Dr. Parvin Pasalar Tehran University of Medical Sciences

2. Objectives To know and explain: Regulation of Bacterial Gene Expression Constitutive ( house keeping) vs. Controllable genes OPERON structure and its role in gene regulation Regulation of Eukaryotic Gene Expression at different levels: DNA methylation Histon modifications(Chromatin Remodeling) Increasing the number of gene copies (gene amplification) Changing the rate of initiation of transcription Alternate splicing mRNA stability Changing the rate of initiation of translation Using of Untranslating Region (UTR)  protein stability Hormonal regulation Cross talk between different regulatory pathways 11-Regulation by protein stability

3. 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 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

5. Different ways for regulation of gene expression in bacteria 1- Promoter recognition: 2-Transcription elongation( Attenuation)

6. OPERON in gene regulation of prokaryotes

9. Lac OPERON an inducible Operon

10. CRP or CAP is positive regulator of Lac and some other catabolic Operons

15. Eukaryotic gene regulation occurs at several levels

16. Heterochromatin is the most tightly packaged form of DNA. transcriptionally silent, different from cell to cell Methylation is related to the Heterochromatin formation Small percentages of newly synthesized DNAs (~3% in mammals) are chemically modified by methylation. Methylation occurs most often in symmetrical CG sequences. Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes. Methylation results in a human disease called fragile X syndrome; FMR-1 gene is silenced by methylation.

21. a. In Drosophila courtship, the male behaviors include: Following, Singing & � b. Regulatory genes (fruitless= fru) in the sex determination pathways control these behaviors. c. Physiologically, the CNS (central nervous system) is responsible for key steps in male courtship behavior.) (fruitless) The sex-specific fru mRNAs are synthesized in only a few neurons in the CNS (500/100,000). The proteins encoded by these mRNAs regulate transcription of a set of specific genes, showing that fru is a regulatory gene. Its expression seems to be confined to neurons involved in male courtship

22. For the iron (Fe 2+) transport protein transferrin receptor. A stem loop structure in the mRNA acts as an iron response element and binds a 90 kDa protein in the absence of iron. The RNA and iron binding regions of the protein overlap so in the presence of iron the 90 kDa binding protein can no longer bind to the mRNA iron response element and the stem loop no longer occurs. Since the stem loop is at the 3� end of the mRNA , the loop is stabilising of the mRNA , protecting it from degradation. In the presence of iron, the loop disappears and the mRNA is degraded by 3� exonucleases.sFor the iron (Fe 2+) transport protein transferrin receptor. A stem loop structure in the mRNA acts as an iron response element and binds a 90 kDa protein in the absence of iron. The RNA and iron binding regions of the protein overlap so in the presence of iron the 90 kDa binding protein can no longer bind to the mRNA iron response element and the stem loop no longer occurs. Since the stem loop is at the 3� end of the mRNA , the loop is stabilising of the mRNA , protecting it from degradation. In the presence of iron, the loop disappears and the mRNA is degraded by 3� exonucleases.s

23. The binding of iron to the 90 kDa protein has opposite effects for ferritin. In this case, the stem loop is at the 5� end. It inhibits translation by preventing ribosomes getting onto the mRNA and thus its disappearance stimulates transcription. Its removal leads to degradation of the mRNA and thus reduces translation. The binding of iron to the 90 kDa protein has opposite effects for ferritin. In this case, the stem loop is at the 5� end. It inhibits translation by preventing ribosomes getting onto the mRNA and thus its disappearance stimulates transcription. Its removal leads to degradation of the mRNA and thus reduces translation.

24. mRNA stability. When milk protein synthesis is stimulated in the mammary epithelium at child birth, the rapid increase in casein level arising from the pituitary hormone prolactin results from increased transcription of the casein gene but also from stabilisation of its mRNA. In fact, the stabilisation of mRNA is an essential component of the rapid build up of casein protein and this sort of regulation is evident in many situations in which the production of a particular protein needs to be increased to a high level. The mechanism is not well understood. The poly A tail protects the mRA from 3� degradation. Histone mRNA (histones are produced during the DNA synthetic phase of the cell cycle) do not have a poly(A) tail and are unstable. The sequence of bases in the 3� untranslated region, especially runs of As and Us can affect the stability of individual mRNAs. However, the enzzymes that break down the RNA are not well characterised.mRNA stability. When milk protein synthesis is stimulated in the mammary epithelium at child birth, the rapid increase in casein level arising from the pituitary hormone prolactin results from increased transcription of the casein gene but also from stabilisation of its mRNA. In fact, the stabilisation of mRNA is an essential component of the rapid build up of casein protein and this sort of regulation is evident in many situations in which the production of a particular protein needs to be increased to a high level. The mechanism is not well understood. The poly A tail protects the mRA from 3� degradation. Histone mRNA (histones are produced during the DNA synthetic phase of the cell cycle) do not have a poly(A) tail and are unstable. The sequence of bases in the 3� untranslated region, especially runs of As and Us can affect the stability of individual mRNAs. However, the enzzymes that break down the RNA are not well characterised.

26. Protein stability. Ubiquitin system. 20 kda protein ubiquitin is activated by ATP It is linked by its C terminus to amino group on a lysine side chain in target protein. Enzyme is ubiquitin protein ligase. Up to 50 molecules of ubiquitin / target protein molecule. Ubiquitinylated protein molecule then degraded by proteosome. A large multiprotien complex (2000 kDa) The level of cell cycle regulatory proteins called cyclins are produced at the G1 to S phase boundary of the cell cycle. The proteins stimulate kinases which trigger DNA synthesis. Once this triggering has occurred, there is no further need for the cyclins and they are degraded by the ubiquitin system. This system allows for rapid removal of proteins. It is selective. Another method for degrading proteins, the lysozome is non-selective. It was originally thought that breakdown of proteins occurred in the lysozomes. However, reticulocytes which do not have lysozomes still break down abnormal proteins. The system involves a 76 amino acid residue protein called ubiquitin, because it is widespread in eukaryotic spp. It is also one of the most conserved proteins known, differing in only 3 AA between human and fruit fly. Attachment is to the C terminus of ubiquitin and this is transferred to the amino group of a lysine side chain of the protein. Many ubiquitins per target protein molecule. Proteasome is a 20 s/u multi protein complexcontaining at least 5 different proteolytic activities in the shape of a bi-capped hollow barrel. UBIQUITIN IS NOT DEGRADED. Protein stability. Ubiquitin system. 20 kda protein ubiquitin is activated by ATP It is linked by its C terminus to amino group on a lysine side chain in target protein. Enzyme is ubiquitin protein ligase. Up to 50 molecules of ubiquitin / target protein molecule. Ubiquitinylated protein molecule then degraded by proteosome. A large multiprotien complex (2000 kDa) The level of cell cycle regulatory proteins called cyclins are produced at the G1 to S phase boundary of the cell cycle. The proteins stimulate kinases which trigger DNA synthesis. Once this triggering has occurred, there is no further need for the cyclins and they are degraded by the ubiquitin system. This system allows for rapid removal of proteins. It is selective. Another method for degrading proteins, the lysozome is non-selective. It was originally thought that breakdown of proteins occurred in the lysozomes. However, reticulocytes which do not have lysozomes still break down abnormal proteins. The system involves a 76 amino acid residue protein called ubiquitin, because it is widespread in eukaryotic spp. It is also one of the most conserved proteins known, differing in only 3 AA between human and fruit fly. Attachment is to the C terminus of ubiquitin and this is transferred to the amino group of a lysine side chain of the protein. Many ubiquitins per target protein molecule. Proteasome is a 20 s/u multi protein complexcontaining at least 5 different proteolytic activities in the shape of a bi-capped hollow barrel. UBIQUITIN IS NOT DEGRADED.

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