CondensedVersionChapter31andVirusesPowerpoint2009 11.45.23 PM

CondensedVersionChapter31andVirusesPowerpoint2009 11.45.23 PM

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Unformatted text preview: Dec.3,2009 CHAPTER 31 Control of Gene Expression CHAPTER 31: CONTROL OF GENE EXPRESSION SELECTED LECTURE TOPICS: The Big Picture Differential gene expression: prokaryotes and eukaryotes Regulation of gene expression: Transcription regulated by DNA-binding proteins E. coli lactose (lac) operon Helix-turn-helix motif: prokaryotic DNA-binding proteins Eukaryotic gene regulation: Transcriptional activation/repression is mediated by protein–DNA and/or protein-protein interactions Evidence of Eukaryotic Differential Gene Expression: Relative amounts of mRNAs (% total RNA) 1,000 or more 0.001 (or less) CONTROL OF GENE EXPRESSION Theme: Control involves sequence-specific DNA binding proteins and/or protein-protein interactions Wide Groove DNA binding protein (dimer) Phage repressor protein bound to target sequence : “operator” Control of Prokaryotic (Differential) Gene Expression: Example: Transcriptional regulation of the lactose operon β-galactosidase is an inducible enzyme (-) inducer [~ 7% of cell’s total protein] (+) inducer Lactose operon regulation: an inducible operon lac repressor a (-) regulator Inducer: (+) regulator CAP protein (cAMP binding protein): a (+) regulator CAP-DNA complex: DNA bends 94o ** improves a weak promoter ++ * (+) +1 (p) Repressor (-) * *** Repressor and CAP protein overlap with RNA polymerase Repressor and corepressors: Regulation of the Tryptophan (trp) Operon trp operator DNA trp repressor (dimer) tryptophan (co-repressor) Helix-turn-helix motif of DNA binding regulatory proteins 34 Angstroms = 1 full turn of DNA helix DNA Exception: β-strand interaction is basis of Met operon repressor DNA binding. Eukaryotic Chromosomes Linear, organized and complex – not just a DNA molecule Levels of Complexity of Chromosome Structure Chromosome Pair Metaphase (1000:1 packing ratio) Nuclear scaffold proteins Solenoid (40:1 packing ratio) Nucleosomes [Histone proteins, (+) charge, high Arg, Lys content] Nucleosome (6-7:1 packing ratio) Eukaryotic Transcription is Regulated and Mediated by: • DNA-Protein Interactions • Protein-protein interactions (see Ch. 29 also) EXAMPLES Steroid hormone responsive genes Enhancer/activator protein complexes interact from a distance with RNA polymerase (at promoters): (Result is efficient initiation and enhanced transcription) 5` Activator protein F 3` Promoter Steroid Hormone - Enhancer System (see Ch.29) +1 1 2 Receptor 3 Binding to RNA Polymerase and transcription initiation Steroid-like hormone receptors: Conserved domains Binds transcription factors Activation (variable) (Estrogen) (Progesterone) (Glucocorticoid) (Vitamin D3) (Retinoic Acid) (Thyroxine) DNA binding Hormone binding EUKARYOTIC TRANSCRIPTION ACTIVATORS Ligand binding [Ex: Steroid hormones] Interacts with other proteins to regulate transcription DNA Different combinations of activators switch on different sets of genes Estrogen receptor is activated by hormone binding followed by coactivator binding, etc. Receptor activation domain and Mediator protein complexes interacts with RNA polymerase (at promoters) to turn on transcription. Dec.8,2009 Viruses: Exs: Polio, Influenza A and HIV Virus properties relevant to HIV and AIDS [Any of these can be drug or vaccine target] • • • • • • • • • • • Retrovirus [RNA to DNA to RNA] (+)-strand virus cDNA integrates in genome (provirus) Membrane virus with viral proteins on surface Polyprotein mRNAs High mutability (cf. influenza virus) Overlapping genes Alternative splicing Complex regulation of HIV gene expression [rev switch] Infects specific cell types via target cell receptors Life cycle style/stages (HIV): • Latent (like lambda phage) • active gene expression and virus production Viruses: LECTURE TOPICS 2008 Types of viruses: RNA and DNA Virus assembly: Simple (TMV) Complex (T4 bacteriophage) RNA viruses: Replication/Infection Strategies (poliovirus) Membrane Viruses Influenza A SARS West Nile Retroviruses (RNA to DNA) Types of Viruses * Some Other SS- RNA viruses: Retroviruses SARS West Nile Hantavirus * RNA virus genome/replication strategies: (+) and (-) refer to “sense” of RNA in virus particle Influenza A [HIV] (+) RNA is a translatable “mRNA” (-) RNA must be “copied” to (+) to be translated Poliovirus: (+) RNA (-) RNA (+) mRNA 1 2 3 4 For new virus or for translation to viral proteins Poliovirus nucleocapsid structure self-assembles (virion) Protein coat structure is icosahedral Poliovirus RNA codes for a polyprotein that is cleaved to form: 1) RNA replicase 2) Protease, some other proteins 3) Proteins for mature virus nucleocapsid [7,500 bases] P1 Membrane viruses: Semliki Forest Virus (ssRNA) Membrane virus infects cell by receptor-mediated endocytosis **Cell and viral membrane fuse New membrane virus buds from cell membrane: Creates new virus that has cell’s membrane as a coat (containing virus proteins) and a nucleocapsid inside Stop Dec 9, 2008 West Nile Virus (+RNA) 11kb Problem in Tucson, some deaths, Arizona and Pima County (Late summer, fall, 2006-2008) What type of virus is this? SARS coronavirus genome (+RNA) Longest known RNA virus genome (29,736 bases) Influenza A Virus: A membrane virus virus strain terminology: H#N# (Ex: H5N1-avian flu) (-) RNAs, 8 different ones Influenza A hemaglutinin mutations can and do lead to loss of immunity conferred by vaccine each year Antigenic drift: Influenza A Hemaglutinin is highly mutable at sialic acid receptor binding sites Influenza A Hemaglutinin mutations can and do lead to loss of immunity conferred by vaccine each year Antigenic drift: Neuraminidase also is highly mutable at sialic acid cleavage sites [Antigenic drift] [Pig infected by bird and human flu strains] [Antigenic shift] Influenza A infects man, birds, pigs, horses, other? Migrating birds can be a source of global transmission * * New combination of RNAs: A new virus strain [antigenic shift variant] Influenza A pandemics and predominant strains H1N1: humans, pigs H = hemagglutinin N = neuraminidase Virus Life Cycles and Infection Strategies Scenario 1: Infect + make more virus (polio, flu) Scenario 2: Tale of two fates (λ bacteriophage) Scenario 3: Tale of a few fates (retroviruses) VIRUS LIFE CYCLE Scenario 2: Bacteriophage lambda (λ) life cycle [A tale of two fates] 1 new 2 HIV does this in human chromosomes [Latent] Scenario 3: HIV (Retrovirus) [A tale of a few fates] ds DNA genome (provirus) integrates in chromosome like λ phage Provirus Retrovirus virion (HIV, RNA tumor viruses) [env] [core] [pol] Retrovirus reverse transcriptase: Has 3 different enzyme activities 1 2 RNase H 3 [1st DNA strand] [Cuts RNA] [2nd DNA strand] Retrovirus RNA ds-DNA by reverse transcriptase Reverse transcriptase has 3 different enzyme activities 1. RNA-directed DNA polymerase (1st DNA strand) 2. RNase H (ribonuclease that removes viral RNA) 3. DNA-directed DNA polymerase (2nd strand DNA) • Product is a double stranded DNA copy of viral genome • HIV genome inserts (integrates) into chromosome (uses viral integrase) Retrovirus genome replication is complex [primer is a tRNA] [Viral RNA] [ds-DNA] Retrovirus RNA tumor* viruses (oncogenic) Avian sarcoma virus genome Reverse transcriptase oncogene * Core proteins proteins in membrane Involved in replication HIV (retrovirus) genome Avian sarcoma retrovirus genome HIV and AIDS: LECTURE TOPICS HIV virus Life Cycle RNA Genome, integration, replication Regulation of HIV gene expression [complex] Relation to other retroviruses Transmisison of HIV and AIDS in humans HIV infection targets in humans Potential AIDS therapies Vaccine for HIV? * HIV mutation rates HIGH (like transcription, 10-4 to 10-5) HIV in/out of cell like a typical membrane virus HIV virus attachment and entry into cell HIV virus particle New HIV virus buds from cell membrane HIV virion membrane protein (GP120) binds to CD4 cell receptor in T4 cells (lymphocytes) HIV virion assembly and budding: - polyprotein cleavage by protease - assembly of nucleocapsid core - budding from cell membrane Fates of HIV target cells Virus properties relevant to HIV and AIDS [Any of these can be drug or vaccine target] • • • • • • • • • • • Retrovirus [RNA to DNA to RNA] (+)-strand virus cDNA integrates in genome (provirus) Membrane virus with viral proteins on surface Polyprotein mRNAs High mutability (cf. influenza virus) Overlapping genes Alternative splicing Complex regulation of HIV gene expression [rev switch] Infects specific cell types via target cell receptors Life cycle style/stages (HIV): • Latent (like lambda phage) • active gene expression and virus production HIV therapies and vaccine possibilities [can target any stage, theoretically] Provirus AZT ...
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