Ch. 16 Regulation of Gene Expression PDF

Ch. 16. Regulation of Gene Expression Outline Transcriptional regulation of gene expression in prokaryotes (16.1) Transcriptional regulation of gene expression in eukaryotes (16.2) Post-transcriptional regulation of gene expression in eukaryotes (16.5) Epigenetics (16.4) By the end of tod...

Ch. 16. Regulation of Gene Expression Outline Transcriptional regulation of gene expression in prokaryotes (16.1) Transcriptional regulation of gene expression in eukaryotes (16.2) Post-transcriptional regulation of gene expression in eukaryotes (16.5) Epigenetics (16.4) By the end of today you should be able to… Explain why the control of gene expression is important and at which levels it occurs in prokaryotes and eukaryotes. Describe the transcriptional regulation of prokaryotic operons using repressor and activator proteins. Cells control gene expression Controlling gene expression can save energy and resources (prokaryotes) and allow for cell differentiation and function (eukaryotes). Prokaryotes (2, 5-8 in figure) use less points of control than eukaryotes (1-8 in figure). The earlier the control, the better. Regulation of gene expression in prokaryotes (16.1) Prokaryotes mainly use transcriptional regulation of gene expression to save energy Control of gene expression allows to respond quickly to new conditions. Controlling rate of transcription, mRNA hydrolysis, rate of translation, protein hydrolysis or inhibiting protein function costs energy. Operons are units of transcriptional regulation in prokaryotes Clusters of genes with a single promoter (and an operator). Usually for structural genes (enzymes or cytoskeletal proteins). All or none are transcribed, depending on the presence of a metabolic product or substrate. in E. coli Operons can be regulated by repressor and/or activator proteins An example of an inducible operon: the lac operon in E. coli An inducible operon is regulated by a repressor protein: transcription is induced by the presence of an inducer, which is the substrate of a metabolic pathway. The lac operon is also regulated by an activator protein: positive control to increase transcription efficiency in the presence of an activator.  lac operon P LacI CAP P O lacZ lacY lacA β-galactosidase β-galactoside permease β-galactoside Hydrolyses lactose to Membrane protein transacetylase glucose and galactose (permease) for Not clear role in + transporting lactose lactose Transglycosylation of into the cell metabolism lactose into allolactose Extracellular space Cell membrane permease Cytoplasm β- Galactosidase (transglycosylation) (Hydrolysis) Lactose (disaccharide) Allolactose (disaccharide)  lac operon P LacI CAP P O lacZ lacY lacA Promoter: Activator protein Operator: RNA polhere binds binds here Repressor protein binds here  lac operon P LacI CAP P O lacZ lacY lacA Catabolite Activator Protein site: Operator: Activator protein binds here Repressor protein binds here  Repressor protein is synthesized from this gene (it has its own promoter) lac operon P lacI CAP P O lacZ lacY lacA Activator protein Repressor protein binds here binds here  Repressor protein is synthesized from this gene (it has its own promoter) lac operon P lacI CAP P O lacZ lacY lacA Repressor binds to operator R Repressor protein is constitutively synthesized  RNA pol The repressor does not allow the RNA pol to transcribe Repressor protein is synthesized from this gene R P lacI CAP P O lacZ lacY lacA R  RNA pol R P lacI CAP P O lacZ lacY lacA R The lac operon is repressed (it is “turned off”) ✕ RNA R pol P lacI CAP P O lacZ lacY lacA R Lactose (inducer) enters the cell! Allolactose present Allolactose binds to repressor protein RNA R pol P lacI CAP P O lacZ lacY lacA R Allolactose binds to repressor protein  Repressor inactivates and leaves the operator RNA R pol P lacI CAP P O lacZ lacY lacA R  Allolactose functions as an inducer of the operon: The lac operon is an inducible operon, induced by the substate of a metabolic reaction (lactose R metabolism). Transcription occurs RNA pol P lacI CAP P O lacZ lacY lacA lac mRNA R β-galactosidase β-galactoside β-galactoside permease transacetylase Sugars What sugar Allolactose lacI Repressor Transcription is the cell present? expressed? bound to of lac using? operator? operon? No lactose Lactose  Inactive adenylyl cyclase Low glucose High glucose Active adenylyl cyclase ATP cAMP pyrophosphate ✓ Lactose ↓ Glucose cAMP R A cAMP binds to the activator protein RNA pol P lacI CAP P O lacZ lacY lacA lac mRNA R β-galactosidase β-galactoside β-galactoside permease transacetylase  Activator protein is active and binds to CAP site R A RNA pol P lacI CAP P O lacZ lacY lacA lac mRNA R β-galactosidase β-galactoside β-galactoside permease transacetylase  R Transcription occurs at high efficiency RNA A pol P lacI CAP P O lacZ lacY lacA lac mRNA R β-galactosidase β-galactoside β-galactoside permease transacetylase Watch animation 16.1 An example of a repressible operon: the trp operon in E. coli A repressible operon is regulated by a repressor protein: transcription is repressed in the presence of a co-repressor, which is the product of a metabolic pathway. The trp operon is always transcribed, except when there is enough tryptophan (product) Transcription is repressed when there is a co-repressor (tryptophan) that binds to an inactive repressor protein, allowing the activated protein to bind to the operator. Genes of enzymes of Watch animation 16.2 tryptophan synthesis pathway Summary of control of transcription of operons 1. An inducible operon is regulated by a repressor protein (e.g., lac operon in E. coli): transcription is induced by the presence of an inducer, which is the substrate of a metabolic pathway. 2. A repressible operon is regulated by a repressor protein (e.g., trp operon in E. coli): transcription is repressed in the presence of a co- repressor, which is the product of a metabolic pathway. 3. An operon is regulated by an activator protein (e.g., lac operon in E. coli): positive control to increase transcription efficiency in the presence of an activator. Another layer of control of gene expression in prokaryotes: Sigma factors (proteins) bind to RNA pol to direct it to specific classes of promoters Credit: Abril et al. 2020 Outline Transcriptional regulation of gene expression in prokaryotes (16.1) Transcriptional regulation of gene expression in eukaryotes (16.2) Post-transcriptional regulation of gene expression in eukaryotes (16.5) Epigenetics (16.4) By the end of today you should be able to… Describe how transcriptional regulation of gene expression occurs in eukaryotes using transcription factors. Explain the post-transcriptional regulation of gene expression in eukaryotes using alternative splicing of exons, translation control by miRNAs and siRNAs, and regulation of protein longevity. Describe how epigenetics can also regulate gene expression using methylation of cytosines at the promoter, and acetylation of histone proteins. Regulation of gene expression in eukaryotes (16.2) Recall that gene expression regulation can occur at 8 different levels in eukaryotes >Transcriptional control: No operons, usually genes are far away from each other, and each has its own promoter. Eukaryotes have many more regulatory sequences than prokaryotes. Promoters are more diverse in eukaryotes and recognize transcription factors (proteins) Transcription factors (TFs) control transcription rate: General TFs: bind to common regulatory sequences in the promoter (e.g., a -10 element known as the TATA box). Specific TFs: bind to uncommon regulatory sequences found only in the promoter of a few genes. These TFs are found only in certain cells, certain cell stages, during cell differentiation, etc. Shared regulatory sequences between different genes allow to regulate multiple genes at once (e.g., response to stress): the same TF can control expression of multiple genes simultaneously*. *Similar to the use of sigma factors in prokaryotes. Example: TATA box in the promoter and general TFs (TFIID, B, F, E, H) TFIIF prevents nonspecific binding to the complex and helps RNA pol II to bind TFIID allows TFB to bind TFIIE stabilizes DNA denaturation TFIIH opens up the DNA TFIIB allows RNA pol II to bind and helps identifying the initiation site Transcription begins Enhancers and silencers are sequences outside of the promoter that further control transcription rate Enhancer (sequence): binds specific TFs that activate or increase rate of transcription. Silencer (sequence): binds specific TFs that repress transcription. Mediator (protein complex) binds specific TFs bound to enhancer or silencer (that are very far from the promoter) to the basal transcription apparatus. Watch animation 16.3 TFs have certain structural motifs that allow effective binding to DNA Structural motifs consist of different structural elements in the TF, and some include zinc. Structural motifs ”fit” within major or Interaction with DNA backbone minor grooves in the DNA (recall that the DNA bases have available atoms for hydrogen bonding). A common structural motif is the helix- turn-helix. Interaction with DNA bases Regulation of gene expression in eukaryotes after transcription (16.5) Recall that gene expression regulation can occur at 8 different levels in eukaryotes Alternative splicing of pre-mRNA (3 in graph) Inhibition of translation of mRNA by miRNAS (6 in graph) Degradation of viral and transposon mRNA induced by siRNAs (5 in graph) Regulation of protein longevity (8 in graph) Alternative splicing of exons in the nucleus can result in different mRNAs (and different proteins) 1 gene Multiple proteins miRNAs and siRNAs participate in regulating translation of mRNAs miRNAs: About 22 bp long. Inhibit translation of transcripts. Pairing with target mRNA does not need to be perfect: each miRNA has many targets. siRNAs: Expressed from viral RNA or transposons in the eukaryotic genome. Target the original sequence of virus or transposon and degrade it: possible defense mechanisms. Protein longevity can be regulated in the cell The amount of protein in a cell is a function of protein synthesis and protein degradation. Targeted proteins for degradation are labelled with a chain of polyubiquitin (protein). Proteasome digests the labelled protein and ubiquitin is recycled. Epigenetics (16.4) Epigenetics are changes in the gene expression not caused by changes in the DNA sequence Epigenetic changes are reversible, but sometimes stable and heritable (usually not in mammals). The environment plays an important role in epigenetic modifications. We will discuss: DNA methylation in the promoter’s Cytosines. Chromatin remodeling by acetylation of histone proteins. DNA methylation adds methyl groups to some of the promoter’s cytosines and silences transcription 5-methylcytosine binds repressor proteins at the promoter: represses gene expression. In mammals, it usually occurs in C adjacent to G, or CpG islands, which are abundant in promoters. DNA Methylation regulates gene expression methyltransferase across tissues and environments. Heavy methylation silences one entire X chromosome in female mammals. DNA methylation is heritable but also reversible DNA methyltransferase During DNA replication, a maintenance methylase adds the methyl groups to C in the new DNA strand. A demethylase can remove methyl Maintenance groups from C. methylase Most DNA methylation is removed during gamete formation in mammals. Demethylase In eukaryotes, chromosomes are supercoiled DNA and histone proteins Histone proteins can be remodeled to control transcription Histones (proteins) have tails at the N terminus with positively charged amino acids such as lysine. The positively charged histones bond with the negatively charged DNA: compact chromatin. Charge in histone tails can be changed by adding acetyl groups Transcription repressed Histone acetyltransferase adds acetyl groups: chromatin opens, and transcription is activated. Histone deacetylase removes acetyl groups: Adding acetyl Removing chromatin closes, and transcription is groups acetyl groups repressed. Histone modifications are mostly removed during gamete formation in mammals. Transcription activated