BIOMG 1350 Lecture 18 - Gene Expression Regulation Part 1

Central Dogma

• DNA to RNA to Protein via transcription and translation.

• Gene expression: the process by which the information encoded in a DNA sequence is translated into a product that has some effect on a cell or organism.

• The fundamental principle of molecular biology describing the flow of genetic information from DNA to RNA to protein. DNA is transcribed into RNA, which is then translated into a sequence of amino acids to produce a protein.

Differential Gene Expression Theory and Cell Differentiation

• Recall the differential gene expression theory: The genome is constant in all somatic cell, only a subset of the genome is expressed in any cell type, unused genes that are not transcribed will retain the potential to be expressed, different cell types express different genes.

• Differential gene expression makes cells different. For example differential gene theory forms the basis of the 3 germ layers in the gastrula and then within those 3 germ layers differential gene expression forms the various cells.

Two Major Ways to Regulate Gene Expression Once Proteins have Been Made

• Protein regulation (molecular switches, for example) and protein degradation (M cyclin degradation, for example) are two ways to regulate gene expression once proteins have already been made—this regulation is fast-acting, but not the most energy efficient.

Gene Expression Control (in regards to the central dogma)

• Gene expression can be controlled at many levels.

• Gene expression is the process by which the information encoded in DNA is translated into a gene product that has some effect on a cell or organism.

• Transcriptional control is relatively slow acting, the most energy efficient, and the most common control point for long term regulation, such as in development.

• Protein activity control is faster acting and readily reversible but isn't as permanent or totally changing. It costs more energy for the cell too.

• Energy cost and speed are the two biggest factors in influencing what kind of gene expression control a cell employs.

7 Points where Gene Expression or Protein Activity can be Controlled

• The various points at which gene expression can be controlled through the central dogma are as follows. There are 7 points of control.

  • Transcriptional Control.

  • RNA Processing Control.

  • mRNA transport and localization control.

  • mRNA degradation control.

  • Translation control.

  • Protein degradation control.

  • Protein Activity Control.

  • From earlier on this list to later,  the speed of regulation goes from slower to faster and the energy efficiency goes from more energy efficient to more energy intensive.

Overview of Prokaryotic Transcription

• RNA polymerase binds to the promoter (stretch of DNA containing certain sequences) with the help of transcription factor(s). One such transcription factor is the sigma factor.

• Promoter defines where RNA polymerase starts to synthesize RNA.

• RNA polymerase synthesizes RNA in the 5' to 3' direction while moving in the 3' to 5' direction using the template strand of DNA.

• RNA polymerase stops at the terminator (specific DNA sequences), falls off from the DNA and then releases the completed RNA.

• Prokaryotes are useful model systems to understand regulation of gene expression.

Sigma Factor

A sigma factor (σ factor) is a bacterial protein that binds to RNA polymerase and helps it recognize specific promoter sequences on DNA to initiate transcription. Different sigma factors allow bacteria to activate different sets of genes in response to environmental conditions.

Promoter

• Promoters tell RNA polymerase where to start transcription. Promoters are upstream of the actual gene that needs to be transcribed. The terminator is downstream, always, of the promoter. Promoters vary depending on the gene being transcribed.

• A promoter is a region of DNA that contains sequence recognized and bound by RNA polymerase to start transcription. They are also located upstream of the transcription start site.

Terminators

• Terminators tell RNA polymerase where to stop transcription. They are always downstream of the promoter and start site. They also are composed of different DNA sequences.

• A terminator is a region of DNA that contains sequences recognized by RNA polymerase as a signal to terminate transcription.

• Sequence of terminator is included in the RNA transcript.

Transcriptional control by Regulation of Promoter Strength, Activators, and Repressors

• Promoters vary in their strength of binding to RNA polymerase with promoter activity being enhanced by transcriptional activators and promoter activity being inhibited by transcriptional repressors.

• Activators and repressors can act in concert to provide highly sensitive transcriptional regulation.

Transcriptional Regulators

• Transcriptional regulators (activators and repressors) are proteins that have DNA binding domains for specific DNA sequences. They can be activators or repressors.

• The DNA binding domain of a transcription factor binds a stretch of unique DNA sequences, allowing for specificity.

• Depending on the exact DNA sequence, the transcriptional regulator will bind tightly, loosely, or not at all.

• Both prokaryotes and eukaryotes use transcriptional regulators but they bind and interact with RNA Polymerase slightly differently.

• They can bind to a family of DNA sequences with varying strength.

Transcriptional Regulator DNA Preference or Affinity

• Depending on the exact DNA sequence, the transcriptional regulator will bind tightly, loosely, or not at all.

• The relative nucleotide preference on one strand for a transcriptional regulator can be shown by base pair letters. The larger the base pair letters the more likely it is for a transcriptional regulator to bind there, and the smaller the base pair letters (or not present) the lower probability for the transcriptional regulator to bind to those base pair sequences.

• They can bind to a family of DNA sequences with varying strength.

Housekeeping Genes

• Housekeeping genes generally have strong promoters and are expressed at stable levels.

• Genes that are constitutively expressed in most or all cell types because they encode proteins essential for basic cellular functions, such as metabolism, protein synthesis, and cell maintenance. They generally have strong promoters and maintain relatively stable expression levels across different cell types and conditions.

Operons

• An operon is a cluster of bacterial genes transcribed from a single promoter. From one operon multiple enzymes that are related can be produced, for example. The operon is controlled by a single promoter and operator.

• Within the operon's promoter is an operator, a DNA sequence that is recognized by one or more transcriptional regulators.

• The operator is like a master switch for transcription of the genes in the operon.

Operator

• An operator is a regulatory DNA sequence in prokaryotes where transcriptional repressors bind to inhibit gene expression. When a repressor binds to the operator, it blocks RNA polymerase from initiating or continuing transcription. Prokaryotes only.

• Operator and promoter are distinct DNA sequences and they can sometimes overlap.

• Note that these are primarily for repressors such as the lac repressor whereas for transcriptional activators, there are seperate sites also near the promoter such as the CAP binding site.

Transcriptional Activator Binding Sites

• An activator-binding site is a regulatory DNA sequence where transcriptional activator proteins bind to increase gene expression. Activators help recruit or stabilize the transcription machinery at the promoter, promoting transcription.

• Like the operator for transcriptional activators. For both prokaryotes and eukaryotes.

• Most sequence-specific transcriptional regulators (activators, repressors) read DNA through the major groove.

Lac Operon

• Transcription of the Lac operon genes allow E coli to utilize lactose when glucose is absent.

• E coli get their energy from the food source they live upon. They prefer to use glucose as an energy source but can break down lactose for energy if no glucose is available. The enzymes needed to break down lactose are expressed within the lac operon.

• Genes in the lac operon are only expressed when glucose is not present and lactose is present. When glucose is present only, when glucose and lactose are present, or if neither are present the lac operon remains off. It only turns on if lactose only is available.

Lac Operon Gene Regulation

• The lac operon provides a simple example of  gene regulation by transcriptional activators and repressors.

• There is a CAP binding site and a RNA polymerase binding site (promoter) with transcription being able to produce B galactoside (turns lactose into glucose and galactose) along with other enzymes which come from the LacZ, LacY, and LacA genes in the Lac operon.

Lac Repressor

• The lac repressor inhibits transcription when lactose is not present. When there is no lactose, there is no allolactose and thus the lac repressor can bind to the operator to prevent transcription of the Lac operon.

• When lactose is present, the allolactose bins to the lac repressor allowing it to detach from the promoter.

• Allolactose is a metabolite of lactose; its levels reflect lactose levels.

• Hoewever, inhibiting repressor binding is not sufficient to allow transcription because lac promoter is weak.

Lac Activator

• CAP activates transcription when glucose is not present.

• When glucose levels are low, cAMP levels go up (part of the stress response) and then this cAMP binds to the CAP activator which is able to bind to the CAP binding site which is upstream of the promoter and operator.

• Once the CAP with cAMP binds to the CAP binding site, it stimulates RNA polymerase transcription of the Lac Operon essentially turning it on.

• CAP activator specifically helps to recruit RNA polymerase to the promoter.

Lac Activator and Repressor Working Together

• An activator and repressor work together to regulate the Lac operon.

• When glucose and lactose is present, the CAP is unbound despite the lac repressor dissociating from the operator.

• When glucose is present but not lactose, the lac repressor remains bound to the operator preventing Lac operon transcription with CAP being unbound as well.

• When glucose is not present and lactose is not present, the lac repressor remains bound to the operator preventing the Lac operon transcription despite CAP being bound to the CAP binding site since cAMP is released in response to the low glucose levels.

• When glucose is not present but lactose is present, the lac repressor dissociates from the operator and the cAMP is able to bind to the CAP with the CAP binding to the CAP binding site allowing the Lac Operon to be transcribed effectively.

Regulation of Gene Expression in Eukaryotes

• Regulation of transcription is more complex for eukaryotes than prokaryotes.

• The promoter contains a TATA box bound by the TATA box binding protein or TBP, which helps recruit general transcription factors.

• This results in RNA polymerase II recruitment at the promoter, which opens the DNA double helix in preparation for gene transcription.

• The mediator also assembles around the general transcription factors, RNA polymerase II and the transcriptional regulators.

• But this is still not enough to start transcription. Specific transcription factors (transcriptional repressors and activators) are needed to regulate the rate of transcription.

Regulation of Gene Expression in Prokaryotes

• Prokaryotes regulate gene expression primarily at the transcriptional level through regulatory proteins called activators and repressors, which bind specific DNA sequences to increase or decrease transcription.

• Genes involved in related functions are often organized into operons, allowing multiple genes to be coordinately regulated by a single promoter and associated regulatory sequences.

TATA Box and TATA Box BInding Protein

• The TATA box is an AT-rich DNA sequence found in the core promoter of many eukaryotic genes, typically located approximately 25–30 base pairs upstream of the transcription start site.

• TATA-binding protein (TBP), a component of the general transcription factor TFIID, binds to the TATA box and bends the DNA, facilitating the recruitment of other general transcription factors and RNA polymerase II to form the transcription preinitiation complex.

3 Types of RNA Polymerases in Eukaryotes and their Function

• Eukaryotic cells contain three distinct RNA polymerase: I, II, III.

  • RNA polymerase I transcribes rRNA to make ribosomes.

  • RNA polymerase II transcribes all mRNAs (this is our focus).

  • RNA polymerase III transcribes tRNAs for translation.

Enhancers

• In eukaryotes, enhancers act to regulate gene expression and they are specific transcription factors.

• Usually eukaryotic activator proteins bind to these enhancers (binding site for activator protein) in order to interact with the polymerase.

• Enhancers are regulatory DNA sequences bound by transcriptional regulators, which usually (but not always) bind activators.

• In development, enhancers allow the transcription of a given gene in specific cell types or at specific developmental stages.

• Enhancers can be located either close or far away (up to tens of thousands of nucleotides) from the transcription start site.

• Enhancer is the larger regulatory DNA region where distal control elements are specific DNA sequences within that enhancer.

Eukaryotic Activator Proteins

• These bind to enhancers which are specific segments of DNA that allow the activator to bind to and regulate gene expression. They are a form of specific transcription factors.

• An activator protein is a transcription factor that binds to specific regulatory DNA sequences, such as enhancers (eukaryotes) or activator-binding sites (prokaryotes), to increase gene expression. It promotes transcription by helping recruit or stabilize the transcription machinery at the promoter.

Enhancer Distance

• Proteins bound to enhancers act from a distance. Through DNA looping, the upstream part of DNA which has the enhancer and activator protein can loop around so it can interact with the mediator and RNA polymerase to start transcription.

• This way, since enhancers can be moved to the place of transcription via DNA looping, enhancers can interact with transcription related proteins regardless of how upstream or downstream they are from the transcription start site.

Mediator

• A 24 subunit complex that serves as an intermediary between transcription regulators and RNA polymerase. They are basically like the exoskeleton that attaches the general transcription factors bound by TATA box binding protein and the proteins on the enhancers to the RNA polymerase.

Role of Regulating Gene Expression in Development

• Enhancers provide context specific gene regulation.

• Regulation of gene expression allows cells with essentially identical genomes to differentiate into specialized cell types by selectively activating or repressing specific genes.

• During embryonic development, spatial and temporal patterns of gene expression control cell fate determination, differentiation, and the formation of tissues and organs. This ensures that the appropriate genes are expressed in the correct cells at the correct times.

Example of Tissue Specific Enhancers and Regulation of Gene Expression

• For example, there could be a limb specific enhancer and a brain specific enhancer.

  • If gene A needs to be expressed in the brain, brain expressed transcription factors bind to the brain specific enhancer and those fold onto the mediator connecting the brain expressed transcription factors to the mediator and thus RNA Polymerase allowing gene A mRNA to be expressed in the brain.

  • If gene A needs to be expressed in the limb, limb expressed transcription factors bind to the limb specific enhancer and those fold onto the mediator connecting the limb expressed transcription factors to the mediator and thus RNA polymerase allowing gene A mRNA to be expressed in the brain.

  • Different cell types contain different transcription factors which activate different enhancers that can turn on the same gene.

Regulation of Shh Expression by a Limb-Specific Enhancer

• Another example is the expression of Shh in the limb.

  • Expression of Shh in the limb is controlled by a limb specific enhancer. Shh mRNA is necessary to produce the Shh morphogens that can regulate cell fate and differentiation.

  • A single base pair change in an enhancer of Shh causes ectopic (abnormal) expression of Shh in the limb, resulting in digit duplication.

  • A G to A nucleotide change in the enhancer of Shh is located 1*10^6 base pairs away from the Shh start codon ATG in the DNA.

Enhancers Across Species (and an example with Shh in Snakes)

• Not all enhancers are found in every single species. Some species lack enhancers or have specific enhancers other species do not.

• The Shh limb enhancer, for example, is conserved in mice, humans, and fish, but not in limbless reptiles such as snakes.

• When testing the function of the Shh limb enhancers in mice we can do the following: Substitute the mouse enhancer with the snake enhancer, and make the snake enhancer more like the human shh limb enhancer.

• Loss of the limb enhancer for Shh is associated with the loss of limb in snakes. Mice that have the snake enhancer for Shh are born without limbs.

Regulation of Gene Expression and Development

• The regulation of gene expression drives development as effectors at the end of intracellular signaling pathways can bind to various enhancers and they can regulate the transcription of certain genes. These genes form proteins which in turn can serve as signals or parts of the cellular signaling pathway.

• Cell signaling, gene expression, cell behavior, and morphogenesis are all connected.

• Regulation of gene expression allows different cells to have different proteins (and thus different characteristics and abilities) which is the key for multicellularity.

• Certain transcription factors in muscle cells verses skin cells for example activate different genes to transcribe and this is dependent on the specific needs of the cell.