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Transcription Introduction - Definitions RNA polymerases: • architecture • catalysis/metal ions

Transcription Introduction - Definitions RNA polymerases: • architecture • catalysis/metal ions • proofreading 2) How to detect DNA protein interactions in vitro: DNA footprinting 3) Recognition o f bacterial promoters by sigma factors 4) The prokaryotic transcriptional cycle

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Transcription Introduction - Definitions RNA polymerases: • architecture • catalysis/metal ions

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  1. Transcription • Introduction - Definitions • RNA polymerases: • • architecture • • catalysis/metal ions • • proofreading • 2) How to detect DNA protein interactions • in vitro: DNA footprinting • 3) Recognition of bacterial promoters by sigma factors • 4) The prokaryotic transcriptional cycle • 5)Transcription regulation in prokaryotes • 6) RNA polymerases in eukaryotes • 8) Assembly of eukaryotic RNA Pol.II on promoters • 9) RNA Pol.IITranscriptional activators • 10) The issue of chromatin of eukaryotic genomes • 11) Transcription regulation by pausing

  2. Nomenclature used to describe a gene Transcriptional Start Site Transcriptional Terminator Stop Codon Start Codon +1 Coding Region = ORF(Open-reading Frame) 5’ 3’ 3’ 5’ 5’ Flanking Region 5’UTR 3’Flanking Region 3’UTR Coding= Non Template = Sense Strand Strand Strand Non-Coding = Template = Antisense Strand Strand Strand * Promoter = The region near the 5’ end of the Transcribed region to which the RNA polymerase Binds prior to the initiation of transcription • UTR • = • UnTranslated Region

  3. Stop Codon Start Codon Transcriptional Start Site Transcriptional Terminator Coding Region = ORF(Open-reading Frame) +1 3’ DNA 5’ Transcription Stops here Transcription Starts here Stop Codon Start Codon RNA produced by transcription 3’ 5’UTR 3’UTR 5’ Translation Stops here Translation Starts here Protein produced by translation of the mRNA Information present In the polypeptide

  4. 5’ 5’ O O Base n Base n O O OH O OH OH : + P P O- O HO P O- O O- O- O O O O Base n+1 P P P O O- HO O O- O- O Base O O O O OH OH OH OH Fundamental properties of RNA Polymerases 1) Catalyze the polymerization of ribonucleotides in the 5’->3’ direction: (rNMP)n+rNTP -> (rNMP)n+1 + PPi 2) Require a template (usually DNA) 3) Do not require a primer rNTPs PPi New rna 3’ 5’ 3’ 5’ 3’ 5’ template

  5. Potato Model of RNA Polymerase about 100 nucs/sec (vs 105 for DNA Polymerase)

  6. RNA polymerases • Single subunit RNAPs • Bacteriophage (T7 for example)-host transcribes it early and then it transcribes T7 late genome • Mitochondria • Chloroplasts (sometimes multi-subunit)’ • MultisubunitRNAPs • Bacteria • 2’ core (sigma specificity) • Archaea/Eukaryotes • similar architecture to bacteria • but many more subunits Euk. : Pol I - large ribosomal RNA Pol II - mRNAs Pol III - various small RNAs

  7. High Resolution Structures of multi subunit RNA polymerases Chemistry Nobel Prize 2006-Roger Kornberg when the apple does not fall far from the tree.. Eukaryotic (yeast RNAPII) prokaryotic Structural Similarities between eukaryotic and prokaryotic RNA polymerases Conserved residues between euk.and bacterial RNA Pol.(orange)

  8. Two metal ion catalysis model for DNA and RNA polymerases 2 metals are present in the active site of single subunit RNA polymerases DNA polymerases What about multi subunit RNA polymerases ?

  9. Initial crystal structures of bacterial RNAP revealed only one divalent cation at the active site… Important for fidelity Bridge helix Only one Mg Is there a second catalytic divalent cation?

  10. Yeast RNAP II +NTP reveals the source of the second Mg Nppp The second Mg comes with the NTP Westover et al (2004) Cell,119:1055

  11. Selection of correct rNTP by RNA polymerases Amino Acids involved are conserved between bacteria (T.th) and eukaryotes (S.c) RNA Pols • ribo selection (N737) • positioning by bridge helix Conserved in RNA Pol.only Conserved in both RNA and DNA Pol.

  12. Proofreading by RNA polymerases involves backtracking and cleavage of a dinucleotide (no 3’-5’ exo domain as in DNA polymerases) important role of the bridge helix in this process

  13. RNA Pol. Holoenzyme: a2bb’s : reduces the affinity of the Polymerase for random DNA sequences, thereby increasing specificity for promoters Core RNA polymerase: a2bb’ Core RNA Polymerase initiates transcription randomly, without specificity Core RNA Polymerase initiates transcription randomly, without specificity a: 36.5 kD, Structural and regulatory role b: 151 kD, Catalytic Site b’: 155 kD, helps Pol. to bind DNA

  14. How to detect DNA-Protein Interactions and promoter recognition ? DNA “Footprinting” • - General regions of Protein binding • DNAse I • - Intimate contacts between proteins and • the major groove • Dimethylsulfate+Piperidine • - Intimate contacts between proteins and • minor grooveOrthophenanthrolin/Cu++ • - Detect regions of single stranded Ts : • KMnO4

  15. 1 = No RNA polymerase/ DNase I 2 = RNA polymerase/ No NTPs/ DNase I 3 = RNApolymerase/ +ATP/ DNase I 4 = RNA polymerase/ +ATP+UTP/ DNase I 5 = RNA polymerase/ +ATP+UTP+GTP/ DNase I 6 = RNA polymerase/ +NTPs/ DNase I Promoter AAUUGAAUGUAAAUGGUACC.. +20 -60 +1 -20 -40 TTAACTTACATTTACCATGG.. Polymerase No NTPs Polymerase + ATP+UTP Polymerase +ATP +UTP +GTP Polymerase + ATP 1 2 3 4 5 6 7 8 DNase I Footprinting of RNA polymerase on the lacUV5 promoter in different conditions - 60 - 40 - 20 +1 +20 +40 +60 +80 Carpoussis & Gralla (1985)

  16. Alignments of the sequences at the site of contact between the RNA polymerase and the promoter reveals conserved sequences

  17. Different sfactors -> Different genes are activated s70:major E.coli factor, used for transcription of most genes, recognizes the consensus promoter TTGACA TATAAT -35 -10 s32: Transcription of heat-shock genes recognizes the consensus promoter: CNCTTGAA CCCCATNT -35 -10 s54: Transcription of genes involved in the response to nitrogen starvation recognizes the consensus promoter: -24 -12 CTGGNA TTGCA

  18. DNA + RNA polymerase (Holoenzyme) -35 +3 -11 OH 5’ppp -35 -11 1234 N N-10 OH N-35 5’ppp OH 5’ppp Closed complex (unstable, t1/2<1s) -35 -10 +1 Local melting - No ATP (s70) - ATP (s54) Open complex (stable, t1/2 =hours in the absence of NTPs) NTPs PPi Initial transcribing complex (tenuous; tendency to abort and reinitiate; RNA <10 nucleotides) NTPs PPi srelease Elongation complex (stable and processive) Transcription Termination Encounter a terminator signal

  19. How does promoter recognition and the transition Closed -> Open Complex occur ? Sigma70 contain 2 DNA recognition regions: Region 4.2 binds -35 element + core flap s70 show in orange Region 3 linker spans the promoter spacer + approaches active site Region 2 binds -10 element + aromatics approach DNA

  20. Structural Basis for Promoter -10 element recognition by Sigma 70 region 2 A-11 recognition T-7 recognition T A-11T A A T-7 -10 element Feklistov & Darst -Cell 2011, 147, 1257-1269 PDB ID = 3UGP

  21. Features of bacterial RNAP that influence transcription initiation and escape RNA exit channel (under flap) Sigma occlusion blocks exit tunnel aromatic residues initiate melting flap & sigma cover exit channel Active site metal Sigma r1 occlusion controls DNA melting Site of entry of riboNTPs

  22. Start here current model for initiation. DNA replaces s1.1 and bending begins melting The polymerase lengthens the open region and begins RNA synthesis. The blocked exit tunnel may force abortive initiation. RNA (red) displaces s3.2 loop The flap opens to allow RNA to exit. This may initiate sigma release.

  23. Visualizing RNA polymerase binding to genes in vivo: “chromatin” immunoprecipitation (technique developed in eukaryotes but adapted to bacterial cells) • Crosslink proteins to DNA in cells • Lyse cells, immunoprecipitate proteins of choice bound to DNA. • Recover bound DNA, amplify and identify the DNA bound by specific proteins using microarray technology Using this tool the goals are: - locate RNA polymerase interaction sites along the E. coli genome. to learn which of these sites are promoters to view how these interactions are changed during regulation.

  24. The beta subunit associates with highly expressed genes/ operons (broad distribution of balong the operon) the sigma factor mostly associates with promoters and does not travel with the core RNA polymerase (sharp peak of enrichment of bin the promoter) Grainger et. al Proc NatlAcadSci U S A. 2005 Dec 6;102(49):17693-8

  25. Transcriptional Regulatory Models

  26. Catabolism of Lactose in E.coli Galactoside permease and b-galactosidase are the two main enzymes of lactose metabolism

  27. Structure of the Lac Operon Nomenclature: LacZ= b-galactosidase LacY = lactose permease Lac A= transacetylase I = Lac Repressor P = Promoter O = Operator Regulation of the Lac Operon transcription

  28. 3 operator sequences in the Lac Operon Repression 82 bp 401 bp

  29. Structural study of the Lac Repressor “70s” model… Space filling view A tetramer of Lac Repressor complexed to looped operator DNA

  30. Interaction of one Helix of the Helix-Turn-Helix domain of the Lac repressor with DNA Interaction of amino acids of the Lac Repressor HTH with the major groove of Operator DNA Spronk et al. Structure 1999, 7:1483–1492 PDB ID = 1CJG

  31. Structural study of the Lac Repressor Conformational change of the Lac Repressor Without inducer (IPTG): transparent image With Inducer (IPTG not shown): overlaid solid image PDB IDs: 1LBG = Lac + DNA 1LBH = Lac + IPTG 1LBI = Lac alone

  32. DNA Binding by CAP a.k.a.CRP CataboliteActivator Protein = Cyclic AMP Receptor Protein High Glucose -> low cAMP -> No binding of CAP Low Glucose -> High cAMP Binding of CAP to Lac promoter CAP facilitates binding of the RNA polymerase to the Lac Promoter

  33. Regulation of transcription of the lacoperon is a mix of negative and positive regulations

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