Gene Regulation Lecture Slides - Fall 24

Gene Regulation: A Continuation of a Complicated Story… BMS 532 BLOCK 4 LECTURE 2  Objectives 1. Define the following terms or phrases: constitutive, coordinate regulation, operator, promoter, regulatory element, regulatory protein, inducible, repressible, aporepressor, iron response...

Gene Regulation: A Continuation of a Complicated Story… BMS 532 BLOCK 4 LECTURE 2  Objectives 1. Define the following terms or phrases: constitutive, coordinate regulation, operator, promoter, regulatory element, regulatory protein, inducible, repressible, aporepressor, iron response element, and attenuation 2. Explain the role of gene regulation in cellular and organismal function with particular emphasis on the post-transcriptional activities and their impact on human health 3. Identify the various levels where regulation can take place and assess outcomes for changes in regulation 4. Explain the concept of gene regulatory networks and the role understanding these networks plays in human genetics 5. Summarize an example of an RNA modification and assess the consequences for changes in RNA regulation or processing on the product generated or its availability 6. Explain the concept of functional heterogeneity and the role of gene expression in conferring heterogeneity in populations of cells 7. Summarize the features of the lac and trp operons including regulation, genes contained, and activity 8. Compare and contrast the lac and trp operons with emphasis on their structures and regulation 9. Compare and contrast transcriptional and translational regulation 10. Explain the role for IRES and iron response elements in translational regulation in eukaryotes with emphasis on how iron deficiency can influence translation 11. Determine the consequence for gene expression (increase, decrease, no change) for environmental conditions as well as DNA mutation with an emphasis on promoters, operators, other regulatory elements, and regulatory proteins LO1, LO2 Reminder… Gene regulation = the process through which levels of gene expression varies under different conditions ◦ Control of gene activity to increase or decrease expression as needed Genes that are unregulated = expressed at all times = Constitutive ◦ Tend to encode for things needed at all times for survival Can be at multiple levels ◦ Transcriptional regulation: most common ◦ Regulate association of polymerase with promoter ◦ Translational regulation LO1, LO2 Post-transcriptional Regulation and Human Disease Errors in post-transcript processing can result in a wide range of tissue-specific disease consequences Areas implicated include: ◦ Processing of mRNA ◦ mRNA export ◦ Localization of mRNA/Assembly of key RNAs ◦ Translation ◦ Turnover/Decay LO1, LO2, LO3, LO9 Gene Expression as a “Pathway” Although gene expression traditionally refers to transcription exclusively, we can view the overall regulation of material in the cell as a pathway of control for functionality Eukaryotes use transcription as the main mechanism for regulation, however, there are multiple additional controls in place ◦ Processing ◦ Translation ◦ Post-translational modification LO4 Gene Regulatory Networks Collection of regulatory relationships between transcription factors and specific mRNA-binding sites ◦ Control both gene expression levels and protein synthesis ◦ microRNAs alongside transcription factors (TFs) = gene regulation ◦ TFs also regulate microRNA production Large scale gene expression data helps to decipher the complex relationships between gene expression and the corresponding regulation of that expression ◦ TFs can be grouped into families based on their DNA-binding domain(s) ◦ Several improvements in this process has increased our understanding in recent years and led to improved mapping of relationships LO5 RNA Modifications in Gene Regulation More than 100 chemical modifications can take place on both coding and noncoding RNA Affect stability, structure, localization, translation, and splicing Previous example: RNA Editing via Base Deamination New Example: Pseudouridylation of RNA ◦ Chemically modifies uridine into pseudouridine via isomerization ◦ Role in stabilizing RNA conformations and destabilizing RNA-protein interactions ◦ Example: tRNA stability and activity for aminoacyl-tRNA synthetases LO1, LO2, LO3 Additional considerations for Gene Regulation Some changes in DNA methylation, histone modification, non-coding RNA biogenesis, and chromatin remodeling can be inherited into the next generation ◦ These play crucial roles in genomic functions and regulation of gene expression The human inactivated X chromosome (Xi) and the Y chromosome have the ability to regulate and modulate the expression of thousands of autosomal and active X genes ◦ While the overall effects of the two chromosomes appear to be similar, transcriptomic differences exist for this regulation particularly in cells outside the reproductive tract ◦ More research is needed to understand the full implications for gene regulation by Xi and Y chromosomes ◦ Epigenetic differences across males and females as well as across cell types are likely playing a role in this LO1, LO2, LO3, LO6 Heritable Gene Expression Profiles The variation of gene expression correlated with differentiation in a multicellular organism is just one level of understanding for heritable gene expression profiles Even within cell populations that are expected to be clonally identical it is possible to find heritable patterns of gene expression and functional heterogeneity that can be maintained ◦ Clonal Identities This is an important implication for cancer genetics ◦ Identified heterogeneous tumors may in fact have a greater degree of functional heterogeneity ◦ Previously believed clonal tumors may in fact have a wide range of functional heterogeneity in the form of multiple clonal identities ◦ Significant implications for consequences of therapeutic interventions and in vitro cancer studies LO1 Revisiting Prokaryotic Friends A quick summary with a few questions… INDUCIBLE REPRESSIBLE Usually encode catabolic enzymes Usually encode anabolic enzymes Why does this make sense? Why does this make sense? ◦ Only want to synthesize the molecules of ◦ Want to synthesize the molecules of building breakdown when the thing you want to break unless you already have all the building down is present materials you need LO1, LO7, LO8 Operon Model The operon consists of several structural genes required for metabolism under the control of a single regulatory domain = coordinate regulation Repressor binding site = operator ◦ A gene may have multiple operators upstream and/or between structural genes controlled by the same promoter Site for transcriptional activation = promoter An operon is induced when RNA polymerase is free to associate and initiate transcription LO7, LO8 Lac Operon Quick Summary Regulates lactose degradation ◦ Lactose permease and β-galactosidase Off (as much as possible) until wanted ON Can be both activated and repressed ◦ Absence of lactose suppresses operon though low levels of activity are still expected ◦ Binding of Lac I (repressor) to operator turns OFF; must inactivate repressor to turn ON ◦ Presence of GLUCOSE shuts OFF operon because GLUCOSE is a preferred fuel ◦ When lots of glucose metabolism = low cAMP and lac operon is OFF (even in absence of repressor) ◦ When glucose metabolism is absent = high cAMP and lac operon can be ON (unless repressor is present) ◦ Presence of lactose induces lac operon activity including promoting proteins that increase lactose uptake into cell Brooker et al McGraw Hill 2021 LO1, LO7, LO8 Trp Operon Quick Summary Makes the enzymes necessary to synthesize tryptophan ON until you have made enough Regulation via both negative feedback and attenuation (folding that prevents activity of the polymerase and/or ribosome; see next slides) Presence of tryptophan generates APOREPRESSOR complex that suppresses transcription Charged tRNA with trp (trp-tRNA/tRNA-trp) can act as a repressor of both transcription and translation (see next slides) LO7, LO8, LO9 Additional Regulation of the trp Operon: Attenuation Attenuation results in the premature termination of mRNA or protein synthesis due to stem and loop formation in the 5’ region of mRNA ◦ trp attenuator sequence ◦ complementary bases pair to form a stem and loop structure Brooker et al McGraw Hill 2021  LO7, LO8, LO9 Regulation of trp via tRNA Availability of charged tRNA molecules can alter both transcription and translation tRNA-trp is present = OFF ◦ Synthesis of the leader peptide results in pairing of mRNA in a manner that blocks the action of the ribosome Low or absent trp-tRNA = ON ◦ mRNA takes on different conformation and ribosome can function to continue synthesis Also affects TRANSCRIPTION and activity of RNA Polymerase or RNA pol association with operon Merino et al 2008 LO9 Additional Translational Regulation Any binding or folding that impacts ribosome association with the mRNA will impact translation Antisense RNA can inhibit translation ◦ Hybridizes with mRNA forming double- stranded molecule that prevents ribosome association LO9, LO10 Regulation of Translation in Eukaryotes Regulation is not only in terms of the timing of translation but also in the location of translation Important in development particularly in terms of spatial patterning of structures Dysregulation of translation is linked to human disease Areas of import include: ◦ tRNA dysfunction ◦ Ribosomopathies ◦ Stress responses ◦ mTOR pathway Translational rates and functional activity can help explain the tissue-specificity of human diseases LO9, LO10 mRNA Transcript Regulation and Translational Regulation Regulation by RNA-binding proteins associating with noncoding elements Can influence translation or alter mRNA degradation Example: Iron assimilation genes ◦ Iron regulatory proteins (IRP) and Iron response elements (IRE) ◦ Low concentrations of Iron free IRP to bind IRE and suppress translation (B of image) ◦ High concentrations of Iron bind IRP keeping IRE open and allowing translation to proceed (E of image) Zhou and Tan 2017 LO9, LO10 CAP-Independent Regulation of Translation Canonical cap-dependent initiation of translation involves the traditional assembly and scanning methodology outlined previously Under stress conditions or physiological challenges, a cap- independent initiation method exists Internal Ribosome Entry Site (IRES) elements ◦ Related to selective cellular protein synthesis ◦ Mostly located in genes associated with stress, mitosis, or apoptosis ◦ Located in 5’ UTRs and can interact with key binding factors (ITAF) to remodel and encourage ribosome recruitment First discovered in poliovirus, these elements have sequence uORF = upstream open reading or structural motifs that recruit translation machinery and, thus, can initiate translation frames LO11 Consequences of Change Changes in the sequence of regulatory elements (particularly where sequence specific-binding occurs) can have significant impacts on regulation outcomes Loss of function has different outcomes depending on the activity or role: ◦ Lack of binding at the operator = no repression ◦ Lack of binding at the promoter = no activity/transcription ◦ Lack of lactose availability = little to no activity (minimal activity) of the lac operon ◦ Iron required for activity and iron present = translation occurs ◦ Iron blocks activity and iron present = translation is blocked Gain of function has different outcomes as well! Questions for your consideration… Which operon example is turned on when needed? (LO4, LO5) Which operon example also has significant translational regulation? (LO4, LO5) Translational regulation in eukaryotes can be thought of as a method that affects the level of translation and/or _________? (LO6) What is the role of mRNA higher order structures in regulation? (LO6) High levels of glucose are available in the environment along with high levels of lactose. What is the most likely consequence for the lac operon in terms of relative activity? (LO4, LO5, LO7) MORE LO7: What is the expected consequence in terms of gene expression for the following changes: constitutive binding of repressor to operator? constitutive binding of cAMP to operon? A change in sequence prevents mRNA folding. If folding was necessary to enable full transcription of the mRNA, what is the consequence in terms of functional product?