Regulation of gene expression . Law of inheritance

Regulation of gene expression

1. Regulation of gene expression Dr Sajad Ahmad

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. l ac 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: Galactosidase permease : ↑ transport of lactose across cell membrane 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. l ac operon 3 elements: Regulator gene – i – synthesizes repressor Operator site- o Structural site- z , y and a

12. l ac 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. p ur 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 c atabolite 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 c ombinational 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: Redundant – a part can/be deleted without loss of function Modular – can ↑ transcription when paired with a variety of DNA binding domains 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 10 4 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

39. 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 .

40. 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)

41. Post transcriptional modification Trp operon : Encodes 5 genes 5’ end has leader seq of 162 nuc before initiation codon Transcript of only 1 st 130 nuc occurs when trp is high Site of termination = attenuator

42. 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

43. Motifs in proteins and gene expression A motif literally means a dominant element.

44. 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 )

45. 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. 1 ST 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.

46. 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.

47. Helix-turn-helix motif

48. 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)

49. 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 C ₂H₂ class Cx class

50. ZINC finger motif

51. Leucine zipper protein : # : periodic repeat of leucine residues in an α -helix. These leucines form hydrophobic interactions with a 2 nd 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.

52. 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 .

53. 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 2 nd is used to bind major grove. So the dimer consists of 4 helices. DNA binding sites are identical in homodimers & unrelated in heterodimers .

54. Examples of motifs

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