Solution of identical DNA fragments radioactively labeled s at one end of one strand Treat with DNase Site of under conditions in DNase cut which each strand is cut once(on average). No cuts are made in the area where RNA polymerase has bound Footprinting: Purified RNA polymerase (or other DNA binding protein) is first mixed with isolated and labeled DNA fragment that is believed to bind to the added protein Before that DNA is cut with nonspecific DNase
Footprinting: Purified RNA polymerase (or other DNA binding protein) is first mixed with isolated and labeled DNA fragment that is believed to bind to the added protein Before that DNA is cut with nonspecific DNase
In the presence of DNA-binding protein In the absence of DNA-binding protein Isolate labeled DNA fragments and denature. Only labeled strands are detected in next step. ** ***水 Separate fragments by polyacrylamide gel electrophoresis and visualize radiolabeled bands on x-ray film Uncut DNA fragment DNA migration Missing bands indicate here RNa polymerase was bound to dNA
In the presence of DNA-binding protein In the absence of DNA-binding protein
Coding strand The An actual footprinting footprints result (RNa polymerase c1o binding to a lac promoter) Regions bound by 20 RNA polymerase 30 -40 50
An actual footprinting result (RNA polymerase binding to a lac promoter) The footprints
UP element F-35 Region Spacer-10 Region Spacer RNA start +1 Consensus NNAAAn AATA TTTNNAAAANNN N TTGACA N17 TATAAT N sequence rnB P1 AGAAAATTATTTTAAATTTCCT N GTGTCA N6 TATAAT Ns A Present only in certain highly expressed genes trp TTGACA NiT TTAACT N,A Sequences of the TTTACA N7 TATGTT NA coding DNa strand is conventional TTGATA N TATAAT N A Shown ara BAD CTGACG N1s TACTGT NA TTTACC N TATAAT Alignment of different promoter sequences from E coli genes: the-10(the Pribnow box)and 35 consensus region were revealed
Alignment of different promoter sequences from E.coli genes: the –10 (the Pribnow box) and –35 consensus region were revealed. Sequences of the coding DNA strand is conventionally shown Add TTTACC N12 TATAAT N7 A Present only in certain highly expressed genes
2276 Biochemistry. vol 30. No 8./ Promoter of E coli Add gene c我A ALIACOOIGOEIOITATAA日 LANGAOICCGACCATCATICATACCACCCIGCCATIAAOIGATATCCATOGCCAC HetleaspTrrLeuProLeumraspIleHlsArgEls 151 CHGATGGCAACATOOCCCCAGACCATOIIGAACLIGGCOGCCAGTATAATATCIOCCTTCCTGCACAATCC LeeaspGlyasnleargrroclnmhrleLeuGlnLeuGlyArsGlnTyrasnlleserLeaProAlaGlnser 226 CTGGAAACACIGACCCCACOLCAGGECAGCCAACGAACCCGATCOCTGAGOHCGACCAAACTGAC LeudlsarLeulerrou1svalGlnvallealassnGluProAspLeavalserrheLemrLyaLeuasp 301 IGGGGCGTAAAGTCIOGCOOICIGATGCCIGIOGCOGOGTGGCATTGAAAACATGAACATGCAGCOOOT TrPGlyvalLyavalLeealaserLeuspAlacysATBArgVaLAlaheGluasnleGIuAsPAleAlRArg 376 CACOGCCTGCACTATGTCCAGCIGCOI-TCACCACGCTACATGCCAATGCCACATCAGCTCCCTGTAGCOOOT EsGIvLeeHisTYralGI Argrbeserrrocly-yrhetAlaMetAlaEiscInLeurrovaLAlaGly S1 C00GAAGOGOIGATOGAGGOGLACOIGAAGOTIGCOGCACCTTGHGTCCAGGOGAAGCTTATOOCCAT alvalGleAlavalneaspclyvalArEGluGlycysArgmrheclyvalGlnAlaLysLealleGlyne 526 ATGAGCCGGACCLLOGAAGCOGCCTGICAGCAAGAGCTGGAGCCCTTTTAGCCCACCGTCACCACATTACCO MetserArgThrbeGleAlAlaCyaGlnclnGlnLeuGleAlapheLeeALaHlsArBAsPGInleThrA HCAA TASAACH A .AsPLeuAlaclyAspGlaLeuGlyPheProGlyserLeahheLeuserElsPheAsnATALaATEAPAla alyurpatsletrvalhtslaGlyGIeAlaAlaGlyProGluserlleTpGInAlalleArgGluL 75. GCGCAGCOZATTGGACATGGOGTAAAAGCCATTGAAGATCGGGOGCTCATGCATTTICTCGCCCAGCAACAAA AlacltArgleclyasGlyualLysAlalleGIeAsPArEAlaLeuhetaspiheLeeAlaGleGlnGlnlle 826 GGTATTGAATCOIGIOICACCICCAATATICAGACCACCACCGTAGCAGAGCTGGCTGCACATCCGCTCAAAA GlyeGlusercyaLeunrserAsnlleGlnThrserThrTalAlaGlnLeuAlaAlaHisProLeulysTh 901 TC0GAGCATGGCATTOGIGCCAGCATTAACAGTCACCATCCCGCCGTACAGGCAGTGGATATCATTCACCAA TheLeudluhssclyieArgAlaserIleAsnThrAsPAspProGlyvalGinGlyvalAspIeleHisGlu 976 TATACCOTGCCGCGCCAGCTCCTGOGTTATOCOGCGAGCAAATCCGCCAGGCACACATTAATGGC AANC TyrmrialALaalarroalaAlaGlyteserArgGluGinIleArgGInAlaGinnleAsnGlyLeaGleMet 1051 GCLCO2CAGOGCLCAGCAAAAACGCGCACTGCCACAAAAAGTOGCCGCCAAGTAACAAAATGGAT A上 erALadlecinLyaArgAlaLeeArscleLysvalALaAlaLysEnd 1126 YGCACACCACATGCAAOGCACAAA se (ada) gene and nanking Putatre regulatory d These incd 10 and-35. an operator which consists of a ng ste(RBS.and by the averted arrows)
Promoter of E.coli Add gene