EM structure of the DNA wrapping in bacterial open transcription initiation complex

2024-11-06 REL RCSB RCSB RCSB 2018-11-19 2019-01-16 2020-05-27 2024-11-06 EM structure of the DNA wrapping in bacterial open transcription initiation complex Florez-Ariza A Cassago A de Oliveira PSL Guerra DG DNA wrapping, bacterial transcription initiation complex, transmission electron microscopy, single particle analysis., TRANSCRIPTION, transcription-dna complex Sosa R Florez-Ariza A Diaz-Celis C Onoa B Cassago A de Oliveira PSL Portugal RV Guerra DG Bustamante C Interactions of Upstream and Downstream Promoter Regions with RNA Polymerase are Energetically Coupled and a Target of Regulation in Transcription Initiation Biorxiv US 2020 doi:10.1101/2020.05.13.070375 2692-8205 6n4c FULLOVERLAP E. coli RNAP-DNA wrapped open complex E. coli RNAP-DNA wrapped open complex 0 1 2 3 4 5 6 7 8

Wrapped transcription initiation open complex assembled between E. coli RNAP-sigma 70 holoenzyme and lambda PR wild-type promoter (+18 to -76)

Escherichia coli BL21 0.50 RNA polymerase sigma factor RpoD Escherichia coli K-12 K12 0.064322277 1 LEVO QLKLLVTRGKEQGYLTYAEVNDHLPEDIVDSDQIEDIIQMINDMGIQVMEEAPDADDLMLAENTADEDAAEAAAQVLSSV ESEIGRTTDPVRMYMREMGTVELLTREGEIDIAKRIEDGINQVQCSVAEYPEAITYLLEQYDRVEAEEARLSDLITGFVD PNAENSIDPELAREKFAELRAQYVVTRDTIKAKGRSHATAQEEILKLSEVFKQFRLVPKQFDYLVNSMRVMMDRVRTQER LIMKLCVEQCKMPKKNFITLFTGNETSDTWFNAAIAMNKPWSEKLHDVSEEVHRALQKLQQIEEETGLTIEQVKDINRRM SIGEAKARRAKKEMVEANLRLVISIAKKYTNRGLQFLDLIQEGNIGLMKAVDKFEYRRGYKFSTYATWWIRQAITRSIAD QARTIRIPVHMIETINKLNRISRQMLQEMGREPTPEELAERMLMPEDKIRKVLKISMETPIGDDEDSHLGIDFIEDTTSA TTESLRAATHDVLAGLTAREAKVLRMRFGIDMNTDYTLEEVGKQFDVTRERIRQIEAKALRKLRHPSRSEVLRSFLDD P00579 DNA-directed RNA polymerase subunit beta Escherichia coli K-12 K12 0.150689672 1 LEVO VYSYTEKKRIRKDFGKRPQVLDVPYLLSIQLDSFQKFIEQDPEGQYGLEAAFRSVFPIQSYSGNSELQYVSYRLGEPVFD VQECQIRGVTYSAPLRVKLRLVIYEREAPEGTVKDIKEQEVYMGEIPLMTDNGTFVINGTERVIVSQLHRSPGVFFDSDK GKTHSSGKVLYNARIIPYRGSWLDFEFDPKDNLFVRIDRRRKLPATIILRALNYTTEQILDLFFEKVIFEIRDNKLQMEL VPERLRGETASFDIEANGKVYVEKGRRITARHIRQLEKDDVKLIEVPVEYIAGKVVAKDYIDESTGELICAANMELSLDL LAKLSQSGHKRIETLFTNDLDHGPYISETLRVDPTNDRLSALVEIYRMMRPGEPPTREAAESLFENLFFSEDRYDLSAVG RMKFNRSLLREEIEGSGILSKDDIIDVMKKLIDIRNGKGEVDDIDHLGNRRIRSVGEMAENQFRVGLVRVERAVKERLSL GDLDTLMPQDMINAKPISAAVKEFFGSSQLSQFMDQNNPLSEITHKRRISALGPGGLTRERAGFEVRDVHPTHYGRVCPI ETPEGPNIGLINSLSVYAQTNEYGFLETPYRKVTDGVVTDEIHYLSAIEEGNYVIAQANSNLDEEGHFVEDLVTCRSKGE SSLFSRDQVDYMDVSTQQVVSVGASLIPFLEHDDANRALMGANMQRQAVPTLRADKPLVGTGMERAVAVDSGVTAVAKRG GVVQYVDASRIVIKVNEDEMYPGEAGIDIYNLTKYTRSNQNTCINQMPCVSLGEPVERGDVLADGPSTDLGELALGQNMR VAFMPWNGYNFEDSILVSERVVQEDRFTTIHIQELACVSRDTKLGPEEITADIPNVGEAALSKLDESGIVYIGAEVTGGD ILVGKVTPKGETQLTPEEKLLRAIFGEKASDVKDSSLRVPNGVSGTVIDVQVFTRDGVEKDKRALEIEEMQLKQAKKDLS EELQILEAGLFSRIRAVLVAGGVEAEKLDKLPRDRWLELGLTDEEKQNQLEQLAEQYDELKHEFEKKLEAKRRKITQGDD LAPGVLKIVKVYLAVKRRIQPGDKMAGRHGNKGVISKINPIEDMPYDENGTPVDIVLNPLGVPSRMNIGQILETHLGMAA KGIGDKINAMLKQQQEVAKLREFIQRAYDLGADVRQKVDLSTFSDEEVMRLAENLRKGMPIATPVFDGAKEAEIKELLKL GDLPTSGQIRLYDGRTGEQFERPVTVGYMYMLKLNHLVDDKMHARSTGSYSLVTQQPLGGKAQFGGQRFGEMEVWALEAY GAAYTLQEMLTVKSDDVNGRTKMYKNIVDGNHQMEPGMPESFNVLLKEIRSLGINIELEDE A0A0A0GWV9 2.7.7.6 DNA-directed RNA polymerase subunit beta' Escherichia coli K-12 K12 0.150320219 1 LEVO EEFDAIKIALASPDMIRSWSFGEVKKPETINYRTFKPERDGLFCARIFGPVKDYECLCGKYKRLKHRGVICEKCGVEVTQ TKVRRERMGHIELASPTAHIWFLKSLPSRIGLLLDMPLRDIERVLYFESYVVIEGGMTNLERQQILTEEQYLDALEEFGD EFDAKMGAEAIQALLKSMDLEQECEQLREELNETNSETKRKKLTKRIKLLEAFVQSGNKPEWMILTVLPVLPPDLRPLVP LDGGRFATSDLNDLYRRVINRNNRLKRLLDLAAPDIIVRNEKRMLQEAVDALLDNGRRGRAITGSNKRPLKSLADMIKGK QGRFRQNLLGKRVDYSGRSVITVGPYLRLHQCGLPKKMALELFKPFIYGKLELRGLATTIKAAKKMVEREEAVVWDILDE VIREHPVLLNRAPTLHRLGIQAFEPVLIEGKAIQLHPLVCAAYNADFDGDQMAVHVPLTLEAQLEARALMMSTNNILSPA NGEPIIVPSQDVVLGLYYMTRDCVNAKGMVLTGPAERLYRSGLASLHARVKVRITEYEKDANGELVAKTSLKDTTVGRAI LWMIVPKGLPYSIVNQALGKKAISKMLNTCYRILGLKPTVIFADQIMYTGFAYAARSGASVGIDDMVIPEKKHEIISEAE AEVAEIQEQFQSGLVTAGERYNKVIDIWAAANDRVSKAMMDNLQTETVINRDGQEEKQVSFNSIYMMADSGARGSAAQIR QLAGMRGLMAKPDGSIIETPITANFREGLNVLQYFISTHGARKGLADTALKTANSGYLTRRLVDVAQDLVVTEDDCGTHE GIMMTPVIEGGDVKEPLRDRVLGRVTAEDVLKPGTADILVPRNTLLHEQWCDLLEENSVDAVKVRSVVSCDTDFGVCAHC YGRDLARGHIINKGEAIGVIAAQSIGEPGTQLTMRTFHIGGAASRAAAESSIQVKNKGSIKLSNVKSVVNSSGKLVITSR NTELKLIDEFGRTKESYKVPYGAVLAKGDGEQVAGGETVANWDPHTMPVITEVSGFVRFTDMIDGQTITRQTDELTGLSS LVVLDSAERTAGGKDLRPALKIVDAQGNDVLIPGTDMPAQYFLPGKAIVQLEDGVQISSGDTLARIPQESGGTKDITGGL PRVADLFEARRPKEPAILAEISGIVSFGKETKGKRRLVITPVDGSDPYEEMIPKWRQLNVFEGERVERGDVISDGPEAPH DILRLRGVHAVTRYIVNEVQDVYRLQGVKINDKHIEVIVRQMLRKATIVNAGSSDFLEGEQVEYSRVKIANRELEANGKV GATYSRDLLGITKASLATESFISAASFQETTRVLTEAAVAGKRDELRGLKENVIVGRLIPAGTGYAYHQDRMRRRAAG A0A369F490 2.7.7.6 DNA-directed RNA polymerase subunit alpha Escherichia coli K-12 K12 0.035275273 1 LEVO TEFLKPRLVDIEQVSSTHAKVTLEPLERGFGHTLGNALRRILLSSMPGCAVTEVEIDGVLHEYSTKEGVQEDILEILLNL KGLAVRVQGKDEVILTLNKSGIGPVTAADITHDGDVEIVKPQHVICHLTDENASISMRIKVQRGRGYVPASTRIHSEEDE RPIGRLLVDACYSPVERIAYNVEAARVEQRTDLDKLVIEMETNGTIDPEEAIRRAATILAEQLEAFVDLRDVRQPEVKEE KPEFDPILLRPVDDLELTVRSANCLKAEAIHYIGDLVQRTEVELLKTPNLGKKSLTEIKDVLASRGLSLGMRLENW P0A7Z4 2.7.7.6 DNA-directed RNA polymerase subunit alpha Escherichia coli K-12 K12 0.034400266 1 LEVO TEFLKPRLVDIEQVSSTHAKVTLEPLERGFGHTLGNALRRILLSSMPGCAVTEVEIDGVLHEYSTKEGVQEDILEILLNL KGLAVRVQGKDEVILTLNKSGIGPVTAADITHDGDVEIVKPQHVICHLTDENASISMRIKVQRGRGYVPASTRIHSEEDE RPIGRLLVDACYSPVERIAYNVEAARVEQRTDLDKLVIEMETNGTIDPEEAIRRAATILAEQLEAFVDLRDVRQPEVKEE KPEFDPILLLPVDDLELTVRSANCLKAEAIHYIGDLVQRTEVELLKTPNLGKKSLTEIKDVLASRGLSLG P0A7Z4 2.7.7.6 DNA-directed RNA polymerase subunit omega Escherichia coli K-12 K12 0.010118352 1 LEVO ARVTVQDAVEKIGNRFDLVLVAARRARQMQVGGKDPLVPEENDKTTVIALREIEEGLINNQILDVRERQEQQEQEAAELQ AVTAIAEGRR P0A800 2.7.7.6 DNA (94-MER) Enterobacteria phage lambda 0.029094616999999996

NT strand DNA (94-MER)

1 (DA)(DA)(DT)(DC)(DT)(DA)(DT)(DC)(DA)(DC)(DC)(DG)(DC)(DA)(DA)(DG)(DG)(DG)(DA)(DT) (DA)(DA)(DA)(DT)(DA)(DT)(DC)(DT)(DA)(DA)(DC)(DA)(DC)(DC)(DG)(DT)(DG)(DC)(DG)(DT) (DG)(DT)(DT)(DG)(DA)(DC)(DT)(DA)(DT)(DT)(DT)(DT)(DA)(DC)(DC)(DT)(DC)(DT)(DG)(DG) (DC)(DG)(DG)(DT)(DG)(DA)(DT)(DA)(DA)(DT)(DG)(DG)(DT)(DT)(DG)(DC)(DA)(DT)(DG)(DT) (DA)(DC)(DT)(DA)(DA)(DG)(DG)(DA)(DG)(DG)(DT)(DT)(DG)(DT) DNA DNA (94-MER) Enterobacteria phage lambda 0.028890528999999998

T strand DNA (94-MER)

1 (DA)(DC)(DA)(DA)(DC)(DC)(DT)(DC)(DC)(DT)(DT)(DA)(DG)(DT)(DA)(DC)(DA)(DT)(DG)(DC) (DA)(DA)(DC)(DC)(DA)(DT)(DT)(DA)(DT)(DC)(DA)(DC)(DC)(DG)(DC)(DC)(DA)(DG)(DA)(DG) (DG)(DT)(DA)(DA)(DA)(DA)(DT)(DA)(DG)(DT)(DC)(DA)(DA)(DC)(DA)(DC)(DG)(DC)(DA)(DC) (DG)(DG)(DT)(DG)(DT)(DT)(DA)(DG)(DA)(DT)(DA)(DT)(DT)(DT)(DA)(DT)(DC)(DC)(DC)(DT) (DT)(DG)(DC)(DG)(DG)(DT)(DG)(DA)(DT)(DA)(DG)(DA)(DT)(DT) DNA singleParticle particle 7.9 NEGATIVE Uranyl Acetate

Negatively stained EM specimens were prepared using a 2% uranyl acetate solution.

CARBON CONTINUOUS

JEOL 2100 FLOOD BEAM BRIGHT FIELD LAB6 200 TVIPS TEMCAM-F416 (4k x 4k) 20.0 1 60393 C1 17.0 FSC 1/2 BIT CUT-OFF IMAGIC 16015 RANDOM ASSIGNMENT IMAGIC ANGULAR RECONSTITUTION IMAGIC IMAGIC emd_0340.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 90 90 90 0 0 0 90 90 90 320.4 320.4 320.4 90.0 90.0 90.0 X Y Z -6.3740783 14.199835 0.000000000759306 1.0 3.56 3.56 3.56 0.42 AUTHOR ::::EMDATABANK.org::::EMD-0340:::: EM map of DNA wrapping in bacterial transcription initiation complex 4YLN PDB experimental model OTHER

Initial fiting was done using manual fitting in Chimera. Yasara software was used for energy minimizaton of the DNA and RNAP coordinates model.