Steps in gene expression: comparison of prokaryotic and eukaryotic cells

Transcription

1 Steps in gene expression: comprison of prokryotic nd eukryotic cells

2 DNA 1 Trnscript RNA 2 3 mrna RNA RNA Processing Trnsport Trnscription control Primry RNA Nucleus Cytoplsm mrna Degrdtion Trnsltion Control 4 Inctive mrna 5 Protein Protein Activity 6 Inctive Protein Six steps t which eukryotic gene expression cn be controlled. In prokryotic cells, genes do not hve introns (no step 2) nd trnscription nd trnsltion re not seprted in spce nd time (no step 3).

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4 Trnscription of DNA into RNA by RNA polymerse. 1. Requires DNA templte, 4 ribonucleotide 5 triphosphtes, Mg De novo synthesis: does not require primer. Low fidelity compred to DNA polymerse: errors 1/ RNA polymerse incorportes ~30 nt/s (much slower thn DNA polymerse). Templte strnd 3. Activity highly regulted in vivo: t initition, elongtion nd termintion. 4. The nucleotide t the 5 end of n RNA strnd retins ll three of its phosphte groups; ll subsequent nucleotides relese pyrophosphte (PPi) when dded to the chin nd retin only their phosphte (red). 5. The relesed PPi is subsequently hydrolyzed by pyrophosphtse to Pi, driving the equilibrium of the overll rection towrd chin elongtion. 6. In most cses, only one DNA strnd is trnscribed into RNA. Non-templte (coding) strnd

5 Trnscription of two genes on different templte strnds. Note tht growth of the trnscript lwys occurs in the 5 -to-3 direction. The convention for designting the 5 nd 3 ends of gene

6 Biochemicl studies of bcteril RNA polymerse 1. DNA binding ssys- DNseI footprinting Gel shift 2. Role of individul subunits- dissocition of holoenzyme by column chromtogrphy

7 DNse I footprinting, common technique for identifying protein-binding sites in DNA. 1. A DNA frgment is lbeled t one end with 32 P (red dot). 2. Portions of the smple then re digested with DNse I in the presence nd bsence of protein tht binds to specific sequence in the frgment. 3. A low concentrtion of DNse I is used so tht on verge ech DNA molecule is cleved just once (verticl rrows). 4. The two smples of DNA then re seprted from protein, dentured to seprte the strnds, nd electrophoresed. The resulting gel is nlyzed by utordiogrphy, which detects only lbeled strnds nd revels frgments extending from the lbeled end to the site of clevge by DNse I.

8 DNseI footprint of RNAP on the lc promoter Lnes 1 nd 2 re DNA sequencing rections for orienttion Lne 3 is the no protein control Lne 4 contined RNAP Footprint: Advntge- cn ssy short-lived interctions; proteins hve very chrcteristic binding ptterns Disdvntge- requires nerly stoichiometric binding

9 Gel shift : electrophoretic mobility shift ssy ( EMSA ) for DNA-binding proteins * Protein-DNA complex * Free DNA probe 1. Prepre lbeled DNA probe 2. Bind protein 3. Ntive gel electrophoresis Advntge: sensitive Disdvntge: requires stble complex; little structurl informtion bout which protein is binding

10 Ion-exchnge chromtogrphy

11 Dissocition of RNAP nd purifiction of s by ion-exchnge chromtogrphy w s b b [NCl] [protein] Crboxymethyl- (-CO 2-2 ) or phospho- (-PO 3-2 ) cellulose s Frction number w b b

12 E. coli RNA polymerse holoenzyme bound to DNA. w Subunit Stoichiometry Role in holoenzyme 2 Binds regultory sequences/proteins b 1 Polymerse; Binds DNA templte b 1 Polymerse; Binds DNA templte s 1 Promoter recognition w 1 RNAP ssembly

13 Thermus quticus core RNA polymerse Blue b Red b Green w Ornge PDB (Protein Dt Bnk) File 1HQM Minkhin, L. et l. Proc.Nt.Acd.Sci.USA 98 pp. 892 (2001)

14 A single RNA polymerse mkes multiple types of RNAs in prokryotes

15 The dissocible sigm subunit gives promoter specificity to prokryotic RNA polymerse (RNAP) w b b + s w s b b Core enzyme No specific promoter binding; tight non-specific DNA binding (K d ~5 x M) Holoenzyme Specific promoter binding; wek non-specific DNA binding (K d ~10-7 M)

16 Holoenzyme Trnscription initition by prokryotic RNA polymerse s b b scnning Promoter Closed complex rntps PPi s b b Core enzyme Open complex; initition 5 pppa mrna b b s

17 Sigm Fctors of E. coli Sigm Fctor Promoters Recognized Promoter Consensus -35 Region -10 Region s70 Most genes TTGACAT TATAAT s32 Genes induced by het shock TCTCNCCCTTGAA CCCCATNTA s28 Genes for motility nd chemotxis CTAAA CCGATAT s38 Genes for sttionry phse nd stress response?? -24 Region -12 Region s54 Genes for nitrogen metbolism nd other functions CTGGNA TTGCA

18 Trnscriptionl elongtion

19 Supercoiling of DNA during trnscription cuses requirement for topoisomerses

20 Rho-independent prokryotic trnscription termintion

21 Rho-dependent trnscription termintion Rho binding site Rho: forms RNA-dependent hexmeric ATPse, trnsloctes long RNA 5 -to-3, promoting dissocition from polymerse termintion Pltt, Ann. Rev. Biochem. 55: 339 (1986)