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2. Regulation of Gene Expression Dr. Ishtiaq Ahmad Khan Dr. Panjwani Center for Molecular Medicine and Drug Research

3. 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

4. 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

5. Modulators of transcription Modulators: (1) specificity factors, (2) repressors, (3) activators 1.Specificity factors: Alter the specificity of RNA polymerase Examples:  -factors (      TBPs  70  32 Heat shock geneHousekeeping gene Heat shock promoter Standard promoter

6. 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 Coding sequence Repressor Operator Promoter Effector (e.g. endproduct)

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

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

9. 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 Coding sequence Activator promoter RNA pol.

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

11. Positive regulation (2) Source: Lehninger pg. 1076 RNA polymerase ActivatorEffector

12. 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

13. The lactose (lac) operon Contains several elements –lacZ gene =  -galactosidase –lacY gene = galactosidase permease –lacA gene = thiogalactoside transacetylase –lacI gene = lac repressor –P i = promoter for the lacI gene –P = promoter for lac-operon –O 1 = main operator –O 2 and O 3 = secondary operator sites (pseudo-operators) PiP ZYA I O3 O1 O2

14. 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 encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. lacY encodes β-galactoside permease (LacY), a membrane-bound transport protein that pumps lactose into the cell. lacA encodes β-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. Theodor Hanekamp © 2003 14

15. 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. 15

16. 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), 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. 16

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18. Regulation of the lac operon PiP ZYA I Q3 Q1 Q2 Inducer molecules: Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside) - synthetic inducer, not metabolized, lacI repressor PiP ZYA I Q3 Q1 Q2 LacZLacYLacA

19. Operons in eukaryotes  Dicistronic transcription units specify a messenger RNA (mRNA) encoding two separate genes that is transported to the cytoplasm and translated in that form. Presumably, internal ribosome entry sites (IRES), or some form of translational re-initiation following the stop codon, are responsible for allowing translation of the downstream gene.  In the other type, the initial transcript is processed by 3΄ end cleavage and trans-splicing to create monocistronic mRNAs that are transported to the cytoplasm and translated.  Like bacterial operons, eukaryotic operons often result in co- expression of functionally related proteins. 19 Blumenthal T, BRIEFINGS IN FUNCTIONAL GENOMICS AND PROTEOMICS. VOL 3. NO 3. 199–211. NOVEMBER 2004

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21. Genes whose function is to specify mitochondrial proteins and those that encode the basic machinery for gene expression, transcription, splicing and translation have a very strong tendency to be transcribed in operons 21

22. Examples in C. elegans  An operon that expresses two subunits of the acetylcholine receptor.  An operon that encodes two proteins needed for modifying collagen, expressed only in collagen-producing cells.  An operon that co-expresses an ion channel protein with a protein that modifies the activity of that channel; 22

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