Gene Regulation PDF

LEARNING OUTCOMES At the end of this lecture, the students should be able to: explain the importance of regulation of gene expression explain the regulation of gene expression in prokaryotes using the lac operon as a model describe the involvement of regulatory proteins and regulatory...

LEARNING OUTCOMES At the end of this lecture, the students should be able to: explain the importance of regulation of gene expression explain the regulation of gene expression in prokaryotes using the lac operon as a model describe the involvement of regulatory proteins and regulatory sites on DNA (enhancers and response elements) Describe the clinical significance of dysregulation in gene expression Nuruliza Roslan, PhD REGULATION OF GENE EXPRESSION  Why the need to regulate gene expression? 1. To adapt to environment changes ◦ Turn the expression of genes on and off 2. To alter expression of genes during development ◦ Fertilised egg → multicellular organism ◦ Adolescence → adulthood  Ways to Regulate Protein Concentration in a Cell Synthesis of primary RNA transcript How to process this RNA into mRNA Posttranscriptional modifications of mRNA Degradation of mRNA Protein synthesis Posttranslational modification of protein Targeting and transport of the protein Degradation of the protein Seven processes that affect the steady-state concentration of a protein. Each process has several potential points of regulation. Regulation of gene expression in prokaryotes  Operons: most common in prokaryotes. An operon consists of: Promoter: binding site for RNA polymerase Operator: binding site of repressor, overlaps the promoter Structural genes The lac operon: 3 structural genes that code for enzymes that break down lactose  A. Transcription of mRNA from bacterial operons Operons: structural genes for proteins involved in performing a related function. The gene in operon are expressed coordinately: all ‘turned on’ or ‘turned off’. When operon is expressed, all of its genes are transcribed. The transcription product: a single polycistronic mRNA – contains multiple sets of start and stop codons. B. Regulation of RNA polymerase binding by repressors Repressors: regulatory proteins that prevent the binding of RNA polymerase to promoter. Inhibition of gene transcription: negative control. Stimulation of gene transcription: positive control. Regulatory mechanisms through controlling repressors 1. Induction: an inducer activates the repressor. 2. Repression: a co-repressor is required to activate the repressor. 1. Induction Inducers stimulates the expression of the lac operon by a: - binding to the repressor - changing its conformation so it can no longer bind to the operator.  Example: induction of the lac operon of E. coli by lactose If milk sugar lactose is available, the cells adapt and begin to produce 3 additional enzymes for lactose met. – encoded by lac operon.  2. Co-repressors A nutrient or its metabolite binds to repressor → activating it. Co-repressor (nutrient or its metabolites) – binds to repressor. Repressor – co-repressor complex bind to operator → prevent RNA pol → prevent gene transcription. Example of a co-repressor : trp operon Encodes 5 enzymes required for synthesis of AA trp Trp – a corepressor that binds to the inactive repressor → conformational change → bind to the operator → inhibit transcription of enzymes => Trp synthesis is inhibited Bacterial Operons in Gene Regulation  C. Stimulation of RNA polymerase binding Lac operon is repressed (turned off). Binding of repressor interferes with the progress of RNA polymerase → blocks transcription → negative regulation When only lactose is available Lac operon is induced. A small amount of lactose is converted to allolactose. Binds to the repressor protein – change its conformation – no longer can bind to the operator. RNA pol initiate transcription → positive regulation. Produces the 3 proteins that allow lactose to be used as energy production.  When both glucose and lactose are available Transcription of the lac operon is negligible, even if lactose is present at a high conc. Adenylyl cyclase is deactivated in the presence of glucose. RNA pol unable to initiate transcription → 3 structural genes not expressed.  D. Regulation of RNA pol binding by sigma factors Sigma (σ) factors bind to RNA pol → stimulate binding to certain promoters → activates transcription. σ70: standard σ factor in E. coli σ32 helps RNA to recognise promoters for the different operons for heat-shock proteins.  E. Attenuation of transcription Some operons are regulated by a process that attenuates transcription after it has been initiated. Example: high levels of tryptophan attenuate transcription of the E. coli trp operon as well as it repress transcription. Attenuation requires coupled transcription and translation, so this mechanism is not applicable to eukaryotic systems. A different hairpin loop forms in the mRNA that does not terminate transcription, and the complete mRNA is transcribed. Generates a hairpin loop in the mRNA that serves as a termination signal for RNA polymerase, and transcription terminates. Regulation of protein synthesis in eukaryotes Multiple Levels: A. DNA and the B. Transcription chromosome D. Initiation of C. Processing of translation & transcripts stability of mRNA  6 7 protein Gene Regulation processing & degradation 1 & 2. transcription - DNA packing - transcription factors 3 & 4. post-transcription 5 - mRNA processing initiation of 4 - splicing translation - 5’ cap & poly-A tail mRNA processing - breakdown by siRNA 5. translation - block start of 2 1 translation initiation of transcription 6 & 7. post-translation - protein processing - protein degradation mRNA mRNA splicing 4 protection 3 A. At the level of DNA and chromosome  1. Chromatin remodeling Displacement of the nucleosome from specific DNA sequences so that transcription of the genes in that sequence can be initiated. Alterations in histone acetyltransferases (HATs) or histone deacetylases (HDACs) → dysregulation of cellular proliferation in certain tumors. Degree of packing of DNA regulates transcription ◦ tightly wrapped around histones ◦ no transcription ◦ genes turned off ▪ heterochromatin darker DNA (H) = tightly packed ▪ euchromatin lighter DNA (E) = loosely packed H E Acetylation of histones enhances access to promoter region and facilitates transcription.  2. Methylation of DNA Cytosine residues in DNA can be methylated to produce 5- methylcytosine. – repress gene transcription Located in CG-rich sequences. A mechanism for regulating gene expression during differentiation, especially during fetal development. guides and restricts differentiation Methylation of DNA blocks transcription factors ◦ no transcription → genes turned off ◦ attachment of methyl groups (–CH3) to cytosine ◦ C = cytosine ◦ nearly permanent inactivation of genes ◦ ex. inactivated mammalian X chromosome = Barr body Prader-Willi and Angelman syndrome Prader-Willi syndrome, Angelman syndrome: deletions of >> fragile X mental retardation protein (FMRP) >>> needed for normal brain development  3. Gene rearrangement Segments of DNA can move from one location to another in the genome. The heavy chain gene from which lymphocytes produce immunoglobulins is generated by combining specific segments.  Disease: Philadelphia chromosome major rearrangements = known as translocations observed in metaphase chromosomes under the microscope produced by a balanced exchange between chromosomes 9 and 22. most of a gene from chromosome 9, the c-abl gene, is transferred to the BCR gene on chromosome 22 >>> Creating a fused BCR-abl gene. The c-abl gene is a tyrosine kinase and its regulation by the BCR promoter results in uncontrolled growth.  4. Gene amplification Certain regions undergo repeated cycles of DNA replication. The newly synthesised DNA is excised and forms small, unstable chromosomes→ double minutes The double minutes integrate into other chromosomes – amplifying the gene in the process.  Gene amplification in cancer Why the interest? ERBB2 Normal Breast cancer Sui, W. et al., 2009  Limited TPD52 co-localization with ERBB2 in SK-BR-3 breast cancer cell line TPD52 ERBB2 Merge Top Middle Roslan, N et al., 2013 (x100 objective, Leica confocal microscope)  5. Gene deletions Occur through errors in DNA replication and cell division. Example: various types of cancers. B. Transcription 1. Gene specific regulatory proteins: -repressors and inducers Transcription factors Repressors: repress transcription Inducers: induce transcription Coactivators Corepressors 2. Transcription factors that are steroid hormone/thyroid hormone receptors Steroid hormones activates or inhibit transcription of specific genes through binding to nuclear receptors. Nuclear receptors bind to DNA regulatory sequences → induce or repress transcriptions of target genes. 2. Transcription factors that are steroid hormone/thyroid hormone receptors 2. Transcription factors that are steroid hormone/thyroid hormone receptors HSP: heat-shock proteins; GRE: glucocorticoid response element; GR: glucocorticoid receptor Activity of the thyroid hormone receptor-retinoid Dimer (TR-RXR) in the presence and absence of Thyroid hormone (T3). HAC: histone acetylase, HDAC: histone deacytelase Disease: Androgen insensitivity syndrome/Testicular feminization syndrome ◦ Mutations in the AR gene cause androgen insensitivity syndrome (AIS). ◦ AR gene makes a protein = androgen receptor. ◦ Androgen receptors allow cells to respond to androgens, which are hormones (such as testosterone ) that direct male sexual development. ◦ Patients produce male sex steroids, but target cells fail to respond due to lack the appropriate intracellular transcription factor receptors ◦ Transcription of the AR genes responsible for masculinization is not activated ◦ AIS = Has XY karyotype but looks like a female 3. Structure of DNA-binding proteins Each of transcription factors has a distinct recognition site (DNA binding domain).  4. Regulation of TF If a cell upregulates or downregulates its synthesis of co- activators→ rate of transcription is increased or decreased. TF activity can be modulated: -changes in the amount of TF synthesized - binding a stimulatory or inhibitory ligand - stimulation of nuclear entry -presence of other TF 5. Multiple regulators of promoters Some TF can activate transcription of many different genes. C. Processing of transcripts  mRNA processing includes three major steps. RNA editing xx D. Initiation of translation & stability of mRNA 4 Mechanisms of Translation Regulation 1. Phosphorylation of translation initiation factors 2. Translational repressors (typically bind to 3’ UTR) 3. Disruption of eIF4E and eIF4G interactions 4. RNA-mediated regulation (gene silencing, eg. miRNA/micro-RNAs)  Micro-RNAs Prevent Translation of mRNA Micro-RNAs (miRNAs) silence genes by binding to mRNAs ◦ can prevent transcription of the mRNA by cleaving it (via endonucleases Drosha or Dicer) or by blocking it Some miRNAs are made briefly during development; they are called small temporal RNAs (stRNAs). Researchers Can Shut Down Genes Artificially via RNA Interference Any dsRNA that corresponds to an mRNA and is introduced into a cell will be cleaved by Dicer into short segments called small interfering RNAs (siRNAs). These will bind to the mRNA to silence its translation. The process is called RNA interference. https://www.syros.com/platform/what-is-gene-control Gene Silencing by RNA Interference  Generation of stably depleted TPD52 cell lines: SK-BR-3 cells Non-target shRNA shRNA-D52-2 shRNA-D52-3 TPD52 GFP (X63 objective, Leica confocal microscope) In summary, To live, cells must be able to respond to changes in their environment. Regulation of the two main steps of protein production — transcription and translation — is critical to this adaptability. Cells can control which genes get transcribed and which transcripts get translated; further, they can biochemically process transcripts and proteins in order to affect their activity. Differences between Prokaryotes and Eukaryotes 1. Eukaryotic cells undergo differentiation and through various developmental stages. 2. Eukaryotes contain nuclei. 3. DNA is complex with histones in eukaryotes. 4. Eukaryotic genes contain introns. 5. Bacterial genes are organized in operons (sets of genes under the control of a single promoter). 6. Each eukaryotic gene has its own promoter.