Chaperones

1. Chaperones • A class of specialized proteins which help in folding of proteins. • One major function of chaperones is to prevent both newly synthesized polypeptide chains and assembled subunits from aggregating into nonfunctional structures. • Most Chaperones are heat shock proteins because the tendency to aggregate increases as proteins are denatured by stress

2. Proteasomes and Ubiquitin • Whenever there is an abnormal protein say a misfolded protein it is targeted for destruction by addition of several UBIQUITIN residues. • Such polyubiquitinated proteins are destroyed in the PROTEASOMES. • Proteasomes are large cytoplasmic complexes that have multiple protease activities capable of digesting damaged proteins.

3. Protein Targeting • Many proteins require signals to ensure delivery to the appropriate organelles.eg • N-terminal hydrophobic signal sequence used to ensure translation on RER • Phosphorylation of mannose residues important for directing an enzyme to a lysosome. Genetic defect affecting this phosporylation produce I-cell disease in which lysosomal enzymes are released into the extracellular space. • Coarse facial features, gingival hyperplasia • Craniofacial abnormalities • Joint immobility • Psychomotor retardation

4. REGULATION OF GENE EXPRESSION Dr. S.Chakravarty MD

5. Concepts Gene: A DNA segment that contains the all genetic information required to encodes RNA and protein molecules. Genome: A complete set of genes of a given species. Gene expression: A process of gene transcription and translation.

6. Introduction • Biomedical importance • Principles of gene regulation • Regulation of Prokaryotic gene expression • Regulation of gene expression in Eukaryotes

7. Introduction • Organisms adapt to environmental changes by altering gene expression. • Gene expression refers to the multistep process that results in production of a functional gene product, either RNA or protein. • The first step in gene expression-transcription is the primary site of regulation in both prokaryotes & eukaryotes.

8. Biomedical Importance • Regulated expression of genes is required for development, cellular differentiation & adaption. • Cell control over structure and function. • Mechanisms that control gene expression are used to respond to hormones & therapeutic drugs.

9. Why gene regulation? • To prevent Wastage of resources • To prevent Overloading of unwanted enzymes • Conservation of energy • Cell differentiation – specificity of function

10. Principles of genes • Inducible gene: gene whose expression increases in response to an inducer or activator, a specific positive regulatory signal. • Constitutive genes: genes that are expressed at a reasonably constant rate and not known to be subject to regulation. Referred to as Housekeeping genes.

11. Types of gene regulation: Positive regulation- expression of genetic information is quantitatively increased by presence of specific regulatory molecule, called positive regulator or activator. Negative regulation- expression of genetic information diminished by presence of a specific regulatory molecule called negative regulator or repressor.

13. Features of Prokaryotic Gene Expression • Operon: -unit of gene expression - includes structural genes, control elements, regulator/inhibitor genes, promoter or operator areas.

14. a. Special DNA sequence For prokaryotic systems: Operon is composed of structural genes, promoter,operator, and otherregulatory sequences. Promoter Structural genes Other requlatory sequence Operator

15. Promoters and Operators • In bacteria, regulated genes have an downstream region adjacent to the promoter called the operator • The operator is a binding site for proteins that help to regulate gene expression Gene DNA Promoter Operator

16. The Lac Operon • Described by Jacob & Monod in 1961. • Based on regulation of lactose metabolism by E.coli. • Structure of lacoperon: 3 structural genes- lac Z-codes - galactosidase lac Y- codes permease lac A- codes thiogalactosidasetransacetylase Regulatory gene-lac I Promoter region-attachment of DNA dependent RNA polymerase Operator site

17. Regulatory Proteins Structure of lac operon Beta galactosidase

18. Metabolism of lactose

19. As the Glucose disappears from surrounding medium • If a cell runs out of glucose, a small molecule (cyclic AMP or cAMP for short) is produced by active adenyl cyclase (converts ATP to cAMP) • This molecule is a ‘hunger signal’ that permits the expression of genes that break down other sugars, including lactose • cAMP binds the activator protein CRP (cAMP receptor protein) or CAP (catabolite activator protein), which can then bind lacP to help activate transcription

21. Regulation of lacoperon

23. Interaction of Inducers with Activators and Repressors

24. 1.Gene regulation on DNA • A) Gene Amplification: • Repeated initiation of DNA synthesis. • Additional copies of gene may be produced. • Seen in response to methotrexate-results in resistance to drug.

25. B)Gene Rearrangement: A segment from the DNA moves from one location to another on the genome. Forms a new combination of genes.

26. C) Gene Loss:- • Certain genes may be lost from a cell- functional protein encoded not produced. • Partial or complete. • Eg. Differentiation of RBCs- nuclei extruded-complete loss of all genes. D)Modification of DNA:- • Methylation of DNA – cytosine methylated by methylase. maintenance of inactive heterochromatin prevents transcription.

27. Chromatin remodeling- helps in basal transcription Histone acetylation and deacetylation: addition of acetyl groups to histones – disruption of nucleosome and DNA separation DNA methylation: addition of methyl groups to DNA – gene silencing

29. DNA methylation • Changes to gene expression without altering the DNA sequences • Methylation of de-oxycytidine residues in the GC rich regions – inactive chromatin • Helps in differentiation of cells destined for a particular function – terminally differentiated cells. Ex: pancreatic cell, hematopoietic cell, neurons etc. • X-chromosome inactivation – 1 of X chromosomes is highly condensed, transcriptionaly inactive heterochromatin

30. A)Chromatin Remodeling: • Heterochromatin-transcriptionally inactive chromatin, densely packed. • Euchromatin- transcriptionally active chromatin, less densely packed. • Differential expression of genes – development of specialized organs, tissues which is achieved by chromatin remodeling.

31. B)Histone covalent modification: • Acetylation of histones H3 & H4 associated with activation or inactivation of gene transcription. C)Regulatory factors: • Enhancer elements- DNA elements, facilitate or enhance initiation at promoter. • Transcription factors- regulatory proteins , bind with high affinity and specificity to the correct region of DNA.

32. Locus Control regions- complex DNA elements controls the expression of a cluster of genes. • Insulators- DNA elements in association with proteins ,prevent an enhancer from acting on a promoter . Serves as transcriptional boundary elements.

33. Structure of a transcription factor • DNA binding domain – bind to specific nucleotide sequences on the DNA • Helix turn helix • Leucine zipper • Zinc fingers • Helix loop helix • Activation domain – • Bind to other transcription factors • HAT and HDAC activity • Stabilize RNA polymerase

34. Types of transcription factors • Basal transcription factors – TFIID, TFIIA They bind to promoter regions (TATA and CAAT box) 2. Activators – steroid receptors, vitamin A receptors (RXR) They bind to enhancer regions and increase transcription 1000 folds • Corepressors (silencers)– bind to repressor region and inhibit transcription • Co activators and co repressors - bind to activators and corepressors and assist them

35. Properties of important transcription factors

36. Basal transcription unit -Eukaryotes

37. 3 unique motifs account for specific protein-DNA interactions: 1. Helix-Turn-Helix motif 2. Zinc Finger motif 3. The Leucine zipper motif.

38. Binding must be of high affinity to the specific site and of low affinity to other DNA. • Small regions of the protein make direct contact with DNA; the rest of the protein, in addition to providing the trans-activation domains, may be involved in the dimerization of monomers of the binding protein, may provide a contact surface for the formation of heterodimers, may provide one or more ligand-binding sites, or may provide surfaces for interaction with coactivators or corepressors. • The protein-DNA interactions are maintained by hydrogen bonds, ionic interactions and van der Waals forces. • The motifs found in these proteins are unique; their presence in a protein of unknown function suggests that the protein may bind to DNA. • Proteins with the helix-turn-helix or leucine zipper motifs form dimers, and their respective DNA binding sites are symmetric palindromes. In proteins with the zinc finger motif, the binding site is repeated two to nine times. These features allow for cooperative interactions between binding sites and enhance the degree and affinity of binding.

40. The Zinc Finger Motif

41. DNA binding & Transactivation Domains of Regulatory proteins

42. The bacterial lac operon is controlled by both glucose and lactose levels. Which of the following conditions would result in the greatest level of transcription from the lacoperon? • Both glucose and lactose present • Glucose present but no lactose • Lactose present but no glucose • No glucose or lactose present

43. The lac operon is negatively controlled by the lactose repressor and positively controlled by which of the following? • Increased concentrations of glucose and cyclic AMP (cAMP) • Decreased concentrations of glucose and cAMP • Increased concentrations of glucose, decreased concentration of cAMP • Decreased concentrations of glucose, increased concentration of cAMP • Increased concentrations of glucose and adenosine triphosphate (ATP)

44. A culture of E. coli is grown in a medium containing glucose and lactose. The expression of the lactose operon over time in the cells is shown in the graph below. Which statement best describes the change that occurred at point A? Lactose was added to the culture cAMP concentration increased in the cells Glucose was added to the culture Repressor protein dissociated from the operator Repressor protein became bound to the operator

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