Molecular Biology of the Cell - Chapter 7 PDF

Alberts Johnson Lewis Raff Roberts Walter Molecular Biology of the Cell Fifth Edition Chapter 7 Control of Gene Expression Copyright © Garland Science 2008  Overview Overview of gene contro...

Alberts Johnson Lewis Raff Roberts Walter Molecular Biology of the Cell Fifth Edition Chapter 7 Control of Gene Expression Copyright © Garland Science 2008  Overview Overview of gene control DNA-binding regulatory proteins Examples of gene control: – Prokaryotes: Lac & Trp operons – Eukaryotes: Chromatin remodeling & position-effect variegation (already discussed, review) gene silencing: DNA methylation dosage compensation alternative splicing of mRNAs (already discussed, review) mRNA stability regulatory RNAs (siRNA & miRNA)  Both cells contain the same genome but they express different RNAs & proteins resulting in profound differences in form and function Figure 7-1 Molecular Biology of the Cell (© Garland Science 2008)  Differences in mRNA expression patterns among different types of cancer cells. Figure 7-3 Molecular Biology of the Cell (© Garland Science 2008)  Differences in protein expression pattern in different human tissues Figure 7-4 Molecular Biology of the Cell (© Garland Science 2008)  Six steps at which eukaryotic gene expression can be controlled Figure 7-5 Molecular Biology of the Cell (© Garland Science 2008)  The outside of the DNA helix can be read by DNA binding proteins without opening up the helix Figures 7-6 & 7-7 Molecular Biology of the Cell (© Garland Science 2008) Table 7-1 Molecular Biology of the Cell (© Garland Science 2008) Figure 7-9 Molecular Biology of the Cell (© Garland Science 2008)  Operon Model François Jacob and Jacques Monod, 1961 – Originally proposed idea that enzyme levels in cells were determined through transcriptional control – Shared the 1965 Nobel Prize in medicine François Jacob Jacques Monod 1920-2013 1910-1976  Operon Model Two controlling elements – Repressor: regulator protein that represses transcription – Operator : site on DNA to which repressor binds Components of an Operon – Set of contiguous structural genes Enzymes involved in lactose catabolism or Trp anabolism – Promoter – Operator Situated between promoter and structural genes – Multigenic mRNAs Offers efficient & coordinated expression of related genes  Operon Model François Jacob and Jacques Monod, 1961 Negative control mechanism for Inducible or Repressible expression – Negative control – repressors turn off transcription – Inducible expression Resting state: off Free Repressor binds operator – Repressible expression Resting state: on Repressor/corepressor bind operator  Operon Components SG = structural gene; point: operon genes grouped together to enable coordinated regulation of sets of genes encoding proteins that carry out a specific function (i.e., breakdown carbohydrates, synthesize amino acids, etc.  Negative & Positive Regulation of operons Lac operon Trp operon Figure 7-37 Molecular Biology of the Cell (© Garland Science 2008)  Operon : Negative control of Inducible system Resting state:? Free Repressor OFF Bound to Operator Example: Lac operon Operon : Negative control of Repressible system Resting state:? ON Free repressor not bound to operator Repressor/co- repressor binds operators Example: Trp operon  A real life example….. Classic example E. coli lactose operon – Negatively controlled inducible operon  Lactose Operon lacI is the regulator gene – Protein product encodes a repressor – Active as a tetramer, four identical polypeptide chains – Binds lac operator sterically prevents RNA polymerase from transcribing lacZ, lacY, and lacA However, low background of lacZ, lacY, and lacA transcription  Lactose Operon A few molecules of b- galactosidase (LacZ) and b- galactosidase permease (Lac Y) are always present. b-galactosidase permease “pumps” lactose into cells Role of LacA transacetylase unknown b-galactosidase cleaves lactose into glucose and galactose.  Lactose Operon Induction of lac operon – Inducer is allolactose, sensor of lactose – Allolactose binds repressor Releases from Operator Transcription is INDUCED  Lactose Operon Lac Z LacY LacA PI LacI Resting State: OFF LacI Allolactose Tetramer Allolactose derepresses: PI LacI Lac Z LacY LacA LacZ,Y, and A transcribed LacI Allolactose Lac Z LacY LacA Glucose induced Repression of lac Glucose prevents expression of lac operon – preferred carbon source – Ensures glucose utilization over lactose Called: Catabolite Repression Mediated by: 1. CAP (catabolite activator protein) regulatory protein 2. cAMP (cyclic AMP) CAP binds cAMP when cAMP present Promoter contains CAP/cAMP binding site  Glucose induced Repression of lac Promoter contains: – CAP/cAMP binding site – RNA polymerase binding site Organization of the promoter-operator region of the lac operon Glucose induced Repression of lac cAMP is the Effector molecule Low glucose ---- high cAMP High glucose ---- low cAMP glucose The adenylcyclase-catalyzed synthesis of cyclic AMP (cAMP) from ATP. cAMP/CAP Complex: Positive Control of Inducible Mechanism When glucose low: CAP binds cAMP CAP/cAMP complex binds promoter and exerts positive control of lac operon transcription – CAP/cAMP must be bound for normal induction – Only complex binds High glucose: cAMP low no complex and no induction Ensures Glucose utilized over other carbon sources  Lac Operon Summary 1. Negative Inducible System 2. Catabolite Repressible system 3. Lac Z,Y,A transcribed only when + lactose and low glucose 4. In the absence of lactose, repressor binds operator inhibits RNA polymerase  Eukaryotes Chromatin remodeling & position-effect variegation (already discussed, review) gene silencing dosage compensation alternative splicing of mRNAs (already discussed, review) mRNA stability regulatory RNAs (siRNA & miRNA)  Gene Silencing How are genes that are needed for neuron function kept silent in liver cells???? One example: DNA methylation  Silencing by Chemical Modification of DNA Bases Methylation of cytosine – ~2-7% of genome – Alters recognition by proteins – Usually found in base pair doublets 5’-mC p G 3’ 3’ G p Cm 5’ – DNA Methyl Transferases C p G unevenly distributed across genome The structure of 5-methylcytosine. – Clusters (CpG islands)  Methylated DNA Methylated CpG associated with repressed transcription Mechanism not fully understood – However, two proteins that repress transcription are known to bind methylated DNA – One of these, MeCP2 changes chromatin structure – Suggests that mCpG bind protein complex that represses nearby genes. – See increased hypermethylation of CpG islands in various cancers  Methylation involved in Imprinting Imprinting – Gene expression is controlled by its parental origin – 20 examples in mice and humans – Methylation established in parental germ line In offspring – Somatic cells develop with same methylation pattern – Germ line development methylation is erased and reestablished based if oogenesis occurs  Methylation established in parental germ line In offspring Somatic cells develop with same methylation pattern Germ line development methylation is erased and reestablished based if oogenesis occurs Imprinting – gene Methylation and “remembers” imprinting of the Igf2 gene in mice. November © 2003 John Wiley and 7, 2006 Sons Publishers  Dosage Compensation Sex chromosomes – XX-female XY-male in humans – Expression of genes on X chromosome will be twice as much in females as males? Gene dosage Three methods to respond to this situation – Inactivation – Hyperactivation – Hypoactivation Three mechanisms of dosage compensation for X-linked genes: inactivation, hyperactivation, and hypoactivation.  Inactivation of X chromosome in mammalian females Begins at site called X inactivation center (XIC) – Spreads in opposite directions towards ends of chromosome XIST gene (X inactive specific transcript) is expressed?!? on inactive chromosome – 17kb RNA – Nuclear localization – binds and coats inactive X chromosome specifically  Fig 24.22 Expression of the XIST gene in the inactive X © 2003 John Wiley and Sons Publishers chromosome of human 36females.  Development Early Embryogenesis – Both X chromosomes express XIST Later – XIST Transcript from one chromosome stabilize and eventually envelop whole X chromosome – XIST transcripts from the other, disintegrate and expression is repressed by methylation of promoter – Random of which X become inactive Active X chromosome represses XIST  Inactive X chromosome During interphase, form darkly staining mass called Barr body During S phase, decondenses and is replicated after all other chromosomes Also have low levels of acetylated Histone H4 – Consistent with the idea that expression in repressed  Hyperactivation of X chromosome in Drosophila males Complex of proteins and RNA bind to specific sites on male X chromosome – These proteins and RNA are only expressed in males Bind 30-40 sites Likely to be involved in chromatin remodeling because – One gene, encodes a helicase – Another gene, is a histone acetyl transferase  Hypoactivation of X chromosomes in Caenorhabditis elegans females Exact mechanism not known but… Protein binds specifically to both X chromosomes in females, not the X in males Results in repression (hypoexpression) of expression on both female X chromosomes – Opposite of mechanism in Drosophila  Alternative Splicing of mRNA Spliceosomes in nucleus remove introns Can modify coding sequence by removing exons – Dependent on RNA/Spliceosome interaction – Economizes on genetic information – Create numerous related yet different proteins  Genes expressed that lead to female development Genes expressed that lead to male development Alternate splicing of transcripts from the Sex-lethal gene in male and female Drosophila. 42  mRNA Stability mRNA in cytoplasm can be translated by several ribosomes simultaneously – Continues until mRNA degraded – Short lived mRNA – Long lived mRNA Lifetime of mRNA controlled by 1. Length of Poly(A) tail 2. 3’UTR sequence – (AUUUA promotes rapid degradation) 3. Metabolic state of cell 4. Regulatory Mechanisms……. RNA Dependent Regulation of Gene Expression Two related mechanisms for inhibition of specific gene expression 1. Directed degradation of mRNA 2. Translational inhibition of specific mRNAs RNAi- Directed Degradation of mRNA in C. elegans and Drosophila First observed while performing (-) control in an anti-sense experiment – Sense strand silenced expression! Introduction of dsRNA homologous to gene of choice results in a much more efficient silencing than either sense or antisense alone Inject dsRNA into worms, – Progeny worms have gene silencing phenotype!  Mechanism of Action-RNAi Initiation – Long dsRNA are processed into 21-23 nucleotide duplexes termed: small interfereing RNAs (siRNA) Cleaved by Dicer – a RNaseIII family member Each strand has a 2-nucleotide 3’overhang RNAi: A Natural Mechanism to Control Protein Expression microRNA (left) siRNA (right)  Mechanism of Action-RNAi Effector step 1. siRNA duplex bound to RNA-induced silencing complex (RISC) 2. Duplex unwound in ATP dependent fashion 3. The antisense strand is preferentially bound to RISC, sense strand diffuses away 4. The RISC/antisense strand complex binds the homologous mRNA transcript – 100% complementarity 5. Target mRNA is cleaved in the middle of the antisense/mRNA duplex by a non-Dicer RNase  Mechanism of Action-RNAi Effector step cont. 6. Cleaved mRNA fragments recognized by cell as unproductive and degraded 7. RESULT- Specific degradation of mRNA! – Amplification of the RNAi signal may occur! The single stranded siRNA or cleaved mRNA may be the template for RNA-dependent RNA polymerase Create more gene specific dsRNA to be cleaved by Dicer and fed into the RNAi pathway!  Translational inhibition of specific mRNAs Mediated by miRNAs (micro RNAs) – >600 microRNA genes in human genome Important in normal and abnormal development – Transcribed as part of longer RNA molecules (