 (bases 38686 to 39224) Cannot confirm this... Transcriptional activation of the origin of coliphage lambda DNA replication is regulated by the host DnaA initiator function. Wegrzyn G; Szalewska-Palasz A; Wegrzyn A; Obuchowski M; Taylor K Department of Molecular Biology, University of Gdansk, Poland. Gene 154: 47-50 (1995) The initiator of phage lambda DNA replication, the lambda O protein, is considered to be an analogue of the initiator of DNA replication (DnaA) of its host, Escherichia coli. Both specifically recognize their origins of replication, ori lambda and oriC, respectively, and organize the assembly of specific replication complexes. However, DnaA has an additional activation function, acting on oriC-proximal DnaA-boxes, and regulating transcription initiated at promoters in and around oriC. Here, we demonstrate that lambda plasmid replication can be synchronized by a temperature shift-down that caused renaturation of the previously denatured DnaAts protein. Moreover, we show that elimination of the activating DnaA function affects transcriptional activation at ori lambda. DnaA may act by binding to DnaA-boxes, situated around the lambda pR promoter; there are no such sequences in ori lambda. Our results being to explain in molecular terms why lambda plasmid replication is DnaA-dependent [Kur et al., J. Mol. Biol. 198 (1987) 203-210] and why the initiation of phage lambda DNA replication is blocked (in E. coli devoid of prophage Rac) after inactivation of DnaA [Wegrzyn et al., Genetics (1995) in press]. AHA!!! but mRNA 38023..38135 /note="mRNA-pr (alt.; via tr0 terminator)" not close... *) (******************************************************************) (* ftsQ-ftsA The effect of DnaA protein levels and the rate of initiation at oriC on transcription originating in the ftsQ and ftsA genes: in vivo experiments. Masters M; Paterson T; Popplewell AG; Owen-Hughes T; Pringle JH; Begg KJ Department of Molecular Biology, University of Edinburgh, Scotland. Mol Gen Genet 216: 475-83 (1989) The DnaA protein of Escherichia coli, essential for initiation at oriC, binds at a defined sequence which occurs at the chromosomal origin, near plasmid replication origins and in the promoters of the dnaA and mioC genes. This sequence also occurs at many other sites on the E. coli chromosome including three sites within the essential cell division genes ftsQ and A. Using an fts-lac fusion phage, lambda JFL100, we show here that fts gene expression responds both to reduced and increased intracellular levels of DnaA protein in a manner consistent with the hypothesis that DnaA protein regulates fts gene expression. Experiments using dnaC and dnaB-ts strains, however, suggest that DnaA control of fts transcription may be indirect, at least in part, with fts responding to the rate of initiation at oriC as well as to changes in DnaA protein level per se. It differs in this respect from dnaA gene expression which is unaffected when initiation of replication is inhibited by DnaB or DnaC inactivation. Strains integratively suppressed with pKN500 behave anomalously; neither fts nor dnaA transcription is significantly increased when DnaA is inactivated in these strains. Probable location (not proven from the paper, only by scan): X02821 1 2489 X02821 1381 -1 8.888622 1.365787 0.086003 organism E.coli; chromosome E.coli; piece X02821; name = "NOT PROVEN ftsQ-ftsA"; GET from 1381 -100 to same +100 direction +; *) (******************************************************************) (* guaBA Regulation of the gua operon of Escherichia coli by the DnaA protein. Tesfa-Selase F; Drabble WT Department of Biochemistry, University of Southampton, UK. Mol Gen Genet 231: 256-64 (1992) The guaBA operon determines production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during guanine nucleotide biosynthesis. Two DnaA boxes, binding sites for the DNA replication-initiating DnaA protein, are present in the gua operon, one at the gua promoter (guaP) and the other within the guaB coding sequence. Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions. In these strains beta-galactosidase acts as a reporter enzyme for transcription initiated at guaP. When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed. Reducing the intracellular level of DnaA, either by sequestration with an oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP. Thus the transcriptional activity of the gua operon is coupled, through the DnaA protein, to the DNA replication cycle. Repression of guaP by DnaA was dependent on the presence of both boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA. 3 2489 X02821 1373 -1 7.463746 -1.850606 0.032113 (mid guaB) organism E.coli; chromosome E.coli; piece X02821; name = "NOT PROVEN guaBA"; get from 1373 -100 to same +100 direction +; *) (******************************************************************) (* ColE1 Masai H; Asai T; Kubota Y; Arai K; Kogoma T EMBO J 13: 5338-45 (1994) DnaA-dependent plasmid replicons such as F, Rts1 and RK2... The effect of dnaA protein and n' sites on the replication of plasmid ColE1. Ma D; Campbell JL Division of Chemistry, California Institute of Technology, Pasadena 91125. J Biol Chem 263: 15008-15 (1988) The role of the dna A protein in the replication of plasmid ColE1 and its derivatives was examined. Wild-type and mutant ColE1 plasmids were compared as to their ability to replicate in an in vitro replication system supplemented with ammonium sulfate fractionated extracts from a dnaA-overproducing strain. Synthesis on plasmid templates containing the wild-type origin of replication was stimulated 1.3-fold by addition of the dnaA-overproducing extract. A larger effect was observed after deletion of the primosome assembly site, the n' site, on the leading strand. On the latter template, synthesis was only about one-half that observed with the wild-type templates, but synthesis could be restored to normal levels by addition of the dnaA-overproducing fractions. When the n' site on the lagging strand of pBR322 was deleted, synthesis in the in vitro replication system was reduced to less than 10% of levels seen with intact templates. dnaA-overproducing extract did not restore activity since the dnaA site was also deleted on these plasmids. To verify that the observed stimulation of wild-type and leading strand n' site mutants was due to the dnaA protein, dnaA protein was purified to greater than 50% homogeneity, and antiserum was prepared. The purified protein stimulated synthesis on the plasmid templates to the same extent as the overproducing extracts, and dnaA antiserum blocked stimulation both by extracts and by the purified protein. Thus, dnaA protein, and, by inference, the dnaA recognition site at the ColE1 origin of replication seem to be important for ColE1 replication. The effect of dnaA protein is enhanced when the n'site is defective, suggesting that the dnaA protein plays a role similar to that of the proteins i, n, n', and n'' in directing primosome assembly, as proposed by Seufert, W., and Messer, W. ((1987) Cell 48, 73-78). Scan of Xer sequences for Ri > 3 ColE1 1 6646 J01566 92 1 3.940043 -3.349390 0.000405 1 6646 J01566 1279 -1 9.747398 -1.721475 0.042582 REFERENCE 1 (bases 1008 to 1370) AUTHORS Tomizawa,J.-i., Ohmori,H. and Bird,R.E. TITLE Origin of replication of colicin E1 plasmid DNA JOURNAL Proc. Natl. Acad. Sci. U.S.A. 74, 1865-1869 (1977) MEDLINE 77193942 This pins the site into the origin! later with 20 sites we get: 1 6646 J01566 1279 -1 19.872823 1.803549 0.035651 1 6646 J01566 1257 1 8.330031 -1.584416 0.056550 organism P.ColE1; chromosome P.ColE1; name "ColE1"; piece J01566; get from 1279 -100 to same +100 direction +; ________________________________________________________________________________ * piece 1, J01566, ColE1, config: linear, direction: -, begin: 1379, end: 1179 * *1370 * *1360 * *1350 5' c a c g t c c a g g c g c t t t t c c g c t t c c t c g c t 3' 3' g t g c a g g t c c g c g a a a a g g c g a a g g a g c g a 5' * *1340 * *1330 * *1320 * *1310 5' c a c t g a c t c g c t a c g c t c g g t c g t t c g a c t g c g g c g a g c g 3' 3' g t g a c t g a g c g a t g c g a g c c a g c a a g c t g a c g c c g c t c g c 5' * *1300 * *1290 * *1280 * *1270 5' g t a c t g a c t c a c a c a a a a a c g g t a a c a c a g t t a t c c a c a g 3' 3' c a t g a c t g a g t g t g t t t t t g c c a t t g t g t c a a t a g g t g t c 5' c a c a g t t a t c c a c a g DnaA g t c DnaA * *1260 * *1250 * *1240 * *1230 5' a a t c a g g g g a t a a g g c c g g a a a g a a c a t g t g a g c a a a a g a 3' 3' t t a g t c c c c t a t t c c g g c c t t t c t t g t a c a c t c g t t t t c t 5' ... a a t c a g DnaA @1279|Ri=22.3|Z= 2.0581|p=0.020 ... t t a g t c c c c t a t t c c g g c DnaA @1257|Ri=10.9|Z= 0.8125|p=0.208 * *1220 * *1210 * *1200 * *1190 5' c c a g g a a c a g g a a g a a g g c c a c g t a g c a g g c g t t t t t c c a 3' 3' g g t c c t t g t c c t t c t t c c g g t g c a t c g t c c g c a a a a a g g t 5' g g c g t t t t t c c a DnaA * *1180 5' t a g g c t c c g c c 3' 3' a t c c g a g g c g g 5' ... t a g g c t c c g DnaA @1196|Ri= 4.5|Z= 2.4153|p=0.008 ________________________________________________________________________________ I searched (irx, blast) for this piece and picked up pBR322: >gb|J01749|SYNPBR322 Cloning vector pBR322, complete genome. Length = 4361 Plus Strand HSPs: Score = 622 (171.9 bits), Expect = 4.0e-44, P = 4.0e-44 Identities = 138/155 (89%), Positives = 138/155 (89%), Strand = Plus / Plus Query: 7 CAGGCGCTTTTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCG 66 |||||||| ||||||||||||||||||||||||||| |||||||||||||| |||||||| Sbjct: 2345 CAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG 2404 ******** Query: 67 AGCGGTACTGACTCACACAAAAACGGTAACACAGTTATCCACAGAATCAGGGGATAAGGC 126 ||||||| ||||| |||| |||||| || |||||||||||||||||||||||| || Sbjct: 2405 AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC 2464 ******** Query: 127 CGGAAAGAACATGTGAGCAAAAGACCAGGAACAGG 161 |||||||||||||||||||||| |||| || ||| Sbjct: 2465 AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG 2499 Score = 241 (66.6 bits), Expect = 1.5e-10, P = 1.5e-10 Identities = 57/68 (83%), Positives = 57/68 (83%), Strand = Plus / Plus Query: 134 AACATGTGAGCAAAAGACCAGGAACAGGAAGAAGGCCACGTAGCAGGCGTTTTTCCATAG 193 || | | |||||||| |||||||| | || |||||| ||| || ||||||||||||||| Sbjct: 2483 AAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAG 2542 Query: 194 GCTCCGCC 201 |||||||| Sbjct: 2543 GCTCCGCC 2550 I marked the two locations of the pBR322 sites. These regions are so similar that it is not fair to include both. Since pBR322 is proven I will use that, with the caveat that the ColE1 sequence may be more "natural" ... note redefinition: +/-8 in coordinates will change these locations. *) (******************************************************************) (* pBR322 This sequence is so similar to ColE1 that I will use it instead of ColE1. Mechanism of DNA A protein-dependent pBR322 DNA replication. DNA A protein-mediated trans-strand loading of the DNA B protein at the origin of pBR322 DNA. Parada CA; Marians KJ Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021. J Biol Chem 266: 18895-906 (1991) pBR322 DNA can be replicated via a DNA A-dependent pathway mediated by its binding to the two DNA A-binding sites (dnaA boxes) present near the plasmid origin. DNA synthesis requires the transcription of RNA II (the leading-strand primer precursor) to generate a specific unwound structure in the region containing the dnaA boxes. In this structure, the DNA containing the dnaA boxes can take the form of either a RNA II-parental H strand pBR322 DNA hybrid opposed by the displaced parental L strand (in the absence of RNase H and DNA polymerase I), or a nascent leading strand-parental H strand DNA duplex opposed by the displaced parental L strand (in the presence of RNase H and DNA polymerase I). These findings defined three types of potential sites for productive DNA A binding: (i) the displaced parental L single strand, (ii) a hairpin formed by the inverted repeat of the two dnaA boxes, and (iii) either the RNA-DNA duplex or the nascent leading strand-parental DNA duplex. By using a combination of: (i) inhibition of the replication of a plasmid carrying oriC by oligonucleotides of various dnaA box sequences and conformation, (ii) a gel mobility shift assay to measure DNA A binding to the same oligonucleotide substrates, (iii) replication of pBR322 DNA templates with either one or no dnaA box, and (iv) photocross-linking to demonstrate DNA A binding to an RNA-DNA hybrid, evidence is presented here that DNA A-mediated pBR322 DNA replication proceeds by a mechanism in which DNA A binds to the duplex side of the unwound origin structures and loads the DNA B protein in trans to the displaced parental L strand DNA. 3 4361 J01749 2439 1 18.562889 1.237510 0.107949 3 4361 J01749 2461 -1 12.519431 -0.387152 0.349322 REFERENCE 15 (bases 2395 to 2495) AUTHORS Fuller,R.S., Funnell,B.E. and Kornberg,A. TITLE The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites JOURNAL Cell 38, 889-900 (1984) Well, the best two cover the origin! misc_binding 2439..2447 /bound_moiety="dnaA" rep_origin 2535 Hiasa.marians1994 discuss DnaA dependent pBR322 DNA replication (page 25001). *) organism C.vector; chromosome C.vector; piece J01749; name "pBR322"; get from 2447 +100 to same -100 direction -; (* Fuller1984 *) name "pBR322"; get from 2453 -100 to same +100 direction +; (* Fuller1984 shows it but they did not note it. Parada.Marians1991 fig 5 and 6; gel shifts and exoIII digestion. *) (******************************************************************) (* plasmid R6K *) organism E.coli; chromosome E.coli; piece V00320; name "R6K GammaOri1"; (* Wu1992 footprint, Wu1994 mutation 4A+7A+9T *) get from 93 +100 to same -100 direction -; name "R6K GammaOri2"; (* Wu1992 footprint, mutations T 7; C 5 *) get from 365 +100 to same -100 direction -; (******************************************************************) (* plasmid R1 L05669 has the same sequence as in Masai.Arai1987, but in the reverse direction. *) organism u.cloning; chromosome u.cloning; piece J01783; name "R1 ori"; (* Masai.Arai1987 footprint *) get from 2000 +100 to same -100 direction -; (******************************************************************) (******************************************************************) (******************************************************************) (******************************************************************) (* UNPROVEN SITES: TITLE [TI] The Escherichia coli dam gene is expressed as a distal gene of a new operon. AUTHOR [AU] Jonczyk P; Hines R; Smith DW ADDRESS [AD] Department of Biology, University of California, San Diego, La Jolla 92093. JOURNAL [JL] Mol Gen Genet 217: 85-96 (1989) ABSTRACT [AB] DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42. Promoter activity was localized using pKO expression vectors and galactokinase assays to two regions, one 1650-2100 bp and the other beyond 2400 bp upstream of the dam gene. No promoter activity was detected immediately in front of this gene; plasmid pDam118, from which the nucleotide sequence of the dam gene was determined, is shown to contain the pBR322 promoter for the primer RNA from the pBR322 rep region present on a 76 bp Sau3A fragment inserted upstream of the dam gene in the correct orientation for dam expression. The nucleotide sequence upstream of dam has been determined. An open reading frame (ORF) is present between the nearest promoter region and the dam gene. Codon usage and base frequency analysis indicate that this is expressed as a protein of predicted size 46 kDa. A protein of size close to 46 kDa is expressed from this region, detected using minicell analysis. No function has been determined for this protein, and no significant homology exist between it and sequences in the PIR protein or GenBank DNA databases. This unidentified reading frame (URF) is termed urf-74.3, since it is an URF located at 74.3 min on the E. coli chromosome. Sequence comparisons between the regions upstream of urf-74.3 and the aroB gene show that the aroB gene is located immediately upstream of urf-74.3, and that the promoter activity nearest to dam is found within the aroB structural gene. This activity is relatively weak (about 15% of that of the E. coli gal operon promoter). The promoter activity detected beyond 2400 bp upstream of dam is likely to be that of the aroB gene, and is 3 to 4 times stronger than that found within the aroB gene. Three potential DnaA binding sites, each with homology of 8 of 9 bp, are present, two in the aroB promoter region and one just upstream of the dam gene. Expression through the site adjacent to the dam gene is enhanced 2- to 4-fold in dnaA mutants at 38 degrees C. Restriction site comparisons map these regions precisely on the Clarke-Carbon plasmids pLC13-42 and pLC29-47, and show that the E. coli ponA (mrcA) gene resides about 6 kb upstream of aroB. *) (* drpA Identification and sequence of the drpA gene from Escherichia coli. Zhou Z; Syvanen M Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis 95616. J Bacteriol 172: 281-6 (1990) The drpA gene of Escherichia coli encodes a factor that is involved in global RNA synthesis. We establish that the drpA gene has been successfully cloned and describe the fine-structure map of three drpA-(Ts) mutations as well as the complete nucleotide sequence of the drpA gene. We identified a major sigma-70 promoter for the drpA gene on the bases of (i) its similarity to the consensus sequence and (ii) S1 protection and primer extension mapping data. In addition, the nucleotide sequence revealed a pair of dnaA boxes and a factor-independent terminator at the 5' end and 3' end of the gene, respectively. The deduced amino acid sequence of the DrpA protein showed a nucleotide-binding pocket found in some ATPases. A total guess of course ... *) (******************************************************************) (******************************************************************) (******************************************************************) (******************************************************************) (******************************************************************) (* NON sites Coliphage 186: need for DnaA is indirect, Williams.Egan1994 M13: need for DnaA is indirect, see reference cited by Williams.Egan1994 This contradicts Fuller1984 that claims specific fragment retention! @article{Williams.Egan1994, author = "S. G. Williams and B. Egan", title = "{DNA} Replication Studies with Coliphage 186: the Involvement of the {{\em Escherichia coli\/}} {DnaA} protein in 186 Replication Is Indirect", journal = "J. Bact.", volume = "176", pages = "6039-6044", year = "1994"} *)