Regulation of Gene Expression PDF

1 REGULATION OF GENE EXPRESSION 9/29/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences  2 Student learning objectives ▪ Reading guide learning objectives....

1 REGULATION OF GENE EXPRESSION 9/29/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences  2 Student learning objectives ▪ Reading guide learning objectives. ▪ Explain how gene expression regulation contributes to cell differentiation and ▪ Differentiate between various levels of gene developmental processes in multicellular regulation. organisms. ▪ Identify and describe the roles of cis- and ▪ Identify key cis and trans regulatory elements trans-acting regulatory elements in gene involved in the regulation of gene expression expression. and their mechanisms of action. ▪ Discuss how prokaryotic cells use gene ▪ Explain how processes such as splicing, regulation to respond to environmental capping, and polyadenylation affect mRNA stresses. stability and translation efficiency. ▪ Describe how alternative sigma factors help ▪ Explain how chromatin remodeling and prokaryotes adapt to environmental changes histone modifications influence gene by regulating transcription of specific sets of accessibility and expression. genes. ▪ Explain epigenetic inheritance based on ▪ Explain the concept of operons. histone modifications and on DNA ▪ Identify how activators and repressors methylation. regulate gene expression in prokaryotes, ▪ Describe the roles of miRNAs and siRNAs in including examples like the lac and trp operon. gene silencing. ▪ Explain what riboswitches are and their role ▪ Describe the action of lncRNAs in gene as regulatory elements. repression and activation.  3 Lecture structure 1. Control of gene expression 4.2 Post-transcriptional Control 2. Regulatory elements 4.3 Control by non-coding RNA 3. Prokaryotic cells 3.1 Principles 3.2 Transcriptional control ▪ Operon – lac and trp operon 3.3 Post-transcriptional – attenuation and riboswitches 4. Eukaryotic cells 4.1 Transcriptional Control A) cis-acting elements B) trans-acting elements C) Cell differentiation and maintenance of identity  4 1. Control of gene expression ▪ A cell generally uses only a fraction of its genome at any given moment in time ▪ Constitutive genes (housekeeping genes) – continuously expressed at a relatively constant level required for normal cell function ▪ Regulated genes – expression changes in response to external and internal signals Outcomes of control of gene expression: ▪ Turn of or off expression of gene ▪ Increase or decrease expression of a gene Gene regulation can occur at various steps… Eukaryotes: … why? Chromatin remodeling Ubiquitinoylation Protein folding, phosphorylation,… *0, 2, and 3 absent in prokaryotes  5 2. Regulatory elements ▪ cis-acting element ▪ Control the expression of genes on the same contiguous piece of DNA - regulatory sequences ▪ trans-acting element ▪ Proteins that bind cis elements - regulatory proteins ▪ Activate transcription ▪ Inhibit transcription Zing finger, basic zipper, basic helix loop helix  6 3. Gene regulation in prokaryotes ▪ Energy efficiency – only express genes when needed ▪ Response to environmental and physical changes Why and when would bacteria regulate gene ▪ Metabolic flexibility – diversity in nutrients expression? ▪ Rapid growth ▪ Stress insults Mechanisms of transcriptional gene expression control: 1. RNA polymerase – 𝜎 (sigma) factor 2. Repression of gene expression ▪ By default, gene expression is ‘on’ ▪ Most genes are under negative control - repression  7 3.1. Bacterial Operons Are Coregulated Gene Clusters Operon – functionally related genes are regulated as a unit ▪ Proteins with related functions – participate in same metabolic pathway → efficient regulation ▪ Polycistronic mRNA Components: ▪ Structural genes ▪ Promoter ▪ Operator – where trans acting elements will bind to ▪ Regulatory gene – separate genes that control the operon expression  8 3.2. How Is Gene Expression Repressed in Bacteria? A. Repressible – anabolic operons ▪ trp operon ▪ Default – on ▪ Repressor Negative ▪ Co-repressor control Tryptophan  9 3.2. How Is Gene Expression Repressed in Bacteria? B. Inducible – catabolic operons ▪ lac operon ▪ Default – off ▪ Repressor – lac repressor Negative ▪ Inducer – lactose control ▪ Activator ▪ Catabolyte activating Positive protein (CAP) control ▪ cAMP CRP=CAP activator  10 3.3. Post-transcriptional control ▪ 3.3.1. Attenuation – transcription elongation control ▪ Fine tuning regulation – determines whether enough tryptophan is present to support protein); regulation of transcription elongation ▪ Ribosome dependent – stalling at 2xTrp codons ▪ Leader sequence 2x Trp codons 2-pairing with-3 Antiterminator hairpin High tryptophan 3-pairing with-4 Terminator hairpin Low tryptophan  11 3.3. Post-transcriptional control ▪ 3.3.1. Riboswitches ▪ 5’ UTR segment ofm RNA that can regulate gene expression in response to specific metabolites ▪ Small metabolites binding (e.g. purine) – terminator hairpin ▪ Ribosome independent – regulation during transcription elongation  12 4. Gene regulation in eukaryotes ▪ Cell differentiation – cell identity ▪ Development processes Why and when would ▪ Homeostasis and responses to environmental cues eukaryotic cell regulate gene ▪ Cell cycle and division expression? ▪ Immune response ▪ Prevention of disease – dysfunction in regulatory mechanisms – abnormal development, pathologies,… Ground state of gene expression – ‘off’ Mechanisms: 1. Transcriptional regulation ▪ Chromatin structure 2. Post-transcriptional regulation 3. Post-translational regulation (already covered) Role of transcription factors and chromatin 4. Control by Non-coding RNA  13 4.1 Transcriptional Control A. cis-acting regulatory elements Upstream of coding sequence: ▪ Promoter (TATA box) Kilobases away from gene: upstream, introns, downstream -restricted to the same chromatin domain ▪ Enhancer ▪ Silencers B. trans-acting regulatory elements ▪ General transcription factors → promoters ▪ Specific transcription factors → enhancers and silencers ▪ Repressors – inhibit transcription ▪ Activators – activate transcription Insulator element  14 4.1 Transcriptional Control – Transcription Factors ▪ Structure: homo- hetero- dimers zinc finger ▪ N-term – DNA binding domain (motifs) basic zipper basic helix loop helix ▪ C-term – activation/repression domain ▪ Function: activate or inhibit (rarely) transcription ▪ Combinatorial control - coordinated interactions of multiple transcription factors Repressors ▪ Co-activators Do not interact ▪ Co-repressors with DNA Activator  15 4.1 Transcriptional Control – Transcription Factors A. ACTIVATORS: stimulate transcription ▪ Recruit other regulators and RNA polymerase: ▪ Participate in assembly of transcription initiation complex (through mediator) ▪ Release RNA polymerase (phosphorylation of CTD-tail) to begin transcription ▪ Promote changes to local chromatin structure – accessibility of promoter ▪ Chromatin remodeling complex ▪ Histon-modifying enzyme acetylases (HAT) ▪ Histone chaperones  16 4.1 Transcriptional Control – Transcription Factors B. REPRESSORS: inhibit transcription ▪ Competitive DNA binding ▪ Masking activation surface of activators ▪ Direct interaction with general transcription factors ▪ Local modification of chromatin: ▪ Chromatin remodeling complex ▪ Histon-modifying enzyme ▪ Deacetylases (HDAT) ▪ Histone methyl transferases or methyl transferase  17 4.1 Transcriptional Control – cell differentiation and maintenance of identity ▪ Cellular differentiation (cellular fate) regulated by transcription factors ▪ Combination of few transcription factors can generate many cell types during development ▪ How are the patterns inherited?  18 4.1 Transcriptional Control – cell differentiation and maintenance of identity 1. Positive feed-back loop – development Epigenetics 2. Chromatin structure ▪ Acetylation, Methylation, phosphorylation and ubiquitin 3. DNA methylation  19 4.1 Transcriptional Control – cell differentiation and maintenance of identity 3. DNA methylation ▪ Cytosine methylation – repression of transcription ▪ Methylated CpG sequences – binding site MBD and histone deacetylases → gene silencing ▪ DNA methylation is reset during gametogenesis  20 4.1 Transcriptional Control – cell differentiation and maintenance of identity 3. DNA methylation Genomic imprinting – maternal or paternal allele silencing ▪ Protected from de-methylation during embryogenesis  21 4.2 Post-transcriptional Control 1. Alternative splicing 2. mRNA editing 3. mRNA stability ▪ tRNA and rRNA – extremely stable ▪ mRNA – variable turnover ▪ Gradual shortening of poly-A tail ▪ 3’→5’ exonuclease ▪ Decapping - 5’→3’ exonuclease ▪ Endonuclease dependent decay  22 4.2 Post-transcriptional Control 4. Translational control ▪ Translational inhibitors: ▪ Repressor proteins – bind to 5’ UTR and 3’ UTR ▪ Phosphorylation of translation factors ▪ Thermosensor – permits translation only at high temperatures  23 4.3 Control by non-coding RNAs A. interference RNA (RNAi) ▪ Small RNA molecules that interfere with gene expression – silencing 1. Micro RNA (miRNA) ▪ Short RNA molecules derived from ssRNA precursor ▪ Bind to complementary mRNA ▪ Inhibit translation ▪ Promote mRNA degradation RNA-induced silencing complex  24 4.3 Control by non-coding RNAs 2. Small interfering RNA (siRNA) ▪ 20-25 nucleotides long derived from a dsRNA precursor ▪ Bind to complementary mRNA strands Function ▪ Degradation of mRNA ▪ RNAi directed heterochromatin formation ▪ RITS complex recruit histone modifying enzymes Inhibition of mRNA translation mRNA degradation  25 4.3 Control by non-coding RNAs B. Long noncoding RNA (lncRNA) ▪ >200 nucleotides and do not encode proteins ▪ Gene regulatory function ▪ Scaffold RNA molecules Xist lncRNA → X chromosome inactivation ▪ Some act on cis ▪ Some act on trans