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Gene Expression

Gene Expression. Prokaryotes vs eukaryotes Gene expression in p rokaryotes Prokaryotic Promotor Lac operon in E. coli Lac operon: Catabolite repression Trp operon attenuation & antitermination Gene expression in eukaryotes Eukaryotic promotor Assembly of a transcriptional complex

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Gene Expression

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  1. Gene Expression • Prokaryotes vs eukaryotes • Gene expression in prokaryotes • Prokaryotic Promotor • Lac operon in E. coli • Lac operon: Catabolite repression • Trp operon attenuation & antitermination • Gene expression in eukaryotes • Eukaryotic promotor • Assembly of a transcriptional complex • Control of Eukaryotic Transcription • chromatin structure & gene expression

  2. Transcription • RNA Polymerase transcribes the DNA code to mRNA. • The mRNA Polymerase `unzips’ the double helix of the DNA to access the code.

  3. Transcription Schematic DNA double helix DNA Rewinding antisense DNA strand 3’ ribonucleotide triphosphates newly synthesized sense mRNA transcript 5’

  4. Translation • Nucleotides are grouped into codons. • Each codon contains three nucleotides. • Each codon corresponds to an aminoacid or a stop.

  5. Ribosome Amino Acids forming Peptide chain P Site A Site E Site tRNA anti-codon AUG GGA codon Translation Met His Tyr Val Pro 3’ CAU UAC GUA CCU 5’ mRNA strand

  6. Eukaryotic vs Prokaryotic Transcription here Chromosomes Prokaryotic Organism Translation here Chromosome Eukaryotic Organism

  7. Transcription and translation in prokaryotes vs eukaryotes

  8. Transcription and translation in prokaryotes vs eukaryotes • In prokaryotes, transcription and translation are tightly coupled. • In contrast, transcription and translation are spatially separated in eukaryotes.

  9. Procaryote: Example: E.coli • Chromosome: contains 4.6 £ 106nucleotide pairs, circular. • DNA encodes approximately 4300 proteins. • Only a fraction of these are made at any time. • Expression is regulated according to available food.

  10. Operon repressor • Active repressor (inducible operon) • Inactive repressor (repressible operon)

  11. Lac operon in E. coli • Operon: a cluster of genes which are regulated collectively • Structural Genes: • Gene z - B-galactosidase - lactose catabolism, breaks down lactose into galactose and glucose • Gene y - galactoside permease - concentrates lactose in cell • Gene a - thiogalactoside transacetylase - not in pathway • Repressor gene: product (R) binds Operator to inhibit transcription (negative gene regulation) • Operator (gene) , controls transcription of three structural genes. • Inducer - Lactose (allolactose), binds repressor product, which no longer binds operator • P: Promoter region - RNA polymerase binding + CAP-cAMP binding. • CAP: Catabolite activator protein, binds promoter region to activate transcription of structural genes (positive gene regulation) • cAMP: Cyclic adenosine monophosphate, complexes with CAP to allow efficient complexing to promoter region.

  12. Breakdown of lactose: Lactose Glucose + Galactose Inducer of lactose operon: Lactose

  13. Gene regulation in procaryotes Lac operon

  14. E. coli on Lactose : Lactose inducer complexes with repressor protein to change shape, Operator not bound, structural gene induced (inducible system) mRNA polycistronic

  15. Positive Regulation: CAP protein acts as a gene activator. a) Substrate glucose - CAP + cAMP, no transcription, repressor binds operator. b) Substrate glucose + lactose - glucose suppresses cAMP, CAP binds inefficiently, little mRNA transcribed. c) Substrate lactose only- cAMP levels elevated, cAMP-CAP complex forms, promotes RNA polymerase association, transcription high, translation high.

  16. Comparison of Negative and Positive Control Negative control = protein gene product functions to turn genes off. Positive control = protein gene product functions to turn genes on.

  17. Tryptophane operon • Five genes which manufacture an enzyme to produce the amino acid tryptophan are adjacent. • Region starts with a promoter. operator promoter E D C B A operon • When tryptophan is present in growth medium, a repressor protein binds to the operator repressing transcription. • Activator proteins also exist.

  18. Trpoperon Chorismate-Anthrqanilate-phosphorybosyl anthranilate -Indole-3-glycerol P- Tryptophan

  19. Tryptophane operon Attenuation & antitermination Attenuation & termination Antitermination

  20. Tryptophane operon antitermination Attenuation & termination Antitermination

  21. Tryptophane operon attenuation When there is HIGH tryptophane, the ribosome translate the segment 1 & 2. This allows the formation of hairpin between section 3 & 4 leading to termination of transcription (Attenuation)

  22. Tryptophane operon antitermination When there is LOW tryptophane, the ribosome stall at the segment 1. This allows the formation of hairpin between section 2 & 3 leading to antitermination

  23. Gene Expression in Eukaryotes Genome : 1.2% coding & 98.8% non-coding Only about 3-5% of all the genes in a human cell are expressed at any given time. The genes expressed can be specific for a particular cell type or tissue. There are several opportunities for control of gene expression…

  24. Gene expression in eukaryotes • Gene expression: • Temporal for development and Spatial for cell specialization • No operon: each gene with own promotor and one or more enhancers • Various transcriptional/regulatory factors

  25. Chromatin Structure

  26. Chromatin Structure

  27. chromatin structure & gene expression

  28. chromatin structure & gene expressionmethylation / acetylation If the cytosine residues of DNA are methylated allows gene expression. Acetylation or methylation of lysine groups of the histones make the genes inaccessible to transcription factors. Histones in the coding region either remain associated with DNA but become hyperacetylated to facilitate RNAPII mobility or alternatively, they are displaced from the coding region,

  29. PRC1: The Polycomb Repressive Complex 1 (PRC1) is a relatively enormous structure with a molecular weight of 2-6 MDa and contains stoichometric amounts of Polyhomeotic (PH), Polycomb (Pc), Posterior sex comb (PSC), and RING protein subunits The PRC2 complex (600kDa) has three highly conserved core proteins (ESC), Enhancer of Zeste E(z), and Suppressor of (Zeste SU(Z)12) found in divergent species such as human

  30. Control of Eukaryotic Transcription • {Transcriptional(1), PostTr(2), Transl.(3), PostTsl(4)} • 1 Chromatin structure • 1 Binding of RNA polymerase to DNA • 2 Processing of RNA • 2 Efficiency of transport channel • 2 Protection/destruction of transcript • 2 Post transcriptional modification • 3 Rate of translation • 4 Protein transport Transcriptional activators and factors Processing or not? Which Exon retained? Calcitonin cytoskeletal tracks for mRNA transport No. of 3’ UTR (AUUUA) Phosphorylation / methylation mRNA in eggs

  31. Eukaryotic promotor • The basal promoter TATA box • Enhancers • Silencers • Insulators a, Simple eukaryotic transcriptional unit. A simple core promoter (TATA), upstream activator sequence (UAS) and silencer element spaced within 100−200 bp of the TATA box that is typically found in unicellular eukaryotes. b, Complex metazoan transcriptional control modules. Containing multiple clustered enhancers interspersed with silencer and insulator elements located 10−50 kb upstream or downstream of a composite core promoter containing TATA box (TATA), Initiator sequences (INR), and downstream promoter elements (DPE).

  32. Assembly of a transcriptional complex D DA DAB DAB-PolF DAB-PolF-EH 6 factors + RNAP II = Pre-Init. Complex 6 Factors are TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH order • TFIID recognizes and binds to the TATA box. TFIID consists of TATA box binding protein - TBP and ~10 TBP associated factors - TAFs.TFIIA binds and stabilizes TFIID binding. • The RNA polymerase II holoenzyme assembles - The holoenzyme consists of the RNA polymerase II complex, and the following transcription factorsTFIIA TFIIB TFIIE TFIIF TFIIH • Finally, the various regulatory factors (Srb-Mediator, Srb10-CDK and Swi-Snf) bind to complete formation of the pre-initiation complex

  33. The transcription activation domain interacts directly with the RNA polymerase II to pre-load RNA polymerase II near the basal transcription complex. Enhancers are brought into close physical proximity to the basal transcription complex through protein-protein interactions between sequence specific enhancer binding proteins and TBP (basal txn complex) as shown below.

  34. Transcriptional Regulation of Gene Expression

  35. Assembly of transcriptional complex-II The carboxy terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated.This results in promoter clearance. RNA polymerase II dissociates from the Transcription factors and other protein complexes that were required for assembly.

  36. Assembly of transcriptional complex-III

  37. Transcriptional Factors Helix-Turn-Helix (HTH) Leucine Zipper Zinc Fingers Helix-Loop-Helix (HLH) Structural Motifs in Eukaryotic Transcription Factors

  38. Representative Transcription Factors

  39. Eukaryotic Transcription initiation • A set of molecules needed are: • TBP (TATA Box Binding Protein) • Auxillary factors binds RNAP & associated Proteins • RNA Polymerase II • Activator proteins

  40. Eukaryotic Transcription • RNA Polymerase transcribes the DNA into mRNA.

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