分子遗传学英文课件WORD版.doc

1-Structures of gene, promoter and enhancerGenotype: The genetic constitution of an organism.Phenotype: The appearance or other characteristics of an organism, resulting from the interaction of its genetic constitution with the environment.Gene: The segment of DNA involved in producing a polypeptide chain. An eukaryotic genes includes a promoter region, regions preceding (5 UTR) and following (3 UTR) the coding region as well as intervening sequences (introns) between individual coding segments (exons).a. Promoter region, responsible for the binding of RNA polymerase and for the subsequent initiation of transcriptionb. The transcriptional initiation site, where the transcription starts and will receive a “cap” of modified (methylated) nucleotides soon after it is transcribed.c. The translation initiation site (ATG), where the translation starts. The sequence between the transcription initiation site and translation initiation site is the 5 untranslated region (5UTR, part of the first exon), which can determine the rate at which translation is initiated.d. Exons, the region of DNA that codes for a protein.e. Introns, the intervening sequences that do not coding for amino acid sequence.f. The translation termination codon, TAA, which becomes UAA in the mRNA. The ribosome dissociates at this codon, and the protein is released.g. 3 untranslated region, part of last exon, containing the sequence AATAAA, which is needed for polyadenylation (addition of a “tail” of about 200-300 adenylate residues on the RNA transcript). This polyA tail 1) confers stability on the mRNA, 2) allows the mRNA to exit the nucleus, and 3) permits the mRNA to be translated into protein. The PolyA tail is inserted into the RNA about 20 bases downstream of the AAUAAA sequence.h. Transcription termination sequence, transcription continues beyond the AATAAA site for about 1000 nucleotides before being terminated.cDNA: A single-stranded DNA complementary to an RNA, synthesized from it by reverse transcription in vitro.Genomic DNA clone: A DNA fragment of the genome carried by a cloning vector.Capping at 5 end with a methylated guanosine and polyadenylation at 3 end may protect the RNA from exonucleases, thereby stabilizing the message and its precursor. The 5 cap is also necessary for the binding of mRNA to the ribosome and for subsequent translation.Promoter structure and functionPromoter: DNA sequences that are typically located immediately upstream from the site where transcription begins- usually several hundreds of base pairs long.Promoter site is required for the binding of RNA polymerase II and the accurate initiation of transcription.Most promoters of genes have similar structures. A typical promoter contains a TATA sequence (also called TATA box) about 30 bp upstream from the initiation site of transcription. Many TATA box regions are flanked by CpG islands (regions of DNA rich in those two nucleotides). Eukaryotic RNA polymerase, whoever, will not bind to this naked DNA sequence. They require the presence of additional proteins, called basal transcription factors, to bind efficiently to the promoter. At least 6 basal transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) are known to be required for proper initiation of transcription by RNA polymerase II.Another critical sequence in a promoter region is CAAT. Mutation in the sequences in TATA or CAAT eliminates or reduces the ability of the promoter to drive transcription.Some promoters do not contain TATA box. In these cases, they usually contain Sp1 sites in the promoter region. Sp1 site (GGGCGG) can be bound by Sp1, a general promoter-binding protein to initiate transcription.Demonstration of promoter function:Promoter + reporter gene- in vitro transfection assay and in vivo assay by transgenic approach. Sited directed mutagenesis could be generated in promoter to test sequence specificity.Enhancer structure and functionEnhancer: DNA sequences that can regulate the utilization of the promoter, controlling the efficiency and rate of transcription from that particular promoter.Enhancers can regulate only cis-linked promoter (on the same chromosome). Enhancer region can be located close to or at a great distance (million base pairs away) to the promoter, being at the 5 or 3 of the gene, or even in the intron.Enhancer can be positive enhancer or negative enhancer. Positive enhancer activates transcription from cis-linked promoter, while negative enhancer (also called silencer) represses transcription from cis-linked promoter.Sometimes one enhancer can act as positive enhancer in some cells but as negative enhancer in others, depending on the other transcription factors present in the cell.Enhancers are required for correct transcription of genes in space (tissue- or cell-specific) and time.The interaction between the proteins bound to the enhancer sites with the transcription apparatus assembled at the promoter regulates transcription.A given gene can have several enhancer sites linked to it.Mutation or deletion of an enhancer can cause elimination or reduction of gene expression spatially and temporally.Function of an enhancer can be demonstrated both in vitro and in vivo.Basic promoter + a typical enhancer + a reporter gene-in vitro assay-enhancement or repression of transcription of reporter gene.Basic promoter + a typical enhancer + reporter gene-transgenic assay-correct expression pattern (spatial and temporal) in animals.2-Transcription factorsTranscription factorsTranscription factor: proteins that bind to the enhancer or promoter region and interact to activate or repress the transcription of a particular gene. A transcription factor contains a DNA-binding domain, a trans-activating domain, and possibly a protein-protein interaction domain.Most transcription factors can bind to specific DNA sequences through the DNA-bindign domains. Slight differences in the amino acids at the binding site can alter the sequence of the DNA to which it binds. Mutations in the DNA-binding domain can change (abolish or increase) its DNA binding ability to its specific DNA binding sequence.Trans-activating domain enables the transcription factor to interact with protein involved in binding RNA polymerase (such as TFIIB or TFIIE) or with enzymes that modify histones.Protein-protein interaction domain allows the transcription factors activity to be modulated by TAFs (TBP-associated factors) or other transcription factors.Transcription factors are grouped based on similarities in structure. Transcription factors within such a family share a common framework in their DNA-binding sites.Some major transcription factor familiesHomeodomain proteins:These proteins are critical for specifying the anterior-posterior body axes throughout the animal kingdom. This homeodomain was first seen in proteins that specify segment identity in Drosophila.These proteins contain a conserved DNA binding domain called homeodomain. It consists of 60 amino acids that fold into three helices. The latter two helices II and III are arranged into a helix-turn-helix confirmation that binds DNA in the major groove of the double helix. -TAAT- is a conserved binding sequence for most homeodomain in vitro. Homeodomain protein binds DNA as monomer.Sub-families: Hox, Pax, POU, and LIM. Basic Helix-loop-helix proteins:This family of proteins contain a basic region (basic amino acids) at the N-terminal and a helix-loop-helix domain. The bHLH proteins bind to DNA through the basic region that proceeds the first a-helix. The helices contain hydrophobic amino acids at every third or fourth position, so that the helix presents a surface of hydrophobic residues to the environment. This enables the the protein to pair by hydrophobic interaction with the same protein or with a related protein that displays such a surface.It was recently found that bHLH homodimers do not bind well to DNA. Heterodimers bind better to DNA and play functions.Ubiquitous bHLH proteins, positive bHLH proteins, negative bHLH proteins.Ubiquitous bHLH protein + positive bHLH protein = activation of transcriptionUbiquitous bHLH or positive bHLH protein + negative bHLH protein = repression.MyoD and myogenin are bHLH transcription factors.E12, E47: ubiquitous bHLH proteinsMyoD family: postive bHLH proteinsId: negative bHLH proteinE12 or E47 + MyoD protein = muscle differentiationId + MyoD protein or E12 or E47 = inhibition of muscle differentiation.The Id protein contains the HLH motif (for dimerization), but lacks of the basic region.Binding of Id to MyoD, E12 or E47 blocks the ability of these proteins to bind DNA.Basic Leucine Zipper proteins:The structure of basic leucine zipper (bZip) transcription factors is very similar to that of the bHLH proteins.The bZip proteins are dimers, each of whose subunits contains a basic DNA-binding domain at the C-terminal, followed closely by an a helix containing a leucine residue at every seventh position. A leucine zipper in one polypeptide interacts with a zipper in another polypeptide to form a dimer.Zinc Finger proteins:These proteins contain a small group of conserved amino acids (two cysteines and two histidines) that binds a zinc ion, and forms a relatively independent “finger-like” DNA binding domain. Each protein contains two or more such domains that are linked in tandem.Two types of DNA-binding proteins have this type of structure:1. The classic “zinc finger” proteins, such as Sp1, Krox-20, Egr-1.2. Nuclear hormone receptors: contain a hormone-binding domain, a DNA-binding domain (zinc-finger domain) which recognizes the hormone-responsive element, a trans-activation domain, which is involved in mediating the signal to initiate transcription. These factors are intracellular receptors that function as transcription factors. They bind hormone such as steroid, thyroid, or retinoic acid in the cytoplasm, translocate to the nucleus, and function via DNA binding. DNA-bending proteins:A set of transcription factors that bind to DNA and bend the bound DNA to faciliate transcription. HMG box proteins: high motility group proteins. Determination of the DNA-binding sites of transcription factors1. Reporter gene construct (a DNA fragment containing the potential enhancer element links to a basal promoter and a reporter gene)2. Gel mobility shift assay (GMSA)3. DNase-I protection assay (DNA footprinting).3-Signaling proteinsSignaling proteins are usually secreted small peptides that act on cell surface to influence cell proliferation and differentiation. Signaling proteins are sometimes called growth factors, because such class of proteins was originally identified based on their functions on stimulating cell growth. Signaling factors bind to their specific membrane-bound receptors, and activate receptors that then transduce signals into cell.A signaling protein, after it is secreted, acts on adjacent cells. This signaling action is called paracrine interaction, and the signaling protein is also called paracrine factor. A signaling protein can also act on the cell that secretes it. This signaling action is called autocrine interaction.A major difference between growth factors and hormone (also called endocrine factors) is that hormones travel through the blood to exert their effects, while growth factors are secreted into the immediate spaces around the cell producing them.Paracrine interaction is critical for embryonic and tissue induction, so that paracrine factors are the “inducing factors” of the embryonic induction. Many of the paracrine factors can be grouped into several major families on the basis of their structures.The fibroblast growth factors (FGFs):This family currently has over 2 dozens structurally related members. They were originally identified as mitogens for fibroblasts in cell culture. Four FGF receptors have been identified. These are receptor tyrosine kinases (RTK). FGFs are associated with several developmental functions, including angiogenesis (blood vessel formation), mesoderm formation, and organ formation and patterning.Receptor tyrosine kinases are proteins that extend through the cell membrane. On the extracellular side is the portion of the protein that binds to the paracrine factor. On the intracellular side is a dormant tyrosine kinase (which can phosphorylate another protein by splitting ATP). When a ligand binds to the receptor, the receptor undergoes a conformational change that enables it to dimerize with another receptor tyrosine kinase, which activated the latent kinase activity of each RTK, and these RTKs phosphorylate each other on particular tyrosine residues. Once RTK is activated, it initiates a signaling cascade by phosphorylating responding proteins. Several families of growth factors use RTK for signaling, including FGFs, epidermal growth factors (EGFs), platelet-derived growth factors (PDGFs), and stem cell factor.The transforming growth factor- (TGF-) superfamily:There are over 30 structurally related members of the TGF- superfamily, which include the TGF- family, the activin family, the bone morphogenetic proteins (BMPs), the Vg1 family, and other proteins. Members of TGF- superfamily are critical for mesoderm induction, organ formation, cell proliferation and growth, and apoptosis.Signaling of TGF- is mediated by heterodimers of transmembrane serine/threonine kinase of type I and type II receptors. The TGF-b ligand binds to a type II TGF-b receptor, which allows binding of receptor II to a type I receptor, and phosphorylating a serine or threonine on the type I receptor, thereby activating it. The activated type I receptor can now phosphorylate the Smad proteins. Phosphorylated Smad proteins binds to Smad4 and form the transcription factor complex that will enter the nucleus and act on gene expression regulation (activation or repression).The Hedgehog family:Vertebrates have at least three members of Hedgehog family, sonic hedgehog (shh), desert hedgehog (dhh), and indian hedgehog (ihh). Several potential Hedgehog receptors have been identified, including Patched (Ptc1 and Ptc2), smoothened (smo), and Hedgehog interacting protein (Hip). Hedgehog proteins are often used by the embryo to induce particular cell type and to create boundaries between tissues.In the presence of Hh ligand, Smo is relieved from the repression of Patched on the cell membrane, which in turn activates a Gli-type transcription factor that moves into the nucleus and turn on target gene. The Wnt family:At least 19 members of Wnt family have been found. They are cysteine-rich glycoproteins, and are involved in mesoderm induction, organ patterning and cell proliferation and growth. Frizzled is an identified receptor for Wnt proteins. In the “canonical” Wnt pathway, Wnt ligand binds to a Frizzled protein which activates the Disheveled protein. Once Disheveled is activated, it inhibits the activity of the Glycogen synthease kinase-3 (GSK-3), which allows b-catenin to be dissociated from the APC protein and enter nucleus. Once inside the nucleus, b-catenin forms heterodimer with an LEF or TCF DNA-binding protein, becoming a transcription factor and activates the Wnt-responsive genes.Wnt ligands can also function through the noncanonical pathways: acting through Rho GTPase which activates the kinases that phosphorylate cytoskeletal proteins and thereby alter cell shape, cell polarity and motility, or acting through phospholipase (PLC) that causes release of calcium ions. The released calcium ions can activate enzymes, transcription factors, and translation factors.The Notch pathwayJuxtacrine signaling: proteins from the inducing cell interact with receptor proteins of adjacent responding cells without diffusing from the cell producing it. Notch proteins (receptors)/delta proteins (ligands); Ephrin/eph receptors.Dominant-negative and dominant-positive effects:One effective way to block function of signaling protein is to generate dominant-negative mutation of its receptor. This mutated receptor will be generated such as the intracellular function domain will be deleted or inactivated. When this mutated form of receptor is expressed in cells, they can bind to ligands, as the wild type does, but fail to transduce ligands signal into cell.A way to enhance the activity of a receptor-mediated signaling is to generate dominant-positive mutation of the receptor. A receptor can be mutated to become activated in the absence of its ligand. For example, an amino acid change within the intracellular domain of a type I TGF-b receptor can generate a constitutively active form, which can phosphorylate responsive proteins in the absence of a TGF-b ligand. 4-Regulation of differential gene expression-IIt has been known that gene expression can be controlled at several levels: activation of gene structure - initiation of transcription - processing the transcripts - transport to cytoplasm - translation of mRNA.Genes do not exist in an uncovered state within the nucleus, readily accessible to RNA polymerase or to any enhancer- or promoter-binding protein. Rather, eukaryotic DNA is associated with proteins. Most are basic polypeptides called histones. This DNA-protein complex is called chromatin. Nucleosome: the basic unit of chromatin structure. It is composed of a histone octamer (two molecules of each of histones H2A, H2B, H3, and H4) wrapped about with two loops of about 146-bp DNA. The length of linker DNA between nucleosome is about 10 to 100 bp. Transcriptional regulation of gene expression: determines which of the nuclear genes are allowed to be transcr