Regulation of gene expression -2


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  • 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.s
  • 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.
  • 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.
  • 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.
  • Regulation of gene expression -2

    1. 1. ‫دانشگاه علوم پزشكي وخدمات بهداش‬ ‫درماني تهران‬ Dr. Parvin Pasalar Tehran University of Medical Sciences
    2. 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
    3. 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
    4. 4. Different ways for regulation of gene expression in bacteria  1- Promoter recognition:  2-Transcription elongation( Attenuation)
    5. 5. OPERON in gene regulation of prokaryotes Definition: a few genes that are controlled collectively by one promoter Its structure: Each Operon is consisted of few structural genes( cistrons) and some cis-acting element such as promoter (P) and operator (O). Its regulation: There are one or more regulatory gene outside of the Operon that produce trans-acting factors such as repressor or activators. Classification: 1- Catabolic (inducible) such as Lac OPERON 2- Anabolic (repressible) such as ara OPERON 3- Other types
    6. 6. General structure of an OPERON
    7. 7. The activity of an Operon in the presence or the absence of repressor No repressor With repressor Figure 8.13
    8. 8. Lac OPERON an inducible Operon In the absence of lac In the presence of lac
    9. 9. CRP or CAP is positive regulator of Lac and some other catabolic Operons In the presence of lac + glucose CRP= Catabolic gene regulatory Protein CRP= cAMP receptor Protein CAP= Catabolic gene Activating Protein
    10. 10. Trp OPERON a repressible example In the absence of Trp In the presence of Trp
    11. 11. Attenuation by different secondary RNA structure Starved: antitermination Nonstarved: termination
    12. 12. The attenuators of some operons 36
    13. 13. Eukaryotic gene regulation occurs at several levels
    14. 14. Control at DNA level by - 1 DNA methylation  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.
    15. 15. Control at DNA level by Histon - 2 (modifications(Chromatin Remodeling • Acetylation by HATs and coactivators leads to euchromatin formation • Methylation by HDACs and corepressors leads to heterochromatin formation
    16. 16. Control at DNA level by gene -3 amplification Repeated rounds of DNA replication yield multiple copies of a particular chromosomal region.
    17. 17. Control at transcription - 4 initiation By using different sequences (promoter, enhancer or silencer sequences) and factors, the rate of transcription of a gene is controlled gene X promoter gene control region for gene X
    18. 18. Control at mRNA splicing - 5 ((alternate splicing (four exons) 1 2 1, 2 & 4 2ll ec 1, 2 & 3 1ll ec 4 3 Calcitonin gene-related peptide 32 amino acids Reduces bone resorption 37 amino acids Vasodilator 61
    19. 19. Alternative splicing: A Role - 5 in Sexual Behavior in Drosophila 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
    20. 20. Control at mRNA stability- 6 • The stem loop at 3’end is an’ iron response element’. • The stem loop is stabilised by a 90 kDa protein in the absence of iron and protects the mRNA from degradation. 90 kDa iron sensing protein ( aconitase) Transferrin receptor mRNA AUG UA A + iron Fe Transferrin receptor mRNA Degraded by 3’ nuclease • In the presence of iron, transferrin receptor protein synthesis is reduced. No iron : mRNA is translated into protein + iron stimulates
    21. 21. Control at mRNA stability- 6 • A stem loop is stabilised by the 90 kDa protein in the absence of iron. • This time, the stem loop is at the 5’ end of the mRNA. No iron AUG Ferritin mRNA • The presence of the stem loop prevents translation of this mRNA by blocking the progress of the ribosomes along the mRNA. + iron Fe AUG UA A + iron stimulates • In the presence of iron, the hairpin is lost, the ribosomes can translate the mRNA and ferritin protein synthesis is increased.
    22. 22. Control at mRNA stability- 6 • Some hormones which enhance the production of proteins also increase the half life of the protein’s mRNA. Estrogen : ovalbumin >24hr Prolactin : casein to 92hr t 1/2 from 2- 5hr to t1/2 from 5 hr
    23. 23. Control at initiation of translation - 7 3’ UTR 5’ UTR AUG UAA Specific sequences make specific secondary structures Specific protein factors bind to these secondary structures
    24. 24. 8-Regulation by protein stability •Ubiquitin-dependent proteolysis. Cyclins control of cell cycle. • Protein molecule is tagged for degradation by attachment of a 20 kDa protein, ubiquitin ATP NH 2 NH 2 + Doomed protein molecule CONH COOH ubiquitin protein ligase CONH 26S proteasome • The stability of a protein depends upon its N-terminal amino acid (the N-end rule). N-terminal : For example arginine , lysine : protein t1/2 = 3 min N-terminal : For example methionine, alanine, : t1/2 >20 hrs.
    25. 25. Regulation by water soluble Hormones Polypeptide hormones bind at the cell surface and activate transmembrane enzymes to produce second messengers (such as cAMP) that activate gene transcription.
    26. 26. Regulation by water soluble Hormones
    27. 27. Regulation by lipid soluble Hormones Steroid hormones pass through the cell membrane and bind cytoplasmic receptors, which together .bind directly to DNA and regulate gene expression