Exam 4 Flashcards
Transcription
Process of copying DNA into RNA
Synthesis is always in the 5’ to 3’ direction
RNA Polymerase synthesizes RNA from the DNA template strand
RNA is complementary to the template strand, and the same as the coding strand (excluding T for U)
The other strand of DNA is called the coding strand
For efficiency several mRNAs may be transcribed from the same template DNA strand at a time
Steps of Transcription
- Initiation - Goal is to form the initiation complex, This requires organizing RNA Polymerase at the promoter, Within the promoter there is something called the TATA box, The transcription starts at the TATA box, A binding protein binds to the TATA box, The TATA binding protein recruits transcription factors
Contains: DNA/TATA Box, TATA binding protein, transcription factors, RNA Polymerase - Elongation - everything between initiation and termination, It is the RNA Polymerase moving on the template strand to build more RNA nucleotides
- Termination - RNA Polymerase recognizes a terminator sequence, Once it recognizes the terminator it falls off or dissociates from the DNA, The RNA is then its own independent molecule that can then be used for whatever, The DNA returns to its original double helix shape
RNA Processing
Starts with a DNA strand, Goes to pre mRNA because it is still in the nucleus with introns, The pre mRNA then gets spliced in the nucleus, This gets rid of the introns and it becomes a mature mRNA strand that can go into the cytoplasm
Steps:
Transcription
Modification - A 5’ mRNA cap and a 3’ poly A tail (tail of 200 A’s) is added to the mRNA strand that cause the mRNA to become more stable and protective
Splicing - The process of plucking out the introns, This is carried out by enzymes, group of proteins called the spliceozomes, These enzymes cut out the introns at multiple sites and reforms the bonds between the exons
Transcription Factors
Are proteins, Control gene expression, Composed of groups of proteins, Bind DNA sequences to initiate transcription, Link gene expression to the environment, Mutations in TF have wide ranging effects
Translation
- Production of protein using mRNA, tRNA, and rRNA (Cytoplasm)
- Folding of the protein into the active 3-D form
Both occur continuously except during M-phase
The process of reading the mRNA base sequence and creating the amino acid sequence of the protein
Occurs on the ribosome
Genetic Code
Is a triplet code - three successive mRNA bases form a codon
There are 64 codons
AUG start (codes for methionine) UAA, UGA, UAG stop
It is non-overlapping
It is degenerate - two or more codons may code for the same amino acid
It is universal (all organisms have the same amino acids)
The 3rd base is often called the wobble base because it doesn’t always affect the amino acid
If you add or subtract a nucleotide in a multiple of 3 it will only add one amino acid
Multiple copies of a protein can be made simultaneously
Things called chaperone proteins prevent the proteins from folding until translation is finished
Steps of Translation
Initiation - Initiation complex is formed, Has the mRNA, a 5’ cap (this recruits the small ribosomal subunit), and initiator sequence (AUG), first tRNA molecule, and the small ribosomal subunit
Elongation - There is an E, P, and A site in the ribosome, mRNA slides between the large and small ribosomal subunits, The A (acceptor) accepts the incoming amino acid using the mRNA template, The P stands for peptidyl site forms the peptide bonds, The E is the exit site, and the tRNA and amino acids exit the ribosome, It moves from the 5’ to the 3’ end
Termination - A stop codon enters the A site, This brings a release factor protein, This binds to the ribosome and causes it to dissociate, The protein then folds into its 3-Dimensional shape
Reading Frame
A sequence of amino acids encoded from a certain starting point in a DNA/RNA sequence
Protein Structure
Protein fold into one or more 3-D shapes or conformations
There are four levels for protein structure:
Primary - It is a linear string of amino acids, also refers to the amino acid sequence
Secondary - When the protein begins to fold, includes alpha helices and beta-pleated sheets, maintained by hydrogen bonds
Tertiary - The final 3-dimensional configuration of proteins, mediated by several types of bonds (hydrophobic interactions, hydrogen bonds, ionic interactions, sulfhydryl bonds which covalently bond)
Quaternary - Two or more tertiary structures combine together to perform a separate function
ex. Hemoglobin
Adult hemoglobin has four globular polypeptide chains
Two alpha chains - 141 amino acids
Encoded on Chromosome 11
Two beta chains - 146 amino acids
Encoded on Chromosome 16
Each globin surrounds an iron-coating heme group
Sickle Cell Anemia
Clinical - Vaso-occlusive events like acute and chronic pain, organ damage, Chronic hemolytic anemia
jaundice - bilirubin is contained within RBCs which helps eliminate things from the body, when the cell bursts open bilirubin is released into the blood causing symptoms of jaundice, it is treated by using a light that makes bilirubin into a form that is safe and can be excreted
Delayed growth and sexual maturation
Infarction of the spleen
Diagnosis - Newborn screening
Inheritance - Autosomal Recessive
Due to a single nucleotide mutation in the beta chain protein (HBB gene), this causes a difference to the primary, secondary, tertiary, and quaternary structure
Protein sequence: Normal - TPEEK Sickle Cell - TPVEK
Gene sequence: Normal - ACT CCT GAG GAG AAG Sickle Cell - ACT CCT GTG GAG AAG
Glutamic acid is a hydrophilic amino acid (regular), valine is a hydrophobic acid amino acid (sickle-cell)
Protein Folding
Protein folding begins as translation proceeds
Enzymes and chaperone proteins assist
Should a protein misfold, an “unfolded protein response” occurs
Protein synthesis slows or even stops
Protein Misfolding
Happens because there is a mutation (all proteins), or just a mistake in one protein folding process
Adds a ubiquitin molecule to the misfolded protein (the more molecules, the greater the abnormality)
Once it surpasses the threshold of ubiquitin molecules, the abnormal protein is added to the proteasome which cuts the polypeptide into individual amino acids, the amino acids are then recycled
Proteasomes also destroy properly-folded proteins that are in excess or no longer needed
Huntington’s Disease
Clinical - Motor, cognitive, and psychiatric disturbances
Mean Age of Onset: 35 to 44 years median
Survival: 15 to 18 years (after diagnosis)
Inheritance - Autosomal Dominant
Trinucleotide (36 sets of CAG) repeat disorder
Molecular - Repeat expansion in the HTT gene, HTT gene makes the huntingtin protein, Protein has long string of glutamines, alerting folding, and blocks proteosomes, altering expression of other genes
Phenylketonuria (PKU)
Defect is in the enzyme phenylalanine hydroxylase which normally breaks down phenylalanine
The mutation is inherited from carrier parents
A localized misfolding occurs
Misfolding spreads until the entire four-subunit enzyme can no longer function
The buildup of phenylalanine causes mental retardation
Gene Expression through Time and Tissue
Changes in gene expression may occur over time and in different cell types
This may occur at the molecular, tissue, or organ/gland level
Epigenetic changes (changes to chemical groups that associate with DNA that get transmitted to future daughter cells)
Example: Globin Chain Switching
Subunits change in response to oxygen levels
Subunit makeup varies over lifetime
Embryo = two epsilon and two alpha chains (idk if this one is right but we don’t need to know it anyway)
Fetal = two gamma and two alpha chains
Adult = two beta and two alpha chains
You get more oxygen during the embryo and fetal periods which allows for more advanced cognition and brain development
Changing Gene Expression in Blood Plasma
Blood plasma contains about 40,000 different types of protein
Changing conditions cause a change in the protein profile of the plasma
Stem cell biology is shedding light on how genes are turned on and off
Pancreas
Originates from a stem cell
This stem cell differentiates into the pancreas
There can be two types of cells it can differentiate into
Either the exocrine cell (digestive) or the endocrine cell (control blood glucose)
The presence of PDX-1 is the transcription factor that controls the difference between which genes these cells will turn on
When it is activated the cells will go down the exocrine pathway
The absence of PDX-1 keeps the cell down the endocrine pathway
Proteomics
Proteomics tracks all proteins made in a cell, tissue, gland, organ or entire body
Proteins can be charted based on the relative abundance of each class of
It is the pie chart thingy
Control of Gene Expression
A protein-encoding gene contains some controls over its own expression level:
Promoter sequence (mutations)
Extra copies of gene
Much of the control of gene expression occurs in two general processes
1. Chromatin remodeling = “On/off” switch
2. MicroRNAs = “Dimmer” switch
Chromatin Remodeling
Histones play major role in gene expression, it exposes DNA when and where it is to be transcribed and shield it when it is to be silenced
The three major types of small molecules that bind to histones (the tail specifically) are (epigenetic):
Acetyl groups (C2H3CO2)
Turns transcription on
Acetyl binding can subtly shift histone interactions in a way that eases transcription
Enhanceosome is what attaches the Acetyl group to the histone tails
It relaxes the DNA by making the histones have a less positive charge
Methyl groups (CH3)
Turns transcription off
Phosphate groups (PO4)
Turns transcription on
Heterochromatin (tight, dark) has less space between the histones than Euchromatin (loose, lighter)
Chromatin remodeling can cause some disease in humans like Rett Syndrome
⭐️Affects transcription
MicroRNAs
MicroRNAs belong to a class of molecules called noncoding RNAs
They are 21-22 bases long
The human genome has about 1,000 distinct MicroRNAs that regulate at least 1/3 of the protein-encoding genes
When a microRNA binds to a “target” mRNA, it prevents translation
⭐️Affects translation
MicroRNAs binding to the mRNA makes it a double stranded molecule that the ribosome cannot translate
Cancer provides a practical application of MicroRNAs because certain MicroRNAs are more or less abundant in cancer cells than in healthy ones
A related technology is called RNA interference (RNAi)
Small synthetic, double-stranded RNA molecules are introduced into selected cells to block gene expression
Maximizing Genetic Information
We have discovered 21,000 genes
100,000 mRNAs
1,000,000 proteins
In other words, one gene makes multiple proteins
We maximize genetic information through multiple processes:
Chromatin remodeling, alternate splicing, MicroRNAs block protein synthesis, protein folding, polypeptides shortened, sugars added, polypeptides aggregate
Alternate splicing
The “genes in pieces” pattern of exons and introns and alternate splicing helps to greatly expand the gene number
Ex. If we have exons A, B, C, and D we could create multiple proteins:
ABD, ACD, BD, AC, ABCD
Ex. Dentinogenesis Imperfecta
This is an example of polypeptide shortening
To make enamel we have to make a set of proteins (DSPP gene)
This makes the DSPP mRNA
The DSPP mRNA makes the DSPP precursor protein that can then be cut into its finalized protein
DPP is what the final product should be, but it is not present in dentinogenesis imperfecta
The DSP protein is made instead which is rare, but degrades enamel rapidly
Sources of DNA in Human Genome
Only 1.5% of human DNA encodes protein
Rest of genome includes:
Viral DNA, Noncoding DNA, Introns, Promoters and other control sequences, Repeated sequences,
Viral DNA
About 8% of our genome is derived from RNA viruses called retroviruses
This is evidence of past infection
Sequences tend to increase over time
Genes Affect on Neurotransmission
Genes control production of myelin
They also control the protein that produces neurotransmitters
They also play a role in reuptake transporter proteins on the presynaptic neuron
They play a role in the receptors on the postsynaptic neuron
Major Depressive Disorder
Affects 6% of the US population
A likely cause is a deficiency of the neurotransmitter serotonin, which affects, mood, emotion, appetite, and sleep
Many antidepressant drugs are selective serotonin reuptake inhibitors (SSRIs)
Epidemiologic studies indicate a strong environmental component
Heritability is 0.37-0.7
Several genes have been found to be associated with MDD
TPH1 - enzyme involved in serotonin synthesis
HTR2C - serotonin receptor
Many antidepressant drugs are SSRIs
Treatment of MDD is trial and error
HTR2A - a gene for a serotonin receptor
It is downregualted by citalopram (SSRI)
Patient with allele A more likely to respond
Autism Spectrum Disorder
Autism affects 1/100-500 children
It strikes four times as many boys as girls
Usually symptoms develop gradually, but may have a regression at 18-24 months
Symptoms must be present before age 3
25% of kids with ASD will progress and gain skill to integrate with peers
Known environmental triggers:
Prenatal Rubella infection, Prenatal valproate use
Complex Autism - presence of facial dysmorphology and/or microcephaly
Essential Autism - absence of facial dysmorphology features and microcephaly
Known genetic causes:
Cytogenetically visible chromosomal abnormalities (~5%)
Copy number variants (CNVs, 10-20%)
i.e. submicroscopic deletions and duplication
Singe gene disorders in which neurological finding are associated with ASD (~5%)
More than 30 genes so far have been associated with autism
Two genes classes that may be involved have been identified
They encode the cell adhesion proteins neurexins (gene: NRXN) and neuroligins (gene: NLGN)
These proteins strengthen synaptic connections in neurons associated with learning and memory
Mutation
Change in a DNA sequence, found in less than 1% of the population
Can either be a loss of function or a gain of function
Can be silent (doesn’t change gene expression or protein)
Can be beneficial in some circumstances
Germline vs. Somatic Mutations
Germline Mutations
Originate in meiosis
Can be passed on to future generations
Affect all cells of an individual
Somatic Mutations
Originate in mitosis
Cannot be passed on to future generations
Affect only cells that descend from changed cell
Collagen and Ehler-Danos Syndrome
A major component of connective tissue
Bone, cartilage, skin, ligament, tendon, and tooth dentin
More than 35 collagen genes encode more than 20 types of collagen molecules
Mutations in these genes causes a multitude of diseases
Collagen has a precise structure
Triple helix of two alpha 1 and one alpha 2 polypeptides
Longer precursor, procollagen is trimmed to from collagen (has a n-terminal and a c-terminal cut off)
Ex. Ehler-Danos Syndrome
Clinical: Variety of symptoms, but. Stretchy skin, joint mobility or laxity are common
Inheritance: Autosomal Dominant
Molecular: COL5A1 gene, Type V collagen protein
In this disorder the n-terminal and the c-terminal are not cleaved
This causes them to not be as tightly packed which makes them more stretchy, but less likely to go back to the same position
Spontaneous Mutations
De novo or new mutations
Comes about when a sperm or egg develops a new mutation (germline)
Not caused by exposure, just errors of DNA replication
Tautomer shift is what causes spontaneous mutations (A paired with G)
This causes the width of the double helix to change aka mismatched pair
Sometimes DNA polymerase proofreads, and finds the errors but other times the mutation can get through
Mutational Hotspots
In some genes, mutations are more likely to occur in regions called hot spots
Areas of DNA symmetry (palindromic, repeated sequences, etc.)
There is a hairpin because the DNA is symmetrical, so the DNA polymerase skips that section entirely
Repeated genes (multiple copies of the same gene)
This occurs during crossing over of meiosis 1
The repeated genes align with one another and cross over
Sometimes one of the chromosomes gets more copies of the repeated gene than the other chromosome because of chromosomal misalignment
Larger genes (think NF1/Neurofibromin)
We each have about 175 spontaneous mutations, but these are not disease causing because: they could be noncoding, they could be recessive, the gene could not be used anymore
Induced Mutations
Caused by mutagens, many are also carcinogens and cause cancer
Examples:
Alkylating agents: remove base (common chemotherapy medicine)
Acridine dyes: add or remove a base
X rays: break chromosomes
UV radiation: create thymidine dimers
Site-directed mutagenesis: changes a gene in a desired way
No mutational hotspots for this type of mutation
Point Mutation
A change of a single nucleotide
Transition - Purine replaces purine or pyrimidine replaces a pyrimidine
Transversion - Purine replaces a pyrimidine or vice versa
Can be:
Missense mutation - replaces one amino acid with another (Sickle Cell Anemia)
Nonsense mutation - changes a codon for an amino acid into a stop codon
A stop codon that is changed to a coding codon lengthens the protein
Duchesse Muscular Dystrophy
Clinical: progressive muscle weakness, heart problems, developmental delay
Inheritance: X-linked recessive (on p arm)
A heterozygous female may still show signs of symptoms due to skewed X-inactivation
Molecular: DMD gene, dystrophin protein
79 exons on the gene so it’s very large
Dystrophin connects muscle cells to the cytoskeleton of other muscle cells
In this disease the dystrophin protein is too short so the muscles aren’t attached to the cytoskeleton
This is a nonsense mutation, transition mutation (pyrimidine exchanged for pyrimidine in this case)
A glutamine amino acid is changed to encode for a stop codon
Splice Site Mutations
Alters a site where an intron is normally removed from mRNA
Can affect the phenotype if:
Intron is translated or Exon is skipped
Frameshift Mutations
The genetic code is read in triplets
Nucleotides changes not in multiples of 3 is a frameshift
Nucleotide changes in multiples of 3 will just add or subtract the amount of amino acids
Deletions and Insertions
Deletions - Removal of DNA, can cause frameshift, if deletion in Y there will be male infertility
Insertions - Addition of DNA, can cause frameshift
Tandem Duplication - The duplicated information is right next to the piece of DNA that was duplicated
Expanding Repeats
Insertion of triplet repeats leads to extra amino acids
Some genes prone to expansion
Number of repeats correlates with earlier onset and more severe phenotype
Anticipation - Expansion of the triple repeat with an increase in severity of phenotype with each generation
Myotonic Dystrophy
A triplet repeat disease
DMPK Gene is affected by a expanding repeat
This first generation - Age of onset is older adulthood, phenotype of mild forearm weakness and cataracts, 50-80 copies of GAC repeat in the mRNA
The second generation - Age of onset mid-adulthood, phenotype of moderate limb weakness, 80-700 copies of GAC repeat in the mRNA
The third generation - Age of onset childhood, phenotype of severe muscle impairment respiratory distress and early death, 700+ copies of GAC repeat in the mRNA
Clinical: increased risk for cataracts, myotonic facies (lack of muscle movement in face), cardiac conduction abnormalities (arrhythmia), lack of muscle movement
Inheritance: Autosomal dominant
Molecular: DMPK Gene
Copy Number Variants
⭐️Abnormal Crossing Over leads to copy number variants
Are sequences that vary in number from person to person
Deletions and Duplications (sometimes even inversions)
Range in size from a few bases to millions of bases
We see repeating segments in about 25% of our genome
Some of these cause disease, but most of them don’t
22q11.2 Deletion
aka DiGeorge or Velocardiofacial Syndrome
Symptoms: Congenital Heart Disease, Cleft lip/palate, facial dysmorphology, learning difficulty, immune deficiency
(Most) People with this syndrome have a 3 million base pair deletion
Molecular: TBX1 Deletion
Inheritance: Autosomal Dominant
There are repeated regions in chromosome 22, which makes the chromosomes align wonky with one another
This causes the deletion on one of the chromosomes and a duplication on the other
The duplicated chromosome may cause some mild symptoms while the deletion chromosome causes severe symptoms
CNVs correlated to cholesterol level might be used to give medical advice
Importance of Position
The degree that a mutation affects a phenotype depends on:
where in the gene the change occurs, how it affects the confirmation or expression of encoded protein
Cystic Fibrosis
Protein: CFTR
Molecular: Missing amino acid or other variant alters conformation of chloride channels in certain epithelial cell plasma membranes, water enters cell drying out secretions
Symptoms: Frequent lung infection, pancreatic insufficiency
Duchenne Muscular Dystrophy
Protein: Dystrophin
Molecular: Deletion eliminates dystrophin, which normally binds inner face of muscle cell to plasma membrane, muscles weaken
Symptoms: Gradual loss of muscle function
Familial Hypercholesterolemia
Protein: LDL receptor
Molecular: Deficient LDL receptors cause cholesterol to accumulate in blood
Signs: High blood cholesterol, early heart disease
Hemophilia B
Protein: Factor IX
Molecular: Absent or deficient clotting factor causes hard-to-control bleeding
Symptoms: Slow or absent blood clotting
Huntington Disease
Protein: Huntingtin
Mutations: Extra bases add amino acids to the protein, which impairs certain transcription factors and proteasomes
Symptoms: Uncontrollable movements, personality changes
Neurofibromatosis Type 1
Protein: Neurofibromin
Molecular: Defect in protein that normally suppresses activity of a gene that causes cell division, leading to abnormal growths
Symptoms: Pigmented skin patches and benign tumors of nervous tissue beneath skin
