Gene Expression and Regulation

• Gene expression can be regulated by chromatin structure, transcription, post-transcription, post-translation, noncoding RNAs, and operons.
• Prokaryotes and eukaryotes alter gene expression based on environmental cues.
• Gene expression in multicellular eukaryotes governs development and creates varied cell types.
• RNA is key to eukaryotic gene expression regulation.

Gene Expression in Prokaryotes

• Natural selection enables bacteria to produce what is needed.
• Bacteria regulate metabolic pathways using feedback inhibition of enzymes, controlled by allosteric regulation.
• Regulation of enzyme production occurs through gene expression regulation, controlled by operons.

Bacterial Operons

• A prokaryotic DNA segment referred to as an operon includes the operator, promoter, and functionally related genes.
• The operator is a regulatory switch, a segment of DNA that controls a cluster of functionally related genes.
• The operator is located within the promoter.
• Operons also include a promoter region, and a group of functionally related genes.

Operon Players

• A repressor protein can switch off the operon.
• The repressor impedes gene transcription by attaching to the operator and obstructing RNA polymerase.
• Repressors are products of separate regulatory genes and exist in active or inactive forms depending on molecules present.
• A corepressor molecule works in tandem with a repressor protein to deactivate an operon.

Negative Gene Regulation

• Operons are switched off by the active form of the repressor.
• Repressible operons:
  • Usually active and regulates gene expression of anabolic enzymes.
  • Synthesis is repressed by high levels of the end product, which acts as a corepressor, activating the repressor.
  • An example is the trp operon.
• Inducible operons:
  • Usually inactive.
  • Regulate expression of catabolic enzymes.
  • Synthesis is induced by a chemical signal (inducer) that inactivates the repressor.
  • An example is the lac operon.

Trp Operon

• E.coli can synthesize tryptophan.
• Transcription is normally on but repressed when tryptophan binds to a regulatory protein.
• Absence of tryptophan:
  • Repressor remains inactive, so it cannot bind the operator.
  • Genes needed for tryptophan synthesis are transcribed.
  • Tryptophan production occurs.
• Presence of tryptophan:
  • Tryptophan acts as a corepressor, binding to the trp repressor protein.
  • The repressor activates and binds the operator,.
  • Trp operon is inactivated, stopping tryptophan output.
• The repressor activates only with its corepressor tryptophan.
• The trp operon is repressed, or turned off, with high tryptophan levels.

Lac Operon

• The lac operon contains genes that code for lactose metabolism enzymes via hydrolysis.
• Lac operons are typically off and activated when lactose acts allosterically on the regulatory protein.
• Lactose absence:
  • Operon is inactivated as the lac repressor is active and binds to the operator.
  • Lactose hydrolysis stops.
• Lactose presence:
  • Allolactose, an inducer, inactivates the repressor.
  • The inducer turns the lac operon on.
  • Genes for lactose hydrolysis are transcribed which initiates lactose breakdown.

Positive Gene Regulation

• Operons switch on via the active form of an activator
• Activator of transcription: a stimulatory protein i.e. Catabolite Activator Protein (CAP).
• The lac operon is active with lactose, and glucose stimulates transcription via positive gene regulation.
• With both glucose and lactose present, E. coli prefers glucose, resulting in low enzyme quantities for lactose breakdown.
• Only when lactose is present and glucose is scarce E.coli synthesizes more enzymes to break down lactose.
• Low glucose levels:
  • Increase cAMP levels, which activates CAP.
  • Activated CAP attaches to the lac operon promoter, increasing affinity of RNA polymerase, which accelerates transcription of the operon to produce proteins involved in lactose to glucose and galactose.
• High glucose levels:
  • Decrease cAMP levels and CAP detaches from the lac operon.
  • Decreases affinity of RNA polymerase, which in turn decreases the low transcription of the lac operon.

Gene Expression in Eukaryotes

• Eukaryotic gene expression affects development and cell specialization.
• Differential gene expression occurs via expression of certain genes via cells with the same genome, resulting in various cell types.
• Organism cells are identical genetically but a human cell only expresses approximately 20% off all genes.
• Only 1.5% of DNA codes for proteins.
• Remaining DNA codes for RNA or remains untranscribed.
• Gene expression abnormalities result in diseases.
• Gene expression can be controlled at many points.
• Regulation can occur through chromatin structure, transcription initiation, as well as post-transcriptional and post-translational regulation.

Chromatin Structure

• The 6.3 Gigabase human DNA is 205cm long, weighs 6.4 picograms.
• DNA is packaged alongside histones which forms chromatin.
• Euchromatin is the active form of loosely packed chromatin, enabling gene expression.
• Heterochromatin is condensed, inactive DNA with silenced genes.
• Chemical modifications to histones and regulatory DNA impact chromatin structure, dictating gene expression.
• Epigenetic inheritance is trait transfer via mechanisms independent of DNA sequence, like chromatin alterations which impact chromatin structure and gene expression.
• Chromatin-modifying enzymes control gene expression via increasing or decreasing the binding of transcription machinery to DNA.
• Consists of histone acetyl-transferases and histone deacetylases.

Nucleosomes

• A nucleosome represents the basic structural unit of DNA packaging in eukaryotes.
• This structure is comprised of a DNA segment as it completes nearly two turns about a core of eight histone proteins (histone octamer comprised of two copies each of histone proteins H2A, H2B, H3, and H4.)

Histone Modification

• Implemented by Histone acetylation enzymes promote transcription remodelling the chromatin structure by recruiting transcriptionmachinery.
• The N terminus of a histone molecule in nucelosomes protrudes outward.
• When acetyl groups (-COCH3) connect to the (+) lysines in histone tails, loosening chromatin and triggering transcription.
• Histone deacetylation Implemented by histone deacetylases (HDACs).
• The removal of Acetyl groups reinstates histone (+) charge, which causes chromatin inactivation and increased binding.

Histone Methylation and Phosphorylation

• Modification occurs via adding groups to amino acids in histones.
• Methylation: methyl groups (-CH3) are added to amino acids, causing chromatin condensation, which result, gene expression inactivation.
• Phosphorylation: a phosphate group to an amino acid next to a methylated amino acid which can result in transcription activation.

DNA Methylation

• The addition of methyl groups (-CH3) to certain DNA bases, usually cytosine, reduces transcription.
• Can cause inactivation of genes over time in cellular differentiation.
• Comparison of similar genes between differing tissue usually shows higher methylation in cells where are unexpressed.

Genomic Imprinting

• Epigenetic event occurs during gamete development, silencing alleles of certain genes by methylation.
• The gene only expresses one allele due to different imprinting in sperm and egg.
• Imprinting impacts 1% if mammalian gene
• IGF2: maternal allele usually silenced via methylation.
• IgF2 is vital for development.
• Disruption of egg formation leads to Beckwith-Wiedemann Syndrome (BWS), in turn causes increased risk of cancer (1:15000 births)

Eukaryotic Gene

• Eukaryotic genes contain promoter, including where RNA polymerase II and transcription factor binds, within each gen upstream of gene
• TATA box is within the promoter.
• Regulation can occur across segments of DNA using control elements via transcription factors. Proximal control elements are close to the promoter, while enhancers, or distal control elements, are far away from gene, or in introns.
• Transcription factors are functional through RNA polymerase II, specific control element.
• Regulation of function of gene expression is possible using controls via cell ypes.

Transcription Factors

• Help RNA polymerase II start transcription.
• Essential for all protein-coding genes.
• Include activators to target enhancers for specified transcription.
• Repressors are are that inhibit transcription.

Enhancers and Transcription Factors

• Enhancers need activators to stimulate trancription, these need general transcription factors, including RNA polymerase itself.

Activators

• Activators are the specific unique transcription factors unique to each gene, that is located in enhancers.
• Transciption requires the binding of general transcription factors to to the TATA box which then triggers RNA Polymerase II to bind to the promoter.
• Transcription is promoted by co-expressed genes as functionaly related genes can be regulated by same promotors.
• Eukaryotics have monocistonic mRNA, and function through the activators even if they are located in different chromosomes.
• Genentic activation needs activated proteins to allow combinational transcription.

Post-Transcriptional Regulation

• Applies to all the stages that are post-transcription.

RNA Processing

• Alternative RNA splicing can create differing molecule structures that generate various functional proteins.
• Beta-thalassemia is caused by issues with RNA splicing.

Degradation

• The length mRNA lasts will dictate protien syntehsis. Eukaryotic mRNA outlasts prokaryotic strands. A 3' end determines mRNA life.
• Noncoding RNA will quicken mRNA degration, resulting in gene expression inhibiton.

Initiation of Translation

• The speed of translation of the mRNA sequence will affect a regulatory response.
• regulatory proteins that bind to sequences or structures within the untranslated 5'UTR region of the mRNA preventing the attachment of the ribosomes, this sequence is affected by noncoding RNA

Protien Modification

• After translation protien function relies on post-translational modification
• Can be modified through cleavage, folding, combining sub-units together or throught chemical modifications

Proteasomes

• Protein degradation marks and destroys protein in order to control cycle
• Proteins that are not functional (misfolded) are also marked to be destroyed
• Proteasomes bind to destroy the molecule with giant enzyme complexes (size = 26S Long lived proteins such as hormones are destroyed within a lysozyme, a part of cell tissue.

gene expression from non-coding RNA

• Most DNA has a purpose, even though its non-coding
• Codes for rRNA and tRNA, as ncRNAs which regulates mRNA translation and regulate gene expression.
• microRNA (miRNA) form a 20-25 bp strand that will bind to mRNA and block translation of that sequence.
  • siRNA are similar but form double stranded RNA molecules of 20-25 bp and cause complete mRNA degradation
  • Therefore the differnece is that miRNA are complete in base paring and highly specific, where complete base pairing of the miRNA with all of the mRNAs, with the effect of mostly translation inhibiting translation.

Genetic Changes that affect the cell cycle and lead to cancer

• Mutated genes (oncognes) affects normal regulation pathways. Tumor viruses will affect genomic cycles as well

Genes Associated with Cancer

• Cancers are abnormal amounts of cells in a unregulated manner The abnormal functions are the effect of a gene regulation malfunction The tumors will also use the regulation to insert it genome in humans to start divison. They come in 2 basic forms as follows

• Oncogenes start cell division, induce development while tumor repressor genes halt cell divison all tumors must have a oncogene to start it development.

Oncogens and proto-oncogens

• Proto-oncogens will be the cell division and grow.
• The signals of this is what is affected by various growth factors and what not that will cause the unregulated divison.
• Proto-oncogens also will mutate to oncogens also affect regulation and divison

Oncogenes Can Be Converted By

• Movement of DNA ( translocation): Active parts will be attached to other parts DNA INSERT UPSTREAM TO ACTIVE promoter: Transcription will cause the encode to greatly increase
• DNA INSERT AN ACTIVE ENCODER FOR: Transcription gene activation increase gene production in the encodement Increase the number of gene copies of the gene EX: HER2 with beast cancers.
• Increases the signaling and proliferation pathways. ( MAP Pathway protein is important in both activation as above)
• Changes within elements, mutations: Causes an already great expression to be greatly increased or expression due to point mutation (ras proteins are also relevant).

Tumor Suppressor

• Loss of function mutations = Inactivation + proteins
• Insert themselves in the DNA The roles with what it effects, are the following:
• Inhibit cell-signaling pathways = cell cycle / apoptosis.
• Repair damage e: BRCA
• Control cell adhesion The p53 gen is what affect cell arrest with cycle

In addition.

• Cell cycle arrest (inhibition of the cell cycle) can be important in the case of damage to a cell’s DNA.
• p53 is a very important tumor suppressor gene called ‘the gatekeeper of the genome’.
• p53 prevents a cell that has DNA damage from passing on mutations to its daughter cells by replicating.
• Mutations in the p53 gene prevent cell cycle arrest→Uncontrolled cell proliferation of damaged cells→Cancer.

In regards to P53 functions

1. There CDK ( enzyme to cause a cycle stage shift through the process cell division in cycle called arrest.

The main summary to keep is that

Tumors need a oncogen to continue while the function to regulate what happens doesn't exists ( tumors are out of control)

How the model Develops

With the correct mutations There are a couple of factors Multistep model means MULTIPLE MUTATIONS With that the active oncogones will increase development and be caused from at least one suppressor or tumor mutagens Certain predisposition can cause increase tumor With the loss of that tumorsuppressant gene it causes tumor suppressor The main part is the polypousis Coli 1 ( APC ). Which is a form that causes tumors