Regulation of Gene Expression Dr. Ishtiaq Ahmad Khan

1. Regulation of Gene Expression Dr. Ishtiaq Ahmad Khan

2. Today’s lecture • Gene expression • Constitutive, inducible, repressible genes • Specificity factors, activators, repressors • Negative and positive gene regulation • Lac operon • Helix-turn-helix motifs • Zinc-fingers • Leucine zippers

3. What is gene expression? • Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product. • A gene is expressed when its biological product is present and active. • Gene expression is regulated at multiple levels.

4. Regulation of gene expression Promoter Gene (red) with an intron (green) Plasmid single copy vs. multicopy plasmids 1. DNA replication 2.Transcription Primary transcript mRNA degradation 3. Posttranscriptional processing Mature mRNA 4. Translation inactive protein Protein degradation 5. Posttranslational processing active protein

5. Condition 2 “turned off” “turned on” 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25 26 19 20 21 22 23 24 induced gene repressed gene constitutively expressed gene inducible/ repressible genes Gene regulation (1) Condition 1 “turned off” “turned on” Chr. I Chr. II Chr. III

6. Condition 4 upregulated gene expression down regulated gene expression Gene regulation (2) Condition 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25 26 19 20 21 22 23 24 constitutively expressed gene

7. Definitions • Constitutively expressed genes: • Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells. • Inducible genes: • Genes that are transcribed and translated at higher levels in response to an inducing factor • Repressible genes: • Genes whose transcription and translation decreases in response to a repressing signal

8. Definitions • Housekeeping genes: • genes for enzymes of central metabolic pathways (e.g. TCA cycle) • these genes are constitutively expressed • the level of gene expression may vary

9. Modulators of transcription • Modulators: (1) specificity factors, (2) repressors, (3) activators • Specificity factors: Alter the specificity of RNA polymerase Examples: s-factors (s70, s32 ), TBPs s70 s32 Standard promoter Heat shock promoter Housekeeping gene Heat shock gene

10. Modulators of transcription 2. Repressors: mediate negative gene regulation may impede access of RNA polymerase to the promoter actively block transcription bind to specific “operator” sequences (repressor binding sites) Repressor binding is modulated by specific effectors Effector (e.g. endproduct) Repressor Operator Coding sequence Promoter

11. Negative regulation (1) Repressor Effector Example: lac operon RESULT: Transcription occurs when the gene is derepressed Source: Lehninger pg. 1076

12. Negative regulation (2) Repressor Effector (= co-repressor) Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism Source: Lehninger pg. 1076

13. Modulators of transcription 3. Activators: mediate positive gene regulation bind to specific regulatory DNA sequences (e.g. enhancers) enhance the RNA polymerase -promoter interaction and actively stimulate transcription common in eukaryotes RNA pol. Activator promoter Coding sequence

14. Positive regulation (1) Activator RNA polymerase Source: Lehninger pg. 1076

15. Positive regulation (2) Activator Effector RNA polymerase Source: Lehninger pg. 1076

16. Operons • a promoter plus a set of adjacent genes whose gene products function together. • usually contain 2 –6 genes, (up to 20 genes) • these genes are transcribed as a polycistronic transcript. • relatively common in prokaryotes • rare in eukaryotes

17. Pi I P O1 Z Y A O3 O2 The lactose (lac) operon • Contains several elements • lacZ gene = b-galactosidase • lacY gene = galactosidase permease • lacA gene = thiogalactoside transacetylase • lacI gene = lac repressor • Pi = promoter for the lacI gene • P = promoter for lac-operon • O1 = main operator • O2 and O3 = secondary operator sites (pseudo-operators)

18. The lac operon consists of three structural genes, and a promoter, a terminator,regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA. • lacZ encodesβ-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. • lacY encodesβ-galactoside permease (LacY), a membrane-bound transport protein that pumps lactose into the cell. • lacA encodesβ-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides. • Only lacZ and lacY appear to be necessary for lactose catabolism.

19. First Level • The lacI gene coding for the repressor lies nearby the lac operon and is always expressed (constitutive). • Hinder production of β-galactosidase in the absence of lactose. • If lactose is missing from the growth medium, the repressor binds very tightly to a short DNA sequence called the lac operator. • The repressor binding to the operator interferes with binding of RNA Pol to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels. • When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose , which is a combination of glucose and galactose, binds to the repressor, causing a change in its shape. • Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to higher levels of the encoded proteins.

20. Second Level • The second control mechanism is a response to glucose, which uses the Catabolite activator protein (CAP) to greatly increase production of β-galactosidase  in the absence of glucose.  • Cyclic adenosine monophosphate  (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose. • It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (a 16 bp DNA sequence upstream of the promoter on the left in the diagram below), • which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase • enabling the cell to hydrolyse (digest) lactose and release galactose and glucose.

22. LacZ LacY LacA Inducer molecules: Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside) - synthetic inducer, not metabolized, lacI repressor Pi Pi I I P P Q1 Q1 Z Z Y Y A A Q3 Q3 Q2 Q2 Regulation of the lac operon

23. Selected DNA binding motifs • Helix-turn-helix • Homeodomain • Zinc Fingers • Cys4 zinc finger • Cys2 His2 zinc finger (e.g. TFIIIA) • Basic domains • Leucine zippers factors (bZIP) • Basic helix-loop-helix (bHLH) • Beta-scaffold factors with minor groove contacts • HMG (High mobility group) proteins

24. Helix-turn-helix motifs • Structure: • about 20 amino acids long • 2 short alpha helicies ( 7 – 9 amino acids long) • DNA recognition helix (binds specific DNA sequence) • Recognition helix and 2nd helix form ~ 90° angle • very short turn ( NOT a beta-turn) • Often glycine at start of the turn (helix breaker)

25. How does the lac repressor bind DNA? LacI repressor (helix-turn-helix domain) DNA recognition helix Second alpha helix turn DNA Source: Lehninger pg. 1082

26. C C C H H H Zn Zn Zn H H H C C C Zinc-Finger Motifs • Several subtypes (Cys4, Cys2-His2 …) • Example: Cys2 His2 type • Zinc does not interact with DNA • Usually multiple zinc-fingers in a row • At least some also bind RNA • Consensus sequence: • [Y,F]-X-C-X2-4-C-XXX-F-XXXXX-L-XX-H-X3-5-H

27. 7 7 7 7 Basic domains • Leucine zippers (bZip): • Basic region of the protein binds to DNA • Mainly act as dimers or other sometimes as other multimers • Special alpha-helices allow formation of coiled-coil structures. • Hydrophobic residues (Leu) align on one side of the helix • Example: Jun and Fos abcdefg Source: Lehninger pg. 1084

28. Leucine zippers Leucines DNA Source: Lehninger pg. 1084

29. Transcription attenuation

30. Some Genes Are Regulatedby Genetic Recombination Example of Salmonella typhimurium

31. Regulation of Eukaryotic Gene Expression

32. Gene Regulation at DNA Level Chromatin Remodeling 1. Changes of DNA Topo structure Formation of ssDNA DNase I hypersensitive site

33. 2. DNA Methylation DNA Methylation

34.  CpG islands ----- are genomic regions that contain a high frequency of CG dinucleotides. ----- CpG islands particularly occur at or near the transcription start site of housekeeping genes.

35. TF RNA pol Unmethylated CpG island TF RNA pol CH3 CH3 CH3 Methylated CpG island Active transcription Repressed transcription

36. TF 3. Histone modification  methylation  acetylation

37. Functions of Histone methylation in transcription • Most well-studied histone modifications are involved in control of transcription. • Actively transcribed genes • Two histone modifications are particularly associated with active transcription: • Trimethylation of H3 lysine 4 (H3K4Me3) at the promotor of active genes • Trimethylation of H3 lysine 36 (H3K36Me3) in the body of active genes • Repressed genes • Three histone modifications are particularly associated with repressed genes: • Trimethylation of H3 lysine 27 (H3K27Me3) • Di and tri-methylation of H3 lysine 9 (H3K9Me2/3) • Trimethylation of H4 lysine 20 (H4K20Me3)

38. Functions of Histone methylation in transcription Acetylated histones and nucleosomes represent a type of epigenetic tag within chromatin. Acetylation removes the positive charge on the histones, thereby decreasing the interaction of the N termini of histones with the negatively charged phosphate groups of DNA. As a consequence, the condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription.

39. 7.3 Transcriptional Regulation 1. Cis-acting element (1) What is cis-acting element?  Concept Cis-acting elements - DNA sequences close to a gene that are required for gene expression

41. 2. What is trans-acting factor?  Concept trans-acting factors - usually they are proteins, that bind to the cis-acting elements to control gene expression.

42. These trans-acting factors can control gene expression in several ways:  may be expressed in a specific tissue  may be expressed at specific time in development  may be required for protein modification  may be activated by ligand binding

43. Domains of trans-acting factors  DNA binding domain DBD  transcription activating domain

44. Post-Transcriptional Regulation 1. Gene Regulation of mRNA Processing exon shuffling alternative gene splicing

45. 2. Gene Regulation of mRNA Editing • 3. mRNA Longevity • mRNA Transport Control • RNA Interference (RNAi) • miRNA •  siRNA

46. 7.5 Translational and Post-translational Regulation 1. Translation Control Blocking mRNA Attachment to Ribosomes 2. Regulation of Protein Processing Protein Modification

47. 3. Regulation of Protein Stability

48. Thank You Very Much