![pur repressor
• Represses gene involved in purine syn (sometimes
pyrimidine syn)
• 31% identical to lac repressor in 3-D str and dimeric
• Analogous to lac repressor
• Binds DNA only when bound to a small molecule
(Corepressor) [thus, behaviour opposite to lac
repressor]
• This corepresoor m/b guanine or hypoxanthine](https://image.slidesharecdn.com/regulationofgeneexpression1-240429060326-4b8a3de5/85/Regulation-of-gene-expression-Law-of-inheritance-16-320.jpg)
Regulation of gene expression . Law of inheritance
- 2. Classification of gene with respect to their Expression Constitutive ( house keeping) genes: 1- Are expressed at a fixed rate, irrespective to the cell condition. 2- Their structure is simpler 3-eg enzymes of glycosis are synthesized by all cells. Controllable genes: 1- Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition. 2- Their structure is relatively complicated with some response elements
- 3. Synthesis of proteins under the influence of genes is called as gene expression. • In eukaryotes the regulation of gene expression is much more complex because of the presence of nuclear membrane, which prevents the simultaneous transcription and translation. • In prokaryotes, the major point of regulation is the control of transcription initiation. • In eukaryotes the regulation of gene expression is controlled at different points.
- 4. Gene regulation in prokaryotes • In bacteria,the gene that encode the proteins required to perform coordinated fn are clustered into operons. • The RNA transcribed from prokaryotic operon is polycistronic a term implying that multiple proteins are encoded in a single transcript.
- 5. Gene expression - Expressed all the time - Expressed only in specific cells or under specific condition Constitutive Regulated
- 6. • In both prokayotes and eukaryotes, regulatory proteins recruit RNAP to sites within genome to initiate transcription • Regulatory sites are usually the binding sites for specific DNA-binding proteins (DNABP)
- 7. Operons • Prokaryotic DNABP bind specifically to regulatory sites in operons
- 8. lac operon • E. coli usually rely on glucose as their source of carbon and energy • However, it can utilize lactose if it is high in the medium
- 9. • No of - galactosidase increases from 10 to several thousand when grown on lactose (by increase in syn) • 2 other enz ↑ together: 1. Galactosidase permease: ↑ transport of lactose across cell membrane 2. Thiogalactosidase transacetylase: detoxification of compounds that are also transported by permease
- 10. • Thus, the expression levels of a given set of enzymes that all contribute to the adaptation to a given change in the environment change together • Such a coordinated unit of gene expression is k/as operon
- 11. lac operon • 3 elements: • Regulator gene – i – synthesizes repressor • Operator site- o • Structural site- z, y and a
- 12. lac repressor • Exist as dimer • Often two dimers are linked to form tetramer • Locates operator site by unique pallindromic sequence via one- dimensional search • 3-D str: 2 domains- - Small (amino-terminal) –binds DNA with helix-turn-helix motif -Large – formation of dimer/ tetramer
- 13. • i = repressor • P= promotor • O= operator
- 14. • inducer forms complex with large domain of repressor c modify the relation b/w the two small domains and the ‘o’ site gets free • Thus inhibition by ‘i’ is lost
- 15. Inducer • Allolactose (not the lactose): - glu and gal are in α- 1,6 linkage (while in α- 1,4 linkage in lactose) - side product of reaction catalysed by - galctosidase enzyme - formed at low levels by few mol of enz that are present before induction • IPTG - used as inducer in vitro
- 16. pur repressor • Represses gene involved in purine syn (sometimes pyrimidine syn) • 31% identical to lac repressor in 3-D str and dimeric • Analogous to lac repressor • Binds DNA only when bound to a small molecule (Corepressor) [thus, behaviour opposite to lac repressor] • This corepresoor m/b guanine or hypoxanthine
- 17. • E.coli genome has >20 regulatory sites, 19 operons and 25 genes
- 18. Catabolite repression • When E.coli is grown on glucose, it has very low levels of catabolic enzymes for metabolizing other sugars • This inhibitory effect of glu is k/as catabolite repression • This occurs as Glu lowers the cAMP conc in E.coli
- 19. CAP • CAP = Catabolite Activator Protein • Also k/as cAMP response protein • Dimer of two identical subunits • Binds at or near the start site for transcription in lac operon ↑ cAMP ↑ CAP – cAMP complex ↑ transcription of lactose and arabinose catabolising genes
- 20. CAP
- 21. Trp Operon • E. coli uses several proteins encoded by a cluster of 5 genes to manufacture the amino acid tryptophan • All 5 genes are transcribed together as a unit called an operon, which produces a single long piece of mRNA for all the genes • RNA polymerase binds to a promoter located at the beginning of the first gene and proceeds down the DNA transcribing the genes in sequence
- 22. Trp operon
- 23. In eukaryotes Organism Size of genome (Mb) E.coli 4.6 Yeast 12 Humans 3000 Gene regulation is much more complex as: 1. Larger genome: 2. Different cell types- gene expression is different in liver and pancreas 3. Transcription and translation are uncoupled
- 24. Basal transcription complex Additional TFs ↑ mRNA synthesis General TF (pre-initiation complex) RNAP-II
- 25. Combinational control • Only a few regulators may affect transcription directly in eukaryotes ( in contrast with prokryotes) • Each factor recruits other proteins to build up large complex • It increases transcription on interaction with transcription machinery • Thus a given reg factor can have different results • The result depends on the presence of other proteins in the same cell • This is k/as combinational control
- 26. Combinational control (cont.) • It is crucial to multicellular organisms • It leads to generation of different cell types in unicellular eukaryotes like yeast.
- 27. Transcriptional factors • Have several domains: Domain Function DNA binding domain bind regulatory sequence at or near promotor Regulatory domain Prevent DNA binding under certain conditions Activation domain Initiates transcription through interaction with RNAP-II or its associated proteins
- 28. • TFs can be grouped into families depending on str of seq specific DNA binding domains. • If binding site lies at a considerable distance from promoter, it is k/as enhancer • The intervening DNA can form loops that bring the enhancer bound activator to the promoter site p E
- 29. Activation domain has multiple interaction partners Transcription Factor RNAP-II Mediator 25-30 subunit part of pre-initiation complex
- 30. Activation domains • Less conserved than DNA binding domains • Some common features: 1. Redundant – a part can/be deleted without loss of function 2. Modular – can ↑ transcription when paired with a variety of DNA binding domains 3. Act synergistically – two activation domains come together create much stronger effect In certain cases, TFs may be repressor rather than activators
- 31. Nucleosomes • Histones forms half of eukaryotic chromosome • DNA+ Histones = chromatin • 5 major histones: H1, 2A,2B,3,4 • The last 4 forms an octamer • Around this octamer 200bp DNA is wrapped • Entire str = Nucleosome • 100 Å str visualised as beads on string in EM
- 32. EM
- 33. • Studies after extensive digestion shows bead consist of 145 bp DNA and histone octamer • It is k/as nucleosome core particle • Each histone has an amino terminal tail – flexible and rich in lys and arg • DNA b/w nucleosome = linker DNA • DNA nucleosome 104 times (7- fold compact) compact
- 34. Chromatin remodelling aids in gene expression • Chromatin packing make DNA less susceptible to clevage by DNAase –I • Regions adj to gene are more susceptible • These are less compacted and more accessible to proteins • k/as hypersensitive sites • These sites are cell type specific developmentally regulated
- 35. • Thus relaxing of chromatin is essential • Yeast DNABP called GAL4 recognizes DNA at 10 sites only; however 4000 such sites are present in its genome • Chromatin binding makes other sites inaccessible in eukaryotes (C.F. prokaryotes)
- 36. Enhancers are cell-specific • Enhancer for CK-MM (m/s isoform) is located b/w -1350 and -1050 bp • Insertion of this enhancer near a gene c is normally not expressed in m/s, l/t ↑ expression • This is not possible for other cell types
- 37. DNA Methylation
- 38. Methylation of DNA correlates with gene inactivation Cytosine→5-methylcytosine Methylcytosine binding proteins associates with DNA Other transcription factors can’t bind DNA bcz of steric hindrance → gene inactivation
- 40. Methylation and DNA imprinting • In DNA Imprinting the different Methylation patterns of DNA inherited from the sperm or egg correlate with choice of allelic expression. • Methylation patterns are conserved after replication by action of hemimethylase( which methylates only one of the 2 strands containing the CG). • Methylation of DNA correlates with deacetylation of Histones.
- 41. Methylation of genes encoding proteins which direct abnormally dividing cell for apoptosis cancer
- 42. In totipotent cell (e.g. fertilized egg ) DNA undergoes fairly global de methylation
- 43. Chromatin str is modulated through covalent modification of histone tails • Coactivators loosen histones from DNA • Effectiveness depends on ability to covalently modify amino terminal tails of histones • Lys residues are acetylated by HAT • B/o this lys looses its charge on amino gp • Thus, affinity to DNA is decreased • In addition acetylated lys interact with bromodomain (acetyl lysine binding domain of certain proteins)
- 44. Post transcriptional modification • Trp operon: • Encodes 5 genes • 5’ end has leader seq of 162 nuc before initiation codon • Transcript of only 1st 130 nuc occurs when trp is high • Site of termination = attenuator
- 45. • When trp is low, 7000 nuc are transcripted • When enough trp is present, a stem loop str form in attenuator region, which l/t release of RNAP from DNA
- 46. Motifs in proteins and gene expression • A motif literally means a dominant element.
- 47. Common motifs in proteins that interact with DNA & regulate transcription • Although their overall AA sequence & composition uniquely identify each TF, the domains involved in each activity can be grouped into a few # motifs : • Helix-turn- helix (HTH) • Zinc finger • Helix-loop-helix,(HLH) • Basic region-leucine zipper (bZIP)
- 48. Helix-turn-helix motif proteins • It is the domain within the protein that allows sequence specific interaction with DNA. • It is of about 20 amino-acids , so is a small part of a larger protein. • 1ST 7AA=α-helix, 4AA= non helical turn, 9AA= α-helix. • 9AA helix: recognition helix that binds in major groove by forming H-bonds with exposed bases.
- 49. • 7AA helix : stabilizes the binding of recognition helix through Hydrophobic interactions. • Both helices include AA, like valine or leucine, that allow these hydrophobic interactions to occur.
- 51. • E.g. of helix-turn-helix includes: • Lactose repressor of E. coli • group of developmentally important transcription factors called homeodomain proteins ( defect in genes for them causes homeosis i.e. leg develops in place of antenna in fruit fly)
- 52. Zinc finger proteins: • # : specific amino acids that coordinate Zn binding. • it binds in the major groove of DNA in a sequence specific manner, mediated by an α-helix formed on one side of finger region. • Depending on AA nature coordinating with Zn 2 subclasses are formed • C₂H₂ class • Cx class
- 54. Leucine zipper protein : • # : periodic repeat of leucine residues in an α- helix. These leucines form hydrophobic interactions with a 2nd protein in which a similar helix allows the formation of a dimer. • so the leucine zipper refers to the protein- protein interaction domain. • α—helical region may continue beyond pr-pr interaction domain, allowing binding to the major grove of DNA.
- 55. • DNA contact surface of protein is basic , bcz of arginine & lysine. • Basic AA stabilizes DNA-protein interaction by combining with –ve charged DNA backbone. • Dimer can be homo or hetero dimer . • Homodimer bind to a site that has a dyad symmetry (2 symmetric half sites), whereas this symmetry is not seen with heterodimers.
- 57. Helix-loop-helix proteins • # : 2 amphipathic α- helical segments separated by an intervening loop. • E.g. myoD, myc, & max TF • 1 helix is used for protein-protein interaction and 2nd is used to bind major grove. • So the dimer consists of 4 helices. DNA binding sites are identical in homodimers & unrelated in heterodimers.
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