Prokaryotic vs. eukaryotic, Apuntes de Bioquímica

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sectetión endoplesmic reticulum >, di Sell camplox 7 vell membrane Prokaryotic vs. euk == lu a ; signal ltochor e Esquence ES ó ; A Ple A Ey proteins mn peroxisome, ES E enlofoplast a E == -_ == Notaphase chromosome MD 30-nm chromnatin fibar of packed nuclsosemes Canclensed scaffold- assocated oran aiddó pe on-estring" form of ehromatin | _—Chromasome = A soaffol Extended scaffold- Short assocatod 300 nm region ol form DÑA double- helix / AACOCICINCO tán Organización genémica y transcripción en precarietas y eucariotas Tránserite primaria Pre-mRNA JA MABURACIÓN TRABUCCHÓN Agisión de caperuza (capping) Poli-A denilasión Bliminación de intrenes (splicing) SF nos y A y e Regulación de la expresión génica Pancreas cell O Glycolysis enzyme gene Crystallin gene eN) Insulin gene Hemoglobin gene Active gene A A Eye lens cell (in embryo) CJ 57 MO Inactive gene Ny Q= E 7 E Sr as Liver cDNAs Liver ANA Kidney RNA Brain RANA | => 1 a mM a uo ds Transcripción: preguntas relevantes -Céóme se eseege qué yen se va a transcribir - Céme se transcrile - Cómo deja de iranscribiras - Céóme se meslula la expresión de les genes (cúando, cuánte y dénda) RNA pelimerasas eucariotas TABLE 6-2 The Three RNA Polymerases in Eucaryotic Cells Relative Activity a-Amanitin Sensitt TYPE OF POLTMERASE GENES TRANSCRIBED | Ens hecatlon Zo RNA polymerase | 585, 185, and 28S RNA genes ANA polymerase 1 Nudeolus Ei peri RANA polyunerase Il all protein-coding genes, plus snoRNA genes ANA polimerase Nucieoriasal E a podesspecif y e DR gas RNA polymerase Il. Nacleop RA polymerase 1 URNA genes, 5S TRNA genes, same sriKiÁ genes send genes for other small NAS TABLE 11-4. Subunits of S, cerevisias s Nuclear ANA Palymerases l, ll, and 112 E, coli core Eukaryotic pu Sd RNA polymerase RNA polymerases ' y "n LS (ora/óp *00) : " Unién TNTPs eN os Pe ALSO B220 Ci60 ! an A A135 B150 Cie | E (y “felike subunits Unién BNA ACÍ0 Ls, ACHO ACIA ACIS CTD (YSPTSPS)a n= 26 en levadura . o l e ensamblaje Common SuBuniTs 0 “ABC274] ñ ABC23 am: clik Same as 1 [ABCHA.S) Same as 1 [a] a !

+ subunits ABCIO% ! ABCIOB ! Srecine Susustrs h 249 B32 cs h . 243 Bl6 os 6) [5] RNP en: A34.5 BI2.6 C37 ¡ Alá BL2,5 C34 0 [e] 4122 GS ! ; A c25 i Mitesendria $ h 9 Common en ! subunits *Subusis are named according to thelr appatca! molecular DD: Clereplastes «weights in kilodalrons based on mobility in SDS ! pelyacrylamide gel electrophoresis preceded by A, B, or C for nn Aa “ubenitz of RNA polymeraces l, M, or El, respective. : Sabonics shared by all thres polymerases are designated ABC. ¡ Tots shared by RNA polymerase | ane II are designated NM: Ml AC. (2) indicate two molecules of the indicated subunie per h - Le poleas ! Additional Souacz: A. Sentegac es al., 1992, in S. L. MeKnighe and Ko Ro +4 01 + enzyme-specific Yamamoto, eds., Transcriptiomal Regulation, Cold Speing subunits | Harbor Laboratory Press, pp. 27-54. Figure 9.5 Yeast RNA polymerase has grooves that Apbl active site loop could be binding sites for nucleic acids. The pink Bpbn paa Metel A beads show a possible path for DNA that is —25 Á . wide and 5-10 Á deep. The green beads show a , Tr, E narrower channel, 12-15 Á wide and 20 Á deep, that So $ could hold ANA. Photograph kindly provided by Roger Komberg. vara aer Se Fourier seee 50% v Tobacco pyrophosphatase ireatmént v Ligate 5 RNA adapter SOACHA Y 1* strand CDNA synthesis (gene-specific or random oli igos) v 2? strand synthesis and amplification (PSR veith gene-speciñe 8 5'adepter oligos) Run PCR products on >2% agarose gel canal api GONA DIS RNA TAP- TAP+ peinar 5 end procesead Send pia trar ner pte, q. CIA? RACE mapping of transcript ends 5" RACE Ligate 3" RNA adapter (canies inverted deo«ythymidine at 3' and do prevent olzomerizalir) 14 RNA ligaso FOH— 5 PI 10 Cut 3 gel-elute bands of interest TA-cloning .—— v 1" strand DNA synthesis (3' adapter-specific oligo) —— M 2" strand synthesis ano amplification (PCR wih gene-specific 4 3' adapter oigos) — — v Run PGR products on >2% agarose gel Análisis de facteres/secuencias de centre! de transcripción Levitra Criaderos ade aid retardo en gal, suUpSr-Te bara Festprimáing run en hiparsena le lbs a DAA sel Iinmunepreepitadén de «e medina misrearays ln via: transfeeción de sálvime en cultiva estaleles y iranelterias delechnres de premetar stransfeeción een factores de iranseripelén Orv —hiylerdl ade levaabura RHA Interferencia Animales y plantas iransgénicss run en ¿3 a 3 IÓ: El |-8 al E Determinación de sitios de hipersensibilidad a DNasal CDNA clenes (preles) PCR product amplification purification printing mRNA target) ona” Tar E HIPO Lacan Puiczecod Te a E Es target te micrearray micrearray excitatien R Ed laser 2 y ¿ scanning ¡sejen . - everlay images ane nermalise y Análisis de facteres/secuencias de centre! de transcripción takina cremategrafíac as afinidad reltarae en gel, suesr-Ta lara Festiprinting run en hipsrcenade laa a DNA cel inmunepracipitación «a crematina MiCTemTayE de via transfeecin de sélules en cultivo estados y irarelterias delechones de premeter estransieeción een faciores de iranseripalón Hr —hyierial abs lev amor RHA interferencia Animales y plantas tranregénicet Transfecciones estables pSuperior,puro a 4354 bps es-transfección cen facteres ele transcripción IN VIVO ASSAY FOR TRANSCRIPTION FACTOR ASSAY Raportar gene 6 oi ES ! dl Xona 2 plasmids Abinding ( site 1: ercodes potential trans factor X ee 2: repcrter gene and binding sites for X protein Plasmid 1 Plasmid 2 pra ! Xtactor memrane Both plasmids introduced Into same cell, hal which lacks X proten. Measure procuctión of reporter activity Meperter gens tanscripts vd Nucleus