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Regulation of gene expression in eukaryotes



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Regulation of gene expression in eukaryotes

  1. 1. Dr. Namrata Chhabra Professor and Head Department of Biochemistry S.S.R. Medical College, Mauritius Biochemistry for medics- Lecture notes 1Biochemistry for medics-Lecture notes
  2. 2. IntroductionIntroduction Gene expression is the combined process of : o the transcription of a gene into mRNA, o the processing of that mRNA, and o its translation into protein (for protein-encoding genes). 2Biochemistry for medics-Lecture notes
  3. 3. Levels of regulation of geneLevels of regulation of gene expressionexpression 3Biochemistry for medics-Lecture notes
  4. 4. Purpose of regulation of genePurpose of regulation of gene expressionexpression Regulated expression of genes is required for 1) Adaptation- Cells of multicellular organisms respond to varying conditions. Such cells exposed to hormones and growth factors change substantially in – o shape, o growth rate, and o other characteristics 4Biochemistry for medics-Lecture notes
  5. 5. Purpose of regulation of genePurpose of regulation of gene expressionexpression 2) Tissue specific differentiation and development The genetic information present in each somatic cell of a metazoan organism is practically identical. Cells from muscle and nerve tissue show strikingly different morphologies and other properties, yet they contain exactly the same DNA. These diverse properties are the result of differences in gene expression. Expression of the genetic information is regulated during ontogeny and differentiation of the organism and its cellular components. 5Biochemistry for medics-Lecture notes
  6. 6. Control of gene ExpressionControl of gene Expression • Mammalian cells possess about 1000 times more genetic information than does the bacterium Escherichia coli. • Much of this additional genetic information is probably involved in regulation of gene expression during the differentiation of tissues and biologic processes in the multicellular organism and in ensuring that the organism can respond to complex environmental challenges. 6Biochemistry for medics-Lecture notes
  7. 7. Mechanism of regulation of geneMechanism of regulation of gene expression- An overviewexpression- An overview Gene activity is controlled first and foremost at the level of transcription. Much of this control is achieved through the interplay between proteins that bind to specific DNA sequences and their DNA binding sites. This can have a positive or negative effect on transcription. 7Biochemistry for medics-Lecture notes
  8. 8. Mechanism of regulation of geneMechanism of regulation of gene expression- An overviewexpression- An overview Transcription control can result in tissue-specific gene expression. In addition to transcription level controls, gene expression can also be modulated by Gene rearrangement, Gene amplification, Posttranscriptional modifications, and RNA stabilization. 8Biochemistry for medics-Lecture notes
  9. 9. Differences between geneDifferences between gene expression in prokaryotes andexpression in prokaryotes and eukaryoteseukaryotes Gene regulation is significantly more complex in eukaryotes than in prokaryotes for a number of reasons: 1) First, the genome being regulated is significantly larger o The E. coli genome consists of a single, circular chromosome containing 4.6 Mb. o This genome encodes approximately 2000 proteins. 9Biochemistry for medics-Lecture notes
  10. 10. 1) Larger genome1) Larger genome • In comparison, the genome within a human cell contains 23 pairs of chromosomes ranging in size from 50 to 250 Mb. • Approximately 40,000 genes are present within the 3000 Mb of human DNA. • It would be very difficult for a DNA-binding protein to recognize a unique site in this vast array of DNA sequences. • More-elaborate mechanisms are required to achieve specificity 10Biochemistry for medics-Lecture notes
  11. 11. 2) Different cell types2) Different cell types Different cell types are present in most eukaryotes. Liver and pancreatic cells, for example, differ dramatically in the genes that are highly expressed. Different mechanisms are involved in the regulation of such genes. 11Biochemistry for medics-Lecture notes
  12. 12. 3) Absence of operons3) Absence of operons The eukaryotic genes are not generally organized into operons as are there in prokaryotes Instead, genes that encode proteins for steps within a given pathway are often spread widely across the genome. 12Biochemistry for medics-Lecture notes
  13. 13. 4) Chromatin structure4) Chromatin structure The DNA in eukaryotic cells is extensively folded and packed into the protein-DNA complex called chromatin. Histones are an important part of this complex since they both form the structures known as nucleosomes and also contribute significantly into gene regulatory mechanisms. 13Biochemistry for medics-Lecture notes
  14. 14. 5) Uncoupled transcription and5) Uncoupled transcription and translation processestranslation processes • In prokaryotes, transcription and translation are coupled processes, the primary transcript is immediately translated. • The transcription and translation are uncoupled in eukaryotes, eliminating some potential gene- regulatory mechanisms. • The primary transcript in eukaryotes undergoes modifications to become a mature functional m RNA. 14Biochemistry for medics-Lecture notes
  15. 15. Mechanism of regulation of geneMechanism of regulation of gene expression- Detailsexpression- Details 1) Chromatin Remodeling • Chromatin structure provides an important level of control of gene transcription. • With few exceptions, each cell contains the same complement of genes (antibody-producing cells are a notable exception). • The development of specialized organs, tissues, and cells and their function in the intact organism depend upon the differential expression of genes. • Some of this differential expression is achieved by having different regions of chromatin available for transcription in cells from various tissues. 15Biochemistry for medics-Lecture notes
  16. 16. 1) Chromatin Remodeling1) Chromatin Remodeling Large regions of chromatin are transcriptionally inactive in some cells while they are either active or potentially active in other specialized cells For example, the DNA containing the -globin gene cluster is in "active" chromatin in the reticulocytes but in "inactive" chromatin in muscle cells. 16Biochemistry for medics-Lecture notes
  17. 17. 17Biochemistry for medics-Lecture notes
  18. 18. Formation and disruption ofFormation and disruption of nucleosome structurenucleosome structure • The presence of nucleosomes and of complexes of histones and DNA provide a barrier against the ready association of transcription factors with specific DNA regions. • The disruption of nucleosome structure is therefore an important part of eukaryotic gene regulation and the processes involved are as follows- 18Biochemistry for medics-Lecture notes
  19. 19. Formation and disruption ofFormation and disruption of nucleosome structure (contd.)nucleosome structure (contd.) i) Histone acetylation and deacetylation  Acetylation is known to occur on lysine residues in the amino terminal tails of histone molecules.  This modification reduces the positive charge of these tails and decreases the binding affinity of histone for the negatively charged DNA.  Accordingly, the acetylation of histones could result in disruption of nucleosomal structure and allow readier access of transcription factors to cognate regulatory DNA elements. 19Biochemistry for medics-Lecture notes
  20. 20. i) Histone Acetylation andi) Histone Acetylation and deacetylationdeacetylation The amino-terminal tail of histone H3 extends into a pocket in which a lysine side chain can accept an acetyl group from acetyl CoA bound in an adjacent site. 20Biochemistry for medics-Lecture notes
  21. 21. Formation and disruption ofFormation and disruption of nucleosome structure (contd.)nucleosome structure (contd.) ii) Modification of DNA  Methylation of deoxycytidine residues in DNA may effect gross changes in chromatin so as to preclude its active transcription.  Acute demethylation of deoxycytidine residues in a specific region of the tyrosine aminotransferase gene—in response to glucocorticoid hormones— has been associated with an increased rate of transcription of the gene. 21Biochemistry for medics-Lecture notes
  22. 22. Formation and disruption ofFormation and disruption of nucleosome structure (contd.)nucleosome structure (contd.) iii) DNA binding proteins The binding of specific transcription factors to certain DNA elements may result in disruption of nucleosomal structure. Many eukaryotic genes have multiple protein-binding DNA elements. The serial binding of transcription factors to these elements may either directly disrupt the structure of the nucleosome or prevent its re-formation. These reactions result in chromatin-level structural changes that in the end increase DNA accessibility to other factors and the transcription machinery. 22Biochemistry for medics-Lecture notes
  23. 23. 2) Enhancers and Repressors2) Enhancers and Repressors • Enhancer elements are DNA sequences, although they have no promoter activity of their own but they greatly increase the activities of many promoters in eukaryotes. • Enhancers function by serving as binding sites for specific regulatory proteins. • An enhancer is effective only in the specific cell types in which appropriate regulatory proteins are expressed. 23Biochemistry for medics-Lecture notes
  24. 24. 2) Enhancers and Repressors2) Enhancers and Repressors (contd.)(contd.) • Enhancer elements can exert their positive influence on transcription even when separated by thousands of base pairs from a promoter; • they work when oriented in either direction; and they can work upstream (5') or downstream (3') from the promoter. • Enhancers are promiscuous; they can stimulate any promoter in the vicinity and may act on more than one promoter. 24Biochemistry for medics-Lecture notes
  25. 25. 2) Enhancers and Repressors2) Enhancers and Repressors (contd.)(contd.) • The elements that decrease or repress the expression of specific genes have also been identified. • Silencers are control regions of DNA that, like enhancers, may be located thousands of base pairs away from the gene they control. • However, when transcription factors bind to them, expression of the gene they control is repressed. • Tissue-specific gene expression is mediated by enhancers or enhancer-like elements. 25Biochemistry for medics-Lecture notes
  26. 26. 2) Enhancers and Repressors2) Enhancers and Repressors (contd.)(contd.) 26Biochemistry for medics-Lecture notes
  27. 27. 3) Locus control regions and3) Locus control regions and InsulatorsInsulators • Some regions are controlled by complex DNA elements called locus control regions (LCRs). • An LCR—with associated bound proteins—controls the expression of a cluster of genes. The best-defined LCR regulates expression of the globin gene family over a large region of DNA. • Another mechanism is provided by insulators. These DNA elements, also in association with one or more proteins, prevent an enhancer from acting on a promoter . 27Biochemistry for medics-Lecture notes
  28. 28. 3) Locus control regions and3) Locus control regions and InsulatorsInsulators Biochemistry for medics-Lecture notes 28
  29. 29. 4) Gene Amplification4) Gene Amplification The gene product can be increased by increasing the number of genes available for transcription of specific molecules Among the repetitive DNA sequences are hundreds of copies of ribosomal RNA genes and tRNA genes. During early development of metazoans, there is an abrupt increase in the need for ribosomal RNA and messenger RNA molecules for proteins that make up such organs as the eggshell. 29Biochemistry for medics-Lecture notes
  30. 30. 4) Gene Amplification (contd.)4) Gene Amplification (contd.) Such requirements are fulfilled by amplification of these specific genes.  Subsequently, these amplified genes, presumably generated by a process of repeated initiations during DNA synthesis, provide multiple sites for gene transcription. 30Biochemistry for medics-Lecture notes
  31. 31. 4) Gene Amplification(contd.)4) Gene Amplification(contd.) Gene amplification has been demonstrated in patients receiving methotrexate for cancer. The malignant cells can develop drug resistance by increasing the number of genes for dihydrofolate reductase, the target of Methotrexate. 31Biochemistry for medics-Lecture notes
  32. 32. 5) Gene Rearrangement5) Gene Rearrangement Gene rearrangement is observed during immunoglobulins synthesis. Immunoglobulins are composed of two polypeptides, heavy (about 50 kDa) and light (about 25 kDa) chains. The mRNAs encoding these two protein subunits are encoded by gene sequences that are subjected to extensive DNA sequence-coding changes. 32Biochemistry for medics-Lecture notes
  33. 33. 5) Gene Rearrangement (contd.)5) Gene Rearrangement (contd.) These DNA coding changes are needed for generating the required recognition diversity central to appropriate immune function. 33Biochemistry for medics-Lecture notes
  34. 34. 5) Gene Rearrangement (contd.)5) Gene Rearrangement (contd.) The IgG light chain is composed of variable (VL), joining (JL), and constant (CL) domains or segments. For particular subsets of IgG light chains, there are roughly 300 tandemly repeated VL gene coding segments, five tandemly arranged JL coding sequences, and roughly ten CL gene coding segments. All of these multiple, distinct coding regions are located in the same region of the same chromosome. 34Biochemistry for medics-Lecture notes
  35. 35. 5) Gene Rearrangement (contd.)5) Gene Rearrangement (contd.) However, a given functional IgG light chain transcription unit contains only the coding sequences for a single protein. Thus, before a particular IgG light chain can be expressed, single VL, JL, and CL coding sequences must be recombined to generate a single, contiguous transcription unit excluding the multiple nonutilized segments. 35Biochemistry for medics-Lecture notes
  36. 36. 5) Gene Rearrangement (contd.)5) Gene Rearrangement (contd.) This deletion of unused genetic information is accomplished by selective DNA recombination that removes the unwanted coding DNA while retaining the required coding sequences: one VL, one JL, and one CL sequence. 36Biochemistry for medics-Lecture notes
  37. 37. 6) Alternative RNA Processing6) Alternative RNA Processing Eukaryotic cells also employ alternative RNA processing to control gene expression. This can result when alternative promoters, intron- exon splice sites, or polyadenylation sites are used. Occasionally, heterogeneity within a cell results, but more commonly the same primary transcript is processed differently in different tissues. 37Biochemistry for medics-Lecture notes
  38. 38. 6) Alternative RNA Processing6) Alternative RNA Processing (contd.)(contd.) Alternative polyadenylation sites in the immunoglobulin (Ig M) heavy chain primary transcript result in mRNAs that are either 2700 bases long (m) or 2400 bases long (s). This results in a different carboxyl terminal region of the encoded proteins such that the (m ) protein remains attached to the membrane of the B lymphocyte and the (s) immunoglobulin is secreted. 38Biochemistry for medics-Lecture notes
  39. 39. 6) Alternative RNA Processing6) Alternative RNA Processing (contd.)(contd.) 39Biochemistry for medics-Lecture notes
  40. 40. 6) Alternative RNA Processing6) Alternative RNA Processing (contd.)(contd.) Alternative splicing and processing, results in the formation of seven unique -tropomyosin mRNAs in seven different tissues. 40Biochemistry for medics-Lecture notes
  41. 41. 7) Class switching7) Class switching In this process one gene is switched off and a closely related gene takes up the function. During intrauterine life embryonic Hb is the first Hb to be formed. 41Biochemistry for medics-Lecture notes
  42. 42. 7) Class switching (contd.)7) Class switching (contd.) It is produced by having two “Zeta” and two “Epsilon” chains. By the sixth month of intrauterine life, embryonic Hb is replaced by HbF consisting of “α2and y2 chains. After birth HbF is replaced by adult type of Hb A 1(97%) and HbA2(3%). Thus the genes for a particular class of Hb are switched off and for another class are switched on. 42Biochemistry for medics-Lecture notes
  43. 43. 7) Class switching (contd.)7) Class switching (contd.) Gene switching is also observed in the formation of immunoglobulins. Ig M is the formed during primary immune response, while Ig G is formed during secondary immune response. 43Biochemistry for medics-Lecture notes
  44. 44. 8) mRNA stability8) mRNA stability Although most mRNAs in mammalian cells are very stable (half-lives measured in hours), some turn over very rapidly (half-lives of 10–30 minutes). In certain instances, mRNA stability is subject to regulation. This has important implications since there is usually a direct relationship between mRNA amount and the translation of that mRNA into its cognate protein. 44Biochemistry for medics-Lecture notes
  45. 45. 8) mRNA stability (contd.)8) mRNA stability (contd.) Changes in the stability of a specific mRNA can therefore have major effects on biologic processes. The stability of the m RNA can be influenced by hormones and certain other effectors. The ends of mRNA molecules are involved in mRNA stability. The 5' cap structure in eukaryotic mRNA prevents attack by 5' exonucleases, and the poly(A) tail prohibits the action of 3' exonucleases. 45Biochemistry for medics-Lecture notes
  46. 46. 9)DNA binding proteins9)DNA binding proteins Steroids such as estrogens bind to eukaryotic transcription factors called nuclear hormone receptors. These proteins are capable of binding DNA whether or not ligands are bound. The binding of ligands induces a conformational change that allows the recruitment of additional proteins called co activators. 46Biochemistry for medics-Lecture notes
  47. 47. 9) DNA binding proteins (contd.)9) DNA binding proteins (contd.) Among the most important functions of co-activators is catalysis of the addition of acetyl groups to lysine residues in the tails of histone proteins.  Histone acetylation decreases the affinity of the histones for DNA, making additional genes accessible for transcription. 47Biochemistry for medics-Lecture notes
  48. 48. 10)Specific motifs of regulatory10)Specific motifs of regulatory proteinsproteins Certain DNA binding proteins having specific motifs bind certain region of DNA to influence the rate of transcription. The specificity involved in the control of transcription requires that regulatory proteins bind with high affinity to the correct region of DNA. 48Biochemistry for medics-Lecture notes
  49. 49. 10) Specific motifs of regulatory10) Specific motifs of regulatory proteins (contd.)proteins (contd.) Three unique motifs—the helix-turn-helix, the zinc finger, and the leucine zipper—account for many of these specific protein-DNA interactions. The motifs found in these proteins are unique; their presence in a protein of unknown function suggests that the protein may bind to DNA. The protein-DNA interactions are maintained by hydrogen bonds and van der Waals forces. 49Biochemistry for medics-Lecture notes
  50. 50. Three unique motifs of DNAThree unique motifs of DNA binding proteinsbinding proteins Helix –turn- helix Leucine zipper Zinc finger 50Biochemistry for medics-Lecture notes
  51. 51. 11) RNA Editing11) RNA Editing Enzyme- catalyzed deamination of a specific cytidine residue in the mRNA of apolipoprotein B-100 changes a codon for glutamine (CAA) to a stop codon (UAA). Apolipoprotein B-48, a truncated version of the protein lacking the LDL receptor-binding domain, is generated by this posttranscriptional change in the mRNA sequence. 51Biochemistry for medics-Lecture notes
  52. 52. 11)RNA Editing (contd.)11)RNA Editing (contd.) 52Biochemistry for medics-Lecture notes
  53. 53. SummarySummary The genetic constitutions of nearly all metazoan somatic cells are identical. Tissue or cell specificity is dictated by differences in gene expression of this complement of genes. Alterations in gene expression allow a cell to adapt to environmental changes. Gene expression can be controlled at multiple levels by chromatin modifications ,changes in transcription, RNA processing, localization, and stability or utilization. Gene amplification and rearrangements also influence gene expression. 53Biochemistry for medics-Lecture notes