Gene Expression: Transcription and Translation

Gene to Protein

• Using a gene to make a protein is known as gene expression
• This includes the synthesis of the protein by transcription of DNA and translation of mRNA
• There are multiple control points for gene expression including transcription factor assembly and DNA accessibility

Transcription - An Overview and Steps

• The first step in gene expression is transcription
• Transcription involves three steps: initiation, elongation, and termination
• Polymerase binds to the promoter during initiation
• During elongation, the polymerase moves downstream, transcribing RNA
• After the terminator is reached the polymerase detaches

Transcription Compared to DNA

• Transcription involves RNA and DNA, that uses different polymerase
• Uses Uracil and not Thymine
• Is single stranded
• Has a different ribose sugar compared to DNA
• DNA contains Thymine and is double stranded

Transcription Initiation

• Requires a distinct set of proteins known as transcription factors, including the TATA box binding protein, to bind onto a promoter
• RNA polymerase II binds along with more transcription factors to form the transcription initiation complex
• The DNA double helix must partially unwind via RNA Pol II, so that one strand can be used as the template for RNA synthesis

Transcription Elongation

• Transcription proceeds on the template strand as RNA polymerase adds complementary RNA nucleotides
• RNA contains uracil (U) in place of thymine (T) found in DNA
• DNA is continuously unwound ahead of the core enzyme and rewound behind it
• The double helix reforms as the transcript leaves the template strand

Termination and Processing

• After transcription of the polyadenylation signal (AAUAAA) nuclear enzymes release the pre-mRNA and RNA polymerase dissociates from the DNA
• The primary transcript is processed in the nucleus through capping, tailing, and spicing
• Capping involves the addition of a modified guanine nucleotide to the 5' end
• Tailing involves the addition of a poly-A tail (50-250 adenine nucleotides) to the 3' end
• Spicing occurs at the spliceosome within the nucleus, where introns are removed and exons are rejoined

Alternative Splicing

• A process by which differing combinations of exons are joined together resulting in multiple forms of mRNA from the same pre-mRNA population
• Introns are generally large, and exons are small, therefore mRNA is shorter than pri-RNA

Translation - An Overview

• Step three is translation
• Mature mRNA transcript exits the nucleus and is bound by the ribosome
• Codons are translated into amino acids
• Enzymes in the cytoplasm load corresponding amino acids onto their tRNA

tRNA

• tRNA molecules within the cytosol, with specific anticodons, carry corresponding amino acids
• Hydrogen bonds form between mRNA and the anticodon of the appropriate tRNA
• The amino acid is added via peptide bonds to the growing polypeptide chain

Translation - Steps

• The steps are initiation, elongation, and termination
• Initiation: The small ribosomal subunit binds to the mRNA at the ribosomal binding site
• tRNA will scan downstream to the AUG start codon at the 5' -cap of mRNA
• Hydrogen bonds form between initiator anticodon and mRNA
• The large ribosomal subunit binds, completing the initiation complex, requiring GTP energy

Elongation

• Codon recognition: base pairs with complementary anticodon
• GTP is invested to increase accuracy/efficiency
• Peptide bond formation: a catalytic RNA peptidyl transferase catalyses peptide bond formation removing it from tRNA in the P site
• Translocation: As the ribosome moves along the mRNA one codon at a time, it moves tRNA from the A site to the P site
• The tRNA from the P-site enters the E site, detaches from the amino acid, and is released to be recharged

Termination

• The ribosome reaches a stop codon on mRNA in the A site, bound by a release factor
• Release factor promotes hydrolysis: the bond between the P-site tRNA and last amino acid hydrolyses, releasing polypeptide
• Ribosomal subunits and other components dissociate, requiring hydrolysis of two GTP molecules

Protein Processing - Structure

• Protein sequence (primary structure) is determined by DNA sequence
• Secondary Structure: held by weak hydrogen bonds to form alpha helices and beta sheets
• Tertiary Structure: 3D shape stabilized by side chain interactions
• Quaternary Structure: Multiple proteins associate together to form a functional protein

Signal Peptide

• A short string of amino acids at the N terminus of the protein (-20aa)
• Encodes where the protein intends to end up
• Secratory proteins are solubilized in lumen, while membrane proteins remain anchored to the membrane

Post-translational Modifications

• Translation is complete, but the protein might not yet be functional
• Modifications include phosphorylation, methylation, acetylation, biotinylation, carboxylation, carbohydrate addition, cleavage, and ubiquitination
• Occur within the Golgi, and others in the cytosol
• Modifications can confer activity or influence the ability to interact with other molecules, or direct a protein to particular locations

Forms of Cell Signaling

• Paracrine Signaling: acts on a cell targeting a nearby cell through diffusion
• Elicits quick responses that last briefly, quickly degraded by enzymes or removed by neighbouring cells
• Synaptic signalling: dendrites receive stimuli, the axon transmits signals (neurotransmission), a neurotransmitter diffuses across the synapse
• Endocrine Signaling: Hormones are ligands released in endocrine signalling and usually have slower responses with long lasting effects
• Autocrine: acts on the cell releasing the signal

Reception

• Signaling proteins/primary messengers bind to receptor proteins resulting in a shape or chemical state change in the protein
• Altered receptors activate another protein, such as G-protein/adenylyl cyclase
• Relay molecules (second messengers) are activated such as cAMP/IP3 and other proteins are activated
• Each activated protein causes a series of changes through a phosphorylation cascade
• Lipids and water soluble molecule receptors can be located in the cytoplasm inside of the nucleus

Receptor Types

• Major receptor types are G-Protein coupled receptors (GPCRs), tyrosine kinases, ligand gated channels, and intracellular receptors
• G-protein-linked receptors bind a ligand and activate a membrane protein called a G-protein
• Seven transmembrane domains, but each receptor has its own specific extracellular domain and G-protein-binding site
• G-proteins are molecular switches that are either on or off depending on GDP or GTP binding

Ligand Gated Channels

• Binding of a ligand at a specific site on a receptor elicits a change in its shape that can open or close it, so ions can pass through
• Electrical signals trigger neurotransmitters
• The nervous system relies heavily on ligand-gated ion channels to propagate action potentials

Transduction through Phosphorylation

• Kinase enzymes transfers a phosphate group from ATP to other proteins
• Activates the affected protein
• Phosphatases are enzymes that dephosphorylate, removing the phosphate, and rendering the protein inactive, but recyclable
• Second messengers include cAMP and Ca ions

Response/Cellular Response/Deactivation

• Cellular responses can include gene expression, protein function alternation, opening or closing an ion channel, alteration cellular metabolism, regulation of cellular organelles or their organization, and rearrangement/movement of cytoskeleton
• Deactivation usually promotes start of reactivation to ensure homeostatic equilibrium
• E.g. Glycogen breakdown results in glucose 1-phosphate that is converted into glucose 6-phosphate that is used in glycolysis to generate ATP

Cellular Energy Harvesting

• Cellular Respiration: when cells break down organic compounds like glucose and release ATP
• C6H12O6+6O2 -> 6CO2+6H2O+Energy
• Occurs in autotrophs and heterotrophs
• Reactions have four stages: glycolysis, pyruvate oxidation, citric acid cycle, and oxidative phosphorylation

Stages 1 - Glycolysis

• Occurs in the cytosol
• Glucose splits
• Two ATP invested
• +2ATP net
• +2CO2

Stages 2 - Pyruvate Oxidation

• Occurs in the matrix
• Pyruvate transformed into acetyl CoA
• +2 CO2

Stages 3 - Citric Cycle - Krebs Cycle

• Occurs in the mitochondrial matrix
• Acetyl CoA + OAA to form Citric Acid
• +2 ATP
• • 4 CO2

Stages 4: Oxidative Phosphorylation ETC & Chemiosmosis

• Occurs on inner membrane
• Electrons removed
• Redox reactions
• H2O
• NADH + FADH reduced
• ATP synthase particles/chemiosmosis
• ATP = 26 - 28
• H+ electrons -turbine spins to activate ATP
• Oxygen pulls down the chain

Hydrolyzed Energy

• ATP is like a rechargeable battery
• When it breaks apart into ADP and phosphate, that enables cells to be energized
• Then ADP can be recharged back to ATP attaching a new phosphate
• AMP can be recharged to ADP

Homeostasis and Glycemia

• Type 1 diabetes = genetic, cells destroyed & insulin not produced
• Type 2 diabetes = non-functional insulin receptors

Normal Conditions & Stimuli

• Fasting state is ~4-6 mmol/L
• Beta cells in pancreas' pancreatic Islets secrete Insulin by receptors
• Stimulus increase in blood glucose
• All body cells respond with glucose uptake, and ATP generation/aa absorption and protein synthesis/triglyceride synthesis in adipose tissue
• All decreases blood glucose concentration restored by homeostasis to normal conditions

Conditions

• Alpha cells in the pancreatic Islets secrete glucagon hormone
• Stimulus decrease in blood glucose
• Liver, skeletal muscle and adipose cells do breakdown of glycogen to glucose/aa to fatty acids/ fat to glucose
• Homeostasis increases blood glucose levels

Cellular Location in the Processes

• Glycolysis occurs in the cytoplasm
• Pyruvate oxidation occurs in matrix
• Citric Acid Cycle occurs here
• Electron Transport Chain/Chemiosmosis occurs in inner membrane

Cell Structure and Components

• Plasma membrane: barrier from external environment; regulates the passage of materials

Nucleus & Nucleolus

• Enclosed by double lipid bilayer nuclear envelope; control cnetre; directs the synthesis of ribosomes and proteins
• Nucleolus: Spherica structure within the cell's nucleus that produces rRNA and transcribes to ribosomes

Cytoplasm, Cytosol and Ribosomes

• Cytoplasm: supports organelles and other cellular components
• Cytosol: fluid portion of the cytoplasm made of water (ions, ATP, proteins, lipids)
• Ribosomes function as a spot for cellular functions and floats freely in the cytoplasm & assists with protein production

Endoplasmic Reticulum (ER)

• Rough ER is continuos with the nuclear envelope with attached ribosomes and assists with production of secreted materials
• Smooth ER extends from the rough ER, lacks ribosomes, and Synthesis lipids/storage

Golgi Apparatus

• It modifies, sorts, packages, and transports newly synthesized proteins from the ER by stacking membranous sacs called cisternae
• Lysosomes bound sacs with hydrolytic enzymes
• Lysosomes store/transport substances/digests macromolecules/breakdwn cells

Mitochondria Structure

• Contains outer and inner mitochondrial membranes with cristae
• Facilitates the generation of ATP through cellular respiration

Cytoskeleton Components

• Microfilaments (actin strands interwined) are small and dynamic: They bear tension and weight by anchoring cytoskeleton to the plasma membrane proteins, and promote amoeboid motility if required
• Intermediate filaments (keratin cables): Act as scaffold for cellular organelles. Less dynamic
• Microtubules: Cell division; support and movement of cilia/flagella; guide movement of vesicle from golgi to membrane; cell shape support
• Microvilli is on plasma membrane

Plasma Membrane Proteins

• They transport with channels or transporters to carry out reactions as part of one enzyme in signalling communication
• Extracellular signalling molecule causes communication of information to the inside of cell
• Molecular signatures (glycoproteins) on molecules of the extracellular section are the basis of tissue typing
• Gap junctions or tight junctions holds molecules
• Fibroneting mediates contact between cell surface integrins and Extracellular matrix to facilitate movement