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Molecular Biology in Medicine 医学分子生物学

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Molecular Biology in Medicine 医学分子生物学. 许正平 [email protected] The greatest intellectual revolution of the last 40 years may have taken place in biology. Can anyone be considered educated today who does not understand a little about molecular biology? - PowerPoint PPT Presentation

Text of Molecular Biology in Medicine 医学分子生物学

  • Molecular Biology in Medicine

    [email protected]

  • Thegreatestintellectualrevolutionof the last 40 years may have taken place in biology. Can anyone be considered educated today who does not understand a little about molecular biology?

    F. H. Westheimer (Harvard University)

  • Genetic Information Transfer Gene Transcription RNA Splicing & Editing RNA Protein Synthesis & Processing Regulation of Gene Expression (miRNARNAi)

  • PCR SDS SDS-PAGE Southern blotting; Northern blotting; Western blotting genechip, microarray, protein chip Gene knock-out/knock-in RNA interference (RNAi)

  • Co-IP pull-downFRET(SPR) ChIPChIP-on-chip X X

  • 3

    Molecular Cell Biology Gene

    More Information: Literature Internet

  • I. Introduction The Central Dogma

  • I. Introduction How many genes in the human genome?Gene Expression

  • I. Introduction FACT 1: an uniform genome in almost every cell of an organismFACT 3: the shape and function of each type of cell are differentFACT 2: the proteome in each type of cell is different

  • I. Introduction the actions and properties of each cell type are determined by the proteins it containsFACT 1: an uniform genome in almost every cell of an organismtranscription of different genes largely determines the actions and properties of cellsFACT 3: the shape and function of each type of cell are differentFACT 2: the proteome in each type of cell is different

  • I. Introduction the types and amounts of the various proteins in a cellthe concentration of mRNA and the frequency at which the mRNA is translatedwhich genes are transcribed and their rateof transcription in a particular cell typeTRUTH: the gene is differentially expressedregulationsame genome in all cells of an organismregulationregulation

  • I. Introduction Gene Expression Occurs by a Two-Stage ProcessTranscription: generates a single-stranded RNA identical in sequence with one of the strands of the duplex DNA Three principal classes of products:message RNA (mRNA)transfer RNA (tRNA)ribosomal RNA (rRNA) Principle: complementary base pairing

    Translation: converts the nucleotide sequence of an RNA into the sequence of amino acids comprising a protein

    each mRNA contains at least one coding region that is related to a protein sequence

  • II. TranscriptionDNA (gene)

    RNA polymerase

    Regulatory ProteinsKey PlayerspromoterAstartpointterminatorTranscription Unittemplateupstreamdownstreamenhancer

  • II. TranscriptionPrimary transcript is the original unmodified RNA product correspondingto a transcription unit. Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription. RNA polymerases are enzymes that synthesize RNA using a DNA template(formally described as DNA-dependent RNA polymerases). Terminator is a sequence of DNA, represented at the end of the transcript,that causes RNA polymerase to terminate transcription. Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene.Key Terms

  • Transcription in eukaryotic cells is divided into three classes. Each class is transcribed by a different RNA polymerase:

    RNA polymerase I:

    RNA polymerase II:

    RNA polymerase III:

    RNA Polymerase II. Transcription

  • Transcription in eukaryotic cells is divided into three classes. Each class is transcribed by a different RNA polymerase:

    RNA polymerase I: rRNA; resides in the nucleolus

    RNA polymerase II: mRNA, snRNA; locates in the nucleoplasm

    RNA polymerase III: tRNA and other small RNAs; nucleoplasm

    II. TranscriptionRNA Polymerase

  • The promoters for RNA polymerases I and II are (mostly) upstreamof the startpoint, but some promoters for RNA polymerase III lie downstream of the startpoint.

    Each promoter contains characteristic sets of short conserved sequences that are recognized by the appropriate class of factors.

    RNA polymerases I and III each recognize a relatively restricted setof promoters, and rely upon a small number of accessory factors. Promoters utilized by RNA polymerase II show more variation in sequence, and are modular in design. Promoter II. Transcription

  • Short sequence elements (cis-acting elements): bind by accessory factors (transcription factors)

    The regulatory region might exist in the promoters of certain eukaryotic genes.

    Location: usually upstream and in the vicinity of the startpoint.

    These sites usually are spread out over a region of >200 bp. common: used constitutivelyspecific: usage is regulated; define a particular class of genes

    These sites are organized in different combinationsCis-acting Element II. Transcription

  • Enhancer element is a cis-acting sequence that increases the utilization of (some) eukaryotic promoters.The components of an enhancer resemble those of the promoter. Involve in initiation, but far from startpoint. Are targets for tissue-specific or temporal regulation.Function in either orientation and in any location (upstream or downstream) relative to the promoter. Enhancertwo characteristics:1. the position of the enhancer need not be fixed.2. it can function in either orientation. II. Transcription

  • The Difference between Promoter and EnhancerThe distinction between promoters and enhancers is operational, rather than imply a fundamental difference in mechanism II. Transcription

    promoter enhancerpositionfixedvariable action directionone wayeither orientationthe density of regulatory elementssparseHeavy (closed packed)redundancy in functionnoyescooperativity between the binding of factorssequentialgreat

  • Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancersas well as a TATA box or other promoter element. Enhancers may be either upstream or downstream and as far away as 50 kb from the transcription start site. In some cases, promoter-proximal elements occur downstream from the start site as well. (b) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is 90 base pairs upstream from the start site. II. Transcription

  • Fact: Regulatory elements in eukaryotic DNA often are many kilobases from start sitesFinding Regulatory Element in Eukaryotic DNA II. Transcription

  • Transcription FactorAny protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor. binds to DNA (trans-acting factor): recognize cis-acting elementsinteracts with other protein: recognize RNA pol, or another factorThe common mode of regulation of eukaryotic transcription is positive: a transcription factor is provided under tissue-specific control to activate a promoter or set of promoters that contain a common target sequence. Regulation by specific repression of a target promoter is less common. II. Transcription

  • Accessory factors are needed for initiation, principally responsible for recognizing the promoter. Interaction with DNA, RNA polymerase, and/or another factors.

    Three groups:General factorsUpstream factorsInducible factorsAnother name: accessory factor II. Transcription

  • general factors: required for the mechanics of initiating RNA synthesis at all promoters; form a complex surrounding the startpoint with RNA pol, and determine the site of initiation. basal transcription apparatus (pol + GF) upstream factors: DNA-binding proteins that recognize specific short consensus elements located upstream of the startpoint. not regulated; ubiquitous; act upon any promoter that contains the appropriate binding site on DNA.

    inducible factors: function in the same general way as the upstream factors. have a regulatory role: control transcription patterns in time and space Accessory Factors II. Transcription

  • II. TranscriptionFour Stages in Transcription

  • On the genome Which gene(s) to be transcribed? Basic events: Protein binding and/or modification 2. On a specific gene If the gene can be transcribed successfully?

    3. On a transcript If the transcript could be correctly spliced? If the transcript could be correctly edited?

    Regulation Levels Key determinant: Cell Signaling!III. Regulation of transcription

  • Potential regulation pointsActivation of gene structure Initiation of transcriptionProcessing the transcript Termination of transcription Transport to cytoplasm

    the overwhelming majority of regulatory events occur at the initiation of transcription III. Regulation of transcription5 potential control points:Active StructureMajor Control PointAlternative Splicing

  • Regulatory Proteinsthe overwhelming majority of regulatory events occur at the initiation of tr

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