MCB Exam 2
Summarize the central dogma of molecular biology, and cite exceptions to the original model.
- DNA -> RNA -> Protein - Reverse transcription info from RNA goes back to DNA - RNA replication -> RNA to RNA (ssRNA virus = covid), uses template for RNA synthesis as protein template
What are the basic structural components of nucleosome?
- DNA does not normally exist as the simple double helix described in the module on DNA structure. Instead, eukaryotic DNA is found packaged with protein, forming a substance called chromatin. - The eight histones of the core particle are arranged as an octomer composed of (H3)2(H4)2 tetramer and a pair of (H2A-H2B) dimers. DNA wraps around the outside of the octomer.
Describe the essential features of DNA structure including the: i) concept of base pairing - weak interactions, ii) antiparallel nature of strands in a double helix, iii) differences in the major and minor grooves of DNA.
- Hydrogen bonds and hydrophobic stacking stabilizes double helix. The hydrophobic effect makes base stacking (A-T, G-C) - Chargaff's rules # of A=T, # of G=C, # purine = pyrimidine Double Helix - Phosphodiester bond connects nucleotides. In a polynucleotide, refers to the bond between the 3' hydroxyl of a sugar group in a nucleotide and a phosphate group attached to the 5' carbon of another sugar group. (connects nucleotides) - in DNA, glycosidic bond connecting N containing base refers to the nitrogen-carbon linkage between the 9' nitrogen of purine bases or 1' nitrogen of pyrimidine bases and the 1' carbon of the sugar group. (connects N to C) - if you remove glycosidic bonds, it causes DNA damage Major and minor • DNA in the B form has a major groove and a minor groove. • Allows access to the hydrogen-bonding capabilities of the exposed bases which provides a means of sequence-specific interactions between DNA and the molecules it must interact with in the processes of replication and transcription. • Several hydrophobic molecules containing flat aromatic and fused heterocyclic rings can insert between the stacked base pairs of DNA. These molecules called intercalating agents. • Intercalating agents (Ethidium bromide, Acridine orange, Actinomycin D) are potential Cancer-inducing reagents (how they inhibit DNA replication??). - They are Hydrophobic molecules, can interact with each other through hydrophobic effect
Describe the enzymes involved in DNA replication and understand their roles. Understand the differences between leading and lagging strand synthesis and importance of DNA proofreading.
- Know enzymes involved o DNA helicase o DNAG primase o SSB o Pol III 3' -> 5' exonuclease, proofreading o Pol I 5' -> 3' exonuclease, remove primer o Ligase o DNA topoisomerase (type 1 (strand), type 2 (loop) remove supercoil of DNA double strand) § bond covalently to the DNA phosphate as they break the phosphodiester linkage between neighboring nucleotides, storing the energy in that bond to use in the process of resealing the bond. § Antimicrobial targets specific · Ciprofloxacin: DNA gyrase (topoisomerase II) · Quinolones: topoisomerase II
Outline the key features of the reactions catalyzed by DNA polymerases. Define template and primer as they relate to DNA polymerases.
- Primer function - provide OH group 3' allows polymerase to add nucleotide and create phosphodiester bond Action of DNA pol • DNA polymerase adds nucleotides to an existing nucleic acids strand. It cannot add nucleotides without an existing nucleic acid and therefore it needs a primer. • The key is the 3' OH group. • DNA polymerase will only synthesize DNA from 5' to 3' - DNA pol III - 3'->5' exonuclease, proofreading - DNA pol III - 5'->3' polymerase activity, DNA synthesis o Leading made out of okazaki fragments
Compare the structure of DNA and RNA, understanding the difference between the constituent bases, sugars, nucleosides and nucleotides
- Similar to DNA Synthesis except - RNA sugar is ribose - The precursors are ribonucleoside triphosphates ATP, GTP, CTP, UTP. - Only one strand of DNA is used as a template. - RNA chains can be initiated de novo (no primer required). - The RNA molecule will be complementary to the DNA template (antisense) strand and identical (except that uridine replaces thymidine) to the DNA nontemplate (sense) strand. - RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5'3' direction. - Highly selective because RNA synthesis corresponds to transcription and corresponds to certain genes - DNA replication requires primer
Briefly describe the different types and functions of RNAs found in eukaryotic cells, including mRNA, tRNA, rRNA, snRNAs, miRNAs and siRNAs
- Transfer RNA (tRNA), 15% o At least one specific type of tRNA for each of the 20 amino acids -adaptors between amino acids and the codons in mRNA o Involved in protein synthesis - Ribosome RNA (rRNA), 80% o structural and catalytic components of ribosomes. o Facilitate the binding and positioning of the mRNA on the ribosomes o Involved in protein synthesis - Messenger RNA (mRNA), 5% o Carry genetic information from DNA to cytosol o Template for protein synthesis - Others non-coding RNAs (snRNA, miRNA, lncRNA) (eukaryotic cells)
Describe the posttranscriptional processing of eukaryotic mRNA including capping, splicing, and polyadenylation
1) 5' Capping: A 7-Methyl guanosine cap is added to the 5' end of the primary transcript by a 5'-5' phosphate linkage; permit the initiation of translation and stabilize the mRNA Unique because linkage of 5'-5' (5' capping) 2) 3' Addition of a poly-A tail: A poly(A) tail (a 20-200 nucleotide polyadenosine tract) is added to the 3'end of the transcript; function of poly A tail stabilize the mRNA and exit of the RNA from nucleus. 3) Removal of intron Non coding sequence needs to be removed 1) by precise endonucleolytic cleavage and ligation reactions catalyzed by special splicing endonuclease and ligase activities (ribozyme). 2) in two-step reactions carried out by spliceosomes Splicing process GU is splice donor site, AG splice acceptor site splicing enzymes: Small nuclear ribonucleoprotein particles (snRNPs), recognize the splice sequence help remove intron sequence starting at GU (splice donor site), end of intron (splice acceptor site) determine where intron is to determine where to remove intron removal of intron makes lariat most introns removed have lariat shape!!!! Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons. The introns are excised from the RNA transcripts prior to their transport to the cytoplasm
Understand the principle of gene therapy. What is the major challenge for gene therapy?
1). Remove some of the patient's blood. Lymphocytes are separated from other cells in the blood. 2). Infect the lymphocyes with a retrovirus modified to carry the normal adenosine deaminase gene - Causes skid - Because patient doesn't carry normal functioning gene, retrovirus delivers normal gene - Once delivered, uses reverse transcription - Can cure patient with specific defect 3). The gene becomes integrated in to the cell's chromosomes and is expressed. Some of the patient's lymphocytes now synthesize adenosine deaminase. 4). Return the cells to the patient, and the patient regains immune function. severe combined immunodeficiency (SCID) Need retroviral vector - Deliver gene of interest into patients cells - Randomly inserts genome into host DNA - Express and duplicate in genome ADA gene therapy was first successful gene therapy Problems with gene therapy - May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis or oncogene - Short lived - Immune response - Viral vectors Multigene disorders
Describe the mechanisms of inhibition of transcription by rifampicin and actinomycin D and alpha-amanitin
1. Rifampin: inhibits initiation and elongation, tuberculosis (TB) Binds to beta subunit of rna polymerase and changes conformation and stops initiation and elongation Mutation: drug resistance Rifampicin (also called Rifampin) present and inhibits transcription by binding to the polymerase on the DNA. Rifampicin itself can only interact specifically with RNA polymerase if the enzyme is free or in a binary complex with DNA 2. Actinomycin D: Actinomycin intercalates between the bases of the DNA double helix, preventing the DNA from being used as a template. Drug that inhibits transcription 3. a-amanitin: synthesized by the poisonous mushroom Amanita phalloides. High specificity for eukaryotic RNA Polymerase II (very important for mRNA) Once it binds to rna pol 2, it inhibits transcription People will get killed by this Doxorubicin treatment for hodgkin's lymphoma that inhibits dna replication and transcription
Understand the pyrimidine dimer formed by ultraviolet light.
1. Thymine dimer: UV (sunlight or tanning lamps) leads to cross-linking of adjacent pyrimidines (thymine) along one strand of DNA
What is mRNA editing? How does mRNA editing regulate gene expression (ApoB48)?
2) RNA Editing of apo-B Pre-mRNA 1) Deamination of cytidine to uridine by cytidine deaminase (C to U): APOBEC-1 (apolipoprotein B-editing catalytic subunit 1) converts cytosine to uracil small intestine - CAA converted to UAA (stop codon in mRNA (mutation) -> makes shorter amino acid - 48) - in liver it is not converted chylomicron transport lipids to lymphatic system apo B-48 is important which is encoded by APOBEC-1
Understand the pathways for the degradation of mRNA.
5' cap and 3' tail stabilize need to remove 5' and 3' leads to exonuclease attacking either 5' or 3' (doesn't matter) inducing degradation of mRNA
Understand the role of cytochrome P450 enzymes in activating carcinogens and mutagens.
Ames Test-assays for carcinogenicity • Salmonella his-- strain: must be grown in the presence of Histidine, and the mutant allele is susceptible to back-mutation to a wild-type allele. • Needs histidine to be activated first • Many non-carcinogens are converted to carcinogens in liver. • Need to combine test compound to rat liver homogenized • Metabolic activation of test compound by rat liver enzyme -> metabolically activated compound -> add salmonella bacteria unable to grow without added histidine in culture medium -> count number of bacterial colonies that have undergone mutation enabling them to grow without added histidine • Small amount of rat liver is added to Ames test to mimic mammalian metabolism. Conversion of procarcinogens to carcinogens by cytochrome p450 1. cigarette smoke (lung cancer) benzo pyrene (procarcinogen) metabolized by cytochrome P450 -> BPDE (ultimate carcinogen) -> DNA (covalently bounds to DNA to form DNA adduct) Rat liver lysates provide cytochrome P450 2. Aspergillus flavus (aflatoxin B1) -cytochromeP450-> alkylating reagents -> DNA After metabolization, both converted to carcinogen and added to DNA causing cancer
Know how aminoacyl-tRNA synthetases (aa-RS's) act to accurately couple the sequence of codons in a mRNA to the sequence of a protein and how compounds targeting aa-RS's are being used as potential antibiotic/antifungal agents (Bactroban, tavaborole (Anacor-AN2690)).
Aminoacyl-tRNA synthetase (aaRS or ARS): attach AA to tRNA has two activities: 1) synthetase activity, 2) editing activity Amino acid not directly added to end of tRNA (IMPORTANT) 1) The amino acid is first activated by reacting with ATP to form aminoacyl-AMP (adenylation reaction) - Attachment of amino acid to tRNA is mediated by amino acid tRNA synthesis (charged tRNA) · Essential for fidelity of translation is maintained by synthetase (editing activity) tRNA synthetase inhibitors as antibiotics - Mupirocin (bactroban) - anti-bacterial -An2690 (tavaborole, keyydin) - anti-fungal
Understand the structure of the lac operon and the functions of the promoter, repressor, operator, and inducer in controlling the production of polycistronic lac mRNA.
Bacteria such as E. coli usually rely on glucose as their source of carbon and energy. However, when glucose is scarce, E. coli can use lactose as its carbon source. - If level of glucose is low and lactose is available, e. coli can use lactose for energy Promoter (site for RNA polymerase), operator (recognized by repressor, neg regulator), CAP binding site (recognized by CAP, pos) - Need to remove negative regulator and have the binding of positive regulator, then lac operon is activated - default is repressor associated with operator, repressor is removed when lactose is present - Repressor removed by allolactose - presence of glucose, operon off because adenylyl cyclase is inhibited by glucose and cAMP isn't made, CAP isn't binded to binding site The operon is regulated by two elements: 1) operator: suppressed by Lac repressor (default). The repressor inhibits transcription by binding to the lac operator (O). Associated with Repressor (negative regulator) binding to the operator is prevented by the inducer. 1) Active form by default 2) CAP binding site: activated by catabolite activator protein (CAP), which stimulates the transcription of lactose gene when bound to cAMP. 1) Positive regulator 2) Sits upstream of promoter - Need to remove negative regulator and need positive regulator there and then lac genes can be activated Adenylyl cyclase is inhibited by glucose and no cAMP is made. - cAMP binds to CAP - E coli prefers glucose o When glucose is available and no lactose, gene is off o Default is repressor binds to operator between promoter and gene, polymerase cannot properly - No glucose o Lac op turned on o Remove repressor with available lactose o Lactose converted to allolactose which binds to repressor protein o Conformation of repressor changes and can't bind to promoter region o cAMP binds to CAP and that binds to active site o remove repressor from promotor o CAP binds to CAP binding site increase positive regulator and decrease negative regulator and moves along template and synthesize genes for lactose - No glucose No lactose o Operon is off o Allolactose removes repressor from operator o Glucose inhibits adenylyl cyclase o Cant bind to CAP binding site o Makes operon off - Both glucose and lactose o Operon off o CAP activated o Repression blocks transcription o No proteins produced - Low glucose, lactose available -> lac genes are strongly expressed - High glucose, lactose not available -> lac genes not expressed - Low glucose, lactose not available -> lac genes not expressed - High glucose, lactose available -> low level of gene expression
Describe the types of DNA damages and the cellular response to DNA damages (Role of Rb and E2F in S phase delay, p53-p21 axis)
Cell senses DNA damage -> ATM/ATR (kinase) activation -> phosphorylates substrates, one being p53 (tumor suppressor) -> activated -> activates downstream of p21 -> inhibits Cdk2/cycline E -> G1 arrest so all cells stop at G1 to maybe fix damage of DNA or apoptosis S phase is phase for dna synthesis -> E2F regulates.... E2F regulated by Rb (tumor suppressor) Normal situation, Rb binds with E2F and inactivates it, when cell sense growth factor, Rb phosphorylated, E2F activated and activates genes for gene transcription in S phase 1. Mismatch 2. Base alteration (methylation coupled with deaminase cause CG to TA mutation) - Change of nucleotide 1. oxidative deamination - 5-methylcytosine ---cytosine deaminase - thymine (C:G->T:A) transition Most common point mutation in cancer cells 2. oxidation: ROS such as hydroxyl radical - when guanine is oxidized it forms 8-oxo-deoxyguanine!!!!!! C-G to A-T transversion 3. Alkylation: addition of hydrocarbon molecule - Alfatoxin B1 metabolized by cytochrome P450 and converted to Alfatoxin B1 epoxide (very reactive) reacts with DNA and purine rings -> DNA adduct (directly interacts with nucleotides).... Pairs with different nucleotides - C-G to A-T pair transversion mutation Methylation is the most common type of alkylation (3-methylcytosine most common!!) 4. depurination: purine or pyrimidine removed from sugar - destabilize covalent bonds resulting in the loss of the purine or pyrimidine base from DNA - AP sites - apurinic or apyriminic abasic sites - misread by dna pol 3. Thymine dimer and break 1. Thymine dimer: UV (sunlight or tanning lamps) leads to cross-linking of adjacent pyrimidines (thymine) along one strand of DNA TUMOR SUPPRESSORS - P53 activated, induces p21 - Rb and cell cycle machinery - E2F regulates cell cycle to s phase
Understand the biotechnologies that are being used in the diagnosis, treatment and prevention Covid-19.
Covid is RNA virus, uses spike protein that interacts with receptor Endocytosis where virus enters host cell RNA replicates to create viral RNA Synthesizes more RNA virus and releases virus outside of cell Immune system tries to fight it off Treatment technology - Anti-spike Ab o Antibody recognizes and blocks entry into cells - Blockage of replication o corticosteroid - Over production of cytokine leads to cytokine storm which causes most deaths o Inhibit it to live Treatments - Remdesivir - blocks replication of virus o Nucleotide analogue, targes RNA polymerase - Corticosteroid o Anti-inflammatory, suppress cytokine storm - Monoclonal antibody drugs o Blocks entry of virus into host cell - 7 vaccine platforms o DNA o RNA o Protein-based o Viral vector (non/replicating) o Virus (in/activated) Covid saliva based test is PCR reaction using viral RNA as template to detect covid
Understand how cytosine nucleotide analogues such as aza-cytosine can be used as drugs to inhibit DNA methylation
Cytidine analog adds cytidine which removes methylation of DNA, inhibits dna replication 5-azacytine - 5' carbon is replaced by "aza"? group 5-Aza-2'-deoxycytidine - Hydroxy group at 2 position is removed - They are converted to the deoxynucleotide triphosphates and are then incorporated in place of cytosine into replicating DNA. - DNMTs (dna methyltransferase) get trapped on DNA containing modified bases such as 5'-azacytosine, 5-fluorocytosine, pseudoisocytosine or zebularine, resulting in the formation of heritably demethylated DNA. - Serve as template for dna replication and can be passed to daughter cells - High levels of drugs cause dna defect myelodysplastic syndrome.
Understand the biogenesis and functions of microRNA. Relate the mechanism of microRNAs to that of RNA interference (RNAi)
DCL/Dicer:Endoribonuclease, cleave double-stranded RNA and pre-microRNA into Small interfering RNA (siRNA) and microRNA miRNA -> translation inhibition, mediated similar to protein and mRNA, mediated by RNA pol II reduce gene expression... targets 3', sequence not perfectly matched, reduce expression of genes by reducing translation of gene siRNA -> RNA cleavage, produced through viral replications and transcription of strands, or rna dependent activity on ssRNA - comes from outside of cell - targets on coding sequence in mRNA, perfectly matched, no mRNA -> no protein translation, antiviral defense in plants fungi and invertebrates
Understand the difference between genomic DNA library and cDNA library.
DNA libraries: Collection of cloned restriction fragments of the DNA of an organism a) Genomic DNA library: is a set of host cells (or phage) that collectively contain all of the DNA sequences from the genome of another organism. - contain all DNA fragments of genome -> genomic DNA - human DNA -cleave with restriction nuclease -> millions of genomic DNA fragments -DNA fragments into plasmids using ligase -> recombinant DNA molecules -introduction of plasmids into bacteria-> genomic library b) cDNA library: is a set of host cells that collectively contain all the DNA sequences produced by reverse transcription of mRNA isolated from the cell (or tissue) type of interest. No introns and no regulatory elements. - Requires reverse transcriptase - mRNA -reverse transcriptase -> cDNA, -cleave with restriction nuclease-> cDNA fragments, -DNA fragments inserted into plasmids using ligase-> recombinant DNA molecules, -introduction of plasmids into bacteria ->cDNA library Cloning amplifies dna in short period of time
Understand the role of DNA polymerase-eta in the damage tolerance and that one type of XP is caused by a mutation in this polymerase.
DNA repair of UV-induced thymine dimer 2) DNA damage tolerance by translesion synthesis. Translesion synthesis can bypass a replication block. Translesion synthesis DNA polymerases can replicate DNA over such lesions to resume publication but the enzymes are error prone. DNA polymerase eta bypasses thymine dimers and uses dimer as template to synthesize new DNA...enzymes are error prone XP is skin cancer associated with dna repair system associated with thymine
Understand the techniques used for the detection of mRNA (RT-PCR, Southern blot, microarray).
Determine the mRNA levels Northern blots Real-time PCR Microarray: contain thousands of immobilized DNA sequencesThe expression levels of thousands of genes can be simultaneously analyzed by using DNA microarrays (gene chips).Different primers represent different genesRed - cancer produces more of this message, green - normal produces more of this message, yellow - both cells produce this message 4. RNA sequencing Real time or quantitative PCR (qPCR) is a technique used to monitor the progress of a PCR reaction in real time. - PCR based acid to detect RNA - Real time PCR requires DNA/RNA as template to create cDNA - Syber green dye interacts with DNA...based on intensity -A relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. -It is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. -This occurs due to the accumulation of the PCR product with each cycle of amplification. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (i.e. SYBR® Green) -It facilitates the monitoring of the reaction as it progresses (real time). Saliva test covid - Collect saliva - Heat at 95 celcius - Add TBE buffer and tween 20 - Run RT-qPCR (unique and specific primer that detects RNA in cells/blood) - Viral RNA is amplified if you have covid - UIUC test difference is that it doesn't go through RNA purification kit and it's not as invasive as nose swab o It is faster and not time consuming
Describe the mechanisms by which diphtheria toxin and ricin inhibit protein synthesis in eukaryotes
Diphtheria Toxin: inactivates translocation by inhibiting eEF-2 (eukaryotes) Ricin: inactivates ribosomes by depurination of 28S rRNA (peptidyltransferase) in 60S ribosomal subunit - Targets on protein translation...protein translation inhibitor which makes it unique - Ricin A-chain (serves as glycosidic to break glycosidic bond) attacks 28S rRNA (peptidyltransferase) (inhibits it) - Covalent bond connects sugar and n base, ricin A-chain breaks glycosidic bond, purine rings detached
Describe direct reversal repair, base and nucleotide excision repair, mismatch repair.
Direct reversal mechanisms are specific to the type of damage incurred and do not involve breaking of the phosphodiester backbone Mismatch repair -> removes mismatched nucleotide and synthesize correct nucleotide Base excision repair - Glycosylase removes damaged base -> ap endonuclease comes in and breaks phosphodiester bond -> creates gap -> dna polymerase fills gap -> dna ligase fills nick Nucleotide excision repair -> specifically important for thymine dimer removal - 1. ID mismatched or mutated DNA strand - 2. Nick the mismatched DNA or mutated strand by endonuclease. Remove the mismatch or mutated DNA strand by exonuclease to create a gap. - 3. 5'-3' DNA polymerase fill the gap - 4. DNA ligase forms the phosphodiester linkage
Describe how telomerase makes DNA of defined sequence in the absence of a DNA template.
Eukaryotic Telomerase, also called telomere terminal transferase, is an enzyme made of protein and RNA subunits that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes. RNA containing An RNA sequence in telomerase acts as a template for DNA. This enzyme adds the telomeric sequence to the 3' end of the chromosome by its reverse transcriptase activity (enzyme using RNA as template to synthesize RNA)
Discuss how expansion of a trinucleotide repeat causes aberrant methylation and affects expression of the FMR1 gene in Fragile X syndrome
Fragile X has insertion of CGG cause hypermethylation silences genes so there is no mRNA 1) Inactivation of Tumor suppressor: meCpG to TpG mutation: account for 1/3 of transition mutation in human. more than 50% of all of the p53 mutations which are acquired in sporadic colorectal cancer occurs at sites of cytosine methylation Hypermethylation of promoters of tumor suppressors 2) Fragile X syndrome: the single most common form of inherited mental retardation (50-200). fragile X mental retardation protein Random insertion of CGG normal less than 50 CGG repeats -> normal FMRP 50<n< 200 CGG inserted -> FXTAS More than 200 CGG -> hyper methylation, silences expression of genes -> no mRNA -> fragile x syndrome - C is site that causes methylation, hyper methylation silences the genes, don't have FMRP genes
Explain the use of SDS gel electrophoresis (SDS-PAGE) in the analysis of proteins
Gel Electrophoresis the phosphodiester backbone of DNA is highly negatively charged. the shorter the DNA fragment, the farther the migration, separated by mass. Bands of DNA in gels can be visualized by staining with ethidium bromide, which fluoresces an intense orange under irradiation by ultraviolet light when bound to a double-helical DNA molecule. - Restriction enzymes cleave DNA into smaller segments of various sizes - DNA segments are loaded into wells in a porous gel. The gel floats in a buffer solution within a chamber between two electrodes - When an electric current is passed through the chamber, DNA fragments move toward the positively charged anode - Smaller DNA segments move faster and farther than larger DNA segments - So many types of DNA fragments Denaturing of proteins by SDS - Charge of protein are different - In order to separate in gel, must denature proteins by adding SDS: sodium dodecyl sulfate (negatively charged chemical) helps destroy noncovalent bonds - Polypeptide becomes negatively charged - 2-mercaptoethanol destroys disulfide bonds - Single strands and then able to separate ... migration of proteins occurs by size (mass) on the SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) Transfer
Understand how hormones regulates gene expression (lipid soluble and water soluble hormones)
Gene Expression Is Regulated by Hormones Transcription is highly selective - can be regulated by stimuli 1) lipid soluble hormones: Glucocorticoid, mineralocorticoid, sex hormone (estradiol), vitamin D, retinoic acid and thyroid hormone. They are hydrophobic signal molecules. They can go through the plasma membrane and go into the cells with these transcription factors. - hydrophobic, needs to by-pass membrane (lipid bilayer), interacts with hormone-receptor complex which bind to DNA and activate expression 2) water soluble hormones: Insulin, Epinephrine, Glucagon (first messenger). They deliver signals outside the cells. Difference between lipid vs water soluble hormone, lipid can go through bilayer, water cannot Transcription by Lipid Soluble Hormone Receptors - estradiol Binds to receptor, transcription factor, expresses gene They can go through the plasma membrane and go into the cells. They bind to hormone receptors and go directly into the nucleus. Hormone receptors bind to specific regions of the DNA called hormone response elements (HRE) and activate gene expression. Nuclear hormone receptors are transcription factors. Nuclear hormone receptors have two highly conserved domains: 1) The DNA binding domain and 2) the ligand binding domain. Transcriptional Regulation by Cell Surface Receptors (for water soluble hormones) - Cannot go through membrane - Need g protein coupled receptor which changes conformation and activates adenylyl cyclase converts ATP to cAMP - cAMP serves as second messenger - activates protein kinase A - phosphorylate inhibition of glycogen synthesis and promotion of glycogen breakdown - First messenger: Insulin, Epinephrine, Glucagon (water soluble Hormones) - Second messenger: cAMP, cGMP, calcium Agonists are ligands that activate a nuclear hormone receptor Ligands that inhibit the receptor are called antagonists Some cancers are dependent on the action of the estradiol-receptor complex. The growth of these cancers can be slowed by administering receptor antagonists, such as tamoxifen and raloxifene
Understand xeroderma pigmentosum, hereditary nonpolyposis colorectal cancer, Familial Breast and Ovarian cancer, their causes and relationships to DNA repair.
Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. Xeroderma pigmentosum (XP), at least eight distinct genes, seven of which are involved in NER. The eight gene encodes DNA polymerase eta Ataxia telangiectasia (AT), small number of cases. Almost 50% of all identified familial breast cancers involve germline transmission of a mutant BRCA1 or BRCA2 allele. By some estimates, 70 to 80% of all familial ovarian cancers are due to mutant germline alleles of BRCA1 or BRCA2.
Understand the basics of the genetic code and the potential effects of point mutations, deletions, and insertions on translation
If you change sequence that encodes amino acids, can cause disease Sickle cell disease (Missense mutation): GAG -> GTG causes val -> mutant protein causes aggregation of hemoglobin Silent mutation - UCA -> UCU both codon for serine Hemoglobin Mckees Rocks (Nonsense mutation): codon changes to termination codon. UAU (ser) -> UAA (termination codon) tyrosine of the b chain is mutated to a stop codon. b Hemoglobins with shorter b chain have high oxygen affinity and decreased oxygen delivery. Cystic Fibrosis: phenylalanine is deleted in CFTR: ATP dependent transport protein, functions as cAMP regulated chloride channel - Deletion of CTT - Phe is deleted CFTR: Cystic fibrosis transmembrane conductance regulator Insertion/deletine of ½ nucleotides cause frame shift Components required for translation Transfer RNA (tRNA) molecules function as an adaptor molecule between a codon and an amino acid. There is at least one tRNA molecule for each amino acid. 1) In a two-dimensional representation, all tRNA molecules appear as a cloverleaf pattern. In 3-D, it appears as a "L" shape. 2) The amino acid-accepting region is the acceptor stem, which contains the 3' CCA terminal region. Many of the nucleotides are involved in hydrogen bonds that form stems and loops. 3) The amino acid is attached to a hydroxyl group of adenosine in the CCA region of the acceptor stem. 4) The 5' end is phosphorylated and the 5' terminal residue is usually pG. 5) The anticodon is in a loop near the center of the sequence. - has anticodon
Describe the subunit structure of RNA polymerase from E. coli and assign functions to the individual subunits.
In E. coli, two DNA sequences that act as a promoter for many genes are the -10 sequence (Pribnow box) and the -35 sequence. Sigma and 2 alpha and 2 beta subunits (holoenzyme!!! Important for initiation) - Sigma - recognize promoter regions on the DNA (initiation) - 2 alpha 2 beta - Core enzyme, involved in elongation and can recognize termination signal and stop transcription • Initiation: the recognition of a specific DNA sequence by RNA polymerase and the 5' of the bond formation • Elongation: RNA polymerase continues the binding, bond formation, and translocation cycle • Termination: The ends of the genes are recognized by the RNA polymerase complex (r-dependent and -independent) The sigma (σ) subunit of the holoenzyme helps the polymerase locate promoter sites. By decreasing the affinity of the polymerase for DNA, the σ subunit allows the enzymes to rapidly scan the DNA for a promoter. The σ subunit also assists the enzyme in identifying the promoter. Once the promoter is located and RNA synthesis begins, the σ subunit dissociates from the enzyme to assist another polymerase in initiation. Sigma only involved in initiation of RNA synthesis, NOT ELONGATION Elongation does not require primer and it has helicase activity (prokaryotic) Extension of nucleotide chain Use ribonucleoside triphosphate, release pyrophosphate
Relate defective DNA repair to cancer and relate tumor-suppressor genes to DNA repair.
Inherited mutations that affect DNA repair genes are strongly associated with high cancer risks in humans. DNA repair important to keep proper function of cells Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. - Defect in DNA mismatch repair Xeroderma pigmentosum (XP), at least eight distinct genes, seven of which are involved in NER. The eight gene encodes DNA polymerase eta - Defect in removal of thymidine thymine repair Ataxia telangiectasia (AT), small number of cases. (double strand DNA breaks) Almost 50% of all identified familial breast cancers involve germline transmission of a mutant BRCA1 or BRCA2 allele. By some estimates, 70 to 80% of all familial ovarian cancers are due to mutant germline alleles of BRCA1 or BRCA2 (double strand DNA breaks)
Name the major initiator codon and the amino acid it encodes. Explain the roles of the nucleotide sequences in 16S rRNA, mRNA, and tRNA in selecting the initiation codon rather than the identical codon that encodes an internal amino acid. Recognize characteristics of the Shine-Dalgarno sequence
Initiation Shrine-Dalgarno pairs with 16S rRNA (exclusive for prokaryotes), positions - interferes with binding of fMet-tRNA and inhibits protein synthesis initiation in bacteria
List the sequence elements of eukaryotic promoters. Contrast the nucleotide sequences and locations of the TATA box of eukaryotes and the 10 sequence of prokaryotes
Initiation: binding of the RNA polymerase holoenzyme to the promoter region which starts RNA synthesis. the TATA, CAAT, and GC boxes and other cis-acting elements in eukaryotic promoters are recognized by proteins other than RNA polymerase itself. The TATA box is usually centered at ∼−25. TATA box closely resembles the bacterial −10 sequence (TATAAT) but is farther from the start site.Pribnow box (in prokaryotes). TATA and CAAT similar to pribnow box. - Difference is eukaryotic gene promoter is RNA polymerase II
Understand how selective estrogen receptors modulators (SERMS) serve as anti-cancer drug
Ligands that activate a nuclear hormone receptor are called agonists, while ligands that inhibit the receptor are called antagonists. Some cancers are dependent on the action of the estradiol-receptor complex. The growth of these cancers can be slowed by administering receptor antagonists, such as tamoxifen and raloxifene. Tamoxifen: antagonist of estrogen receptor, competitively binds to receptor, anti-breast cancer drug. Such antagonist to the estrogen receptor is called selective estrogen receptors modulators (SERMS). - Inhibits activity of estrogen receptor Breast cancer caused by ERalpha expression Estrogen interacts with receptor and activates genes. Over expressed in more than 70% of breast cancer cells - ERalpha serves as target for inhibition of treatment for breast cancer cells (SERMS) by inhibiting estrogen receptor
Understand the use of inhibitors of DNA synthesis as drugs, e.g. in cancer.
Nucleoside analog: modification on the hydroxyl group at the 3'-carbon of the deoxyribose Remdesivir- inhibits RNA synthesis through RNA polymerase (Covid is single strand RNA virus) Cisplatin is a chemotherapeutic agent that reacts with DNA such that nitrogens of adjacent purines replace the chloride atoms. This modification disrupts DNA structure by crossing linking two purines and leads to cell death. Drugs for cancer target all highly proliferative cells Bone marrow, gut epithelium, skin and hair follicles, no G0, very short G1(side effects from chemotherapy)
Understand the roles of oncogenes and tumor suppressors in carcinogenesis
Oncogenes are cancer-causing genes Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle (gain-of-functions) Proto-oncogene -> point mutation - > if mutation of DNA damage acts on promoter region of oncogene -> cause normal growth stimulating protein in excess....if in the cell -> hyperactive or degradation-resistant protein • Tumor-suppressor genes help prevent uncontrolled cell growth • Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset (loss-of-function, defect on two copies) • Tumor-suppressor proteins: Repair damaged DNA, Control cell adhesion, inhibit the cell cycle in the cell-signaling pathway • Inhibit cell division, promote apoptosis, inhibit immortality, inhibit angiogenesis, inhibit metastasis • P53 - mutation leads to no activation -> goes to S phase, RB - inhibits E2F in S phase, prevents cell into S phase. If mutated, cells proliferate unlimited Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division, Ras: oncogene, GTP-bound state of Ras is the "on" state, and the GDP-bound state is the "off" state. Involved in signaling transduction Mutations in the p53 gene prevent suppression of the cell cycle. p53: tumor suppressor Loss of tumor suppressor gene APC -> activation of ras oncogene -> loss of tumor suppressor gene p53 -> invasion of surrounding tissue and blood vessel -> metastasis Cancerous cell usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes
Distinguish among substitution, insertion, and deletion mutations.
Point mutations Substitution - change of a nucleotide or few nucleotides from one type to another Transition - Pyrimidine to a pyrimidine o Cytosine <-> thymine (cytosine methylated first, deamination removes amino group to convert cytosine to thymine) Most often occurs - Purine to a purine o Adenine <-> guanine Transversion - Purine to pyrimidine - Pyrimidine to purine Through DNA replication Insertion - an insertion of one or few nucleotides in the DNA sequence Deletion - a deletion of one or few nucleotides in the DNA sequence
Outline the effects of the acetylation and deacetylation of histone tails on chromatin structure and gene transcription
Post transition modification lysine acetylation has 2 functions - neutralize charge to reduce affinity of tail for DNA, acetylated lysine serves as docking site for BRD which is needed for RNA pol II to stimulate transcription of eukaryotic cells Nucleosome core wrapped by DNA, connected by linkers and histone H1 (contributes to formation of chromosome) Reversible acetylation and gene expression -Lysine bears a positively charged ammonium group at neutral pH. (acetylation occurs on lysine bears of the pair of histone) -The addition of an acetyl group neutralizes the ammonium group to an amide group while adding a negative charge. HAT: histone acetyltransferase HDAC: deacetylase 2 functions 1. This charge neutralization dramatically reduces the affinity of the tail for DNA and decreases the affinity of the entire histone complex for DNA, loosening the histone complex from the DNA. 2. lysine serves as a docking site Bromodomain specifically recognizes the acetylated lysine - BRD serves as docking site for unique motif - Recognizes lysine - Help create/activate RNA pol II -> activates transcription - BRDi - Acetylation occurs on lysine (pos charged AA) becomes neutralized acetylated lysine - Mediated by histone acetyltransferase (HAT) - Also makes closed chromatin -> open chromatin which allows gene expression - Deacetylase can help remove acetylated lysine (HDAC) - Makes open chromatin -> closed chromatin - Changes structures and control gene expression - Postranslational modification of histones - Epigenetic writer - Chromatin enzymes responsible for the deposition of covalent modifications on histones and DNA - Helps modification of tail - Epigenetic eraser - Remove acetyl group from lysine - Enzymes that catalyze the removal of the covalent modifications on histones and DNA - Most reversible - Epigenetic reader Proteins with specialized binding domains that recognize and bind to covalent modifications of histones and DNA
where does gene expression occur in prokaryotes and eukaryotes?
Prokaryotes - gene expression occurs at transcription Eukaryotes - gene expression occurs pre/post and at transcription
Describe common post-translational modifications found in proteins and on what amino acid side chains they occur.
Protein Modifications Phosphorylation - (Tyr, Ser, Thr) Hydroxylation - (Proline) Glycosylation - (O-linked as with Ser/Threo- OH or N-Linked as in asparagine) ubiquitination (occurs on lysine) etc. Important in function of certain polypeptides Histone tails modifications - Me is repressive, ac is active
Outline the processes of protein synthesis: i) Initiation, ii) elongation, and iii) termination in eukaryotes and understand the difference of initiation between prokaryotic and eukaryotic cells.
Protein synthesis consists of three parts: initiation, elongation and termination. Initiation requires the cooperation of ribosomes, tRNA, mRNA, and proteins called initiation factors. There are three tRNA binding sites on the ribosome. The A (aminoacyl) site binds the incoming tRNA. (amino acid site) The P (peptidyl) site binds the tRNA with the growing peptide chain. (associated with tRNA) The E (exit) site binds the uncharged tRNA before it leaves the ribosome. (empty, uncharged tRNA position) The acceptor end of the tRNAs in the A site and P site are near one another at a site on the 50S subunit where the peptide bond is formed. Major differences initiatior - prokaryotic (fMet - tRNA), eukaryotes Met-tRNA Site selection - prokaryote shine-dalgarno mediatied internal initiation, eukaryote - 1) scanning 2) IRES mediated internal entry Order of events - prokaryote 1) mRNA binding 2) fMet-tRNAi binding, eukayote 1) Met-tRNA, binding 2) mRNA binding Toxins prok - resistant euk - sensitive
Describe the principles of western blot and ELISA
Proteins on an SDS-polyacrylamide gel are transferred to a polymer sheet and stained with fluorescent antibody. The fluorescent antibody is excited by light, and the band corresponding to the protein to which the antibody binds is visualized with an appropriate detector. HPR: Horseradish peroxidase Proteins Can Be Detected and Quantified with the Use of an Enzyme-Linked Immunosorbent Assay (ELISA) (covid rapid test) 1) Blood samples containing Covid-19 viral proteins (Spike) - Code for antibody - Antibody recognize spike - Add antigen (blood sample) - If antigen contains spike proteins, it interacts with antibodies - Secondary antibody recognizes antigen - Helps to visual complex formations, detect is antigen sticks 2) Blood samples containing antibodies against Covid-19 proteins the production of color indicates the quantity of antigen. - Code antigen - Run sample - If sample has antibody, antibody recognizes antigen - Secondary antibody is enzyme link which recognizes primary antibody and conformation occurs after adding substrate - Detect whether patient contains antibodies - Production of color indicates the quantity of antigen Uses antibodies instead of probes which recognizes proteins of interest Identifies expression of certain proteins
Understand the principles of PCR reaction and its applications and the unique feature of Taq DNA polymerase.
Quantify mRNA synthesized from samples Requires reverse transcription to convert RNA to cDNA Visualize PCR produce with SYBR Green dye 1. Increase temperature to denature DNA 2. reduce temp for anneal primer 3. increase temp to extend primer Taq DNA polymerase - heat resistant enzyme (hot springs, heat resistance, doesn't denature) allows DNA replication to occur in PCR Application - SARS COV2 Applications - DNA fingerprint - Classification of organisms - Pre-natal diagnosis - Detection of pathogens COVID Human genome project
Understand that targeting sequences/signal sequences act to facilitate protein targeting to specific subcellular or extracellular locations. How is SRP involved in this targeting?
RER Rough endoplasmic reticulum SRP: Signal recognition particle 1) inhibits translation after binding in cytosome 2) docks the ribosome on the ERE then peptide synthesis continues and signal peptidase is from SRP, stay in ERE lumen andm makes different types of modifications and eventually transferred to golgi apparatus. Translation of protein begins in the cytosol. As the signal peptide emerges from the ribosome, an SRP (signal-recognition particle) binds to it and to the ribosome and inhibits further synthesis of protein. SRP binds to its receptor in the RER membrane, docking the ribosome on the RER. The SRP is released and protein synthesis resumes. As the signal peptide moves through a pore into the RER, a signal peptidase removes the signal peptide. Synthesis of the nascent protein continues, and the completed protein is released into the lumen of the RER . Each protein synthesized on the RER is destined for one of the following targets: 1) RER resident; 2) Golgi resident; 3) plasma membrane resident; 3) endosomes/lysosomes; 4) the extracellular space (secreted) 1) Proteins synthesized on the RER travel in vesicles to the cis face of the Golgi complex. 2) After the membranes fuse, proteins enter the Golgi complex and bud from the trans face of the Golgi complex in vesicles. 3) The vesicles may become lysosomes or secretory vesicles depending on their contents. 4) Secretory proteins are released from the cell when the secretory vesicles fuse with the cell membrane. 5) Proteins with hydrophobic regions embedded in the membrane of the secretory vesicles may become cell membrane proteins
what are the different RNA polymerases
RNA pol I - rRNA RNA pol II - mRNA RNA pol III - tRNA
Describe the utility of restriction endonucleases in the analysis of DNA. What is unique for the sequences recognized by restriction endonucleases?
Restriction endonucleases - Cleaves virus - Unique feature is they recognize palindrome sequence - A palindrome o When read in the 5' -> 3' direction, the sequence on the top strand is identical to that of the bottom strand going 5'-> 3' o Break down of phosphodiester bond o TaqI - four base, cohesive ends § Creates sticky ends o Hae III: four base, blunt ends § Creates blunt ends
Describe how recombinant DNA technology is used to clone and express genes
Restriction enzymes and DNA ligase are key tools for forming recombinant DNA molecules Reverse transcriptase is important in technology complementary DNA (cDNA) is created from mRNA - cDNA vs normal DNA - cDNA does not have intron or regulatory regions 2 unique closing vectors - need antibiotic resistance (produce protein) - one restriction enzyme site (help create large amounts of interest DNA)
Understand the rationale and the application of reverse transcriptase in recombinant DNA biology.
Reverse transcriptase - popular way to synthesize and obtain DNA / recombinant DNA. Very important to get cDNA. Attachments of covid 19 - Isolate mRNA from pancreas - Reverse transcriptase with mRNA as template - Create cDNA - Create hybrid DNA plasmid - Create large amounts of cDNA, can be used to create recombinant A complementary DNA (cDNA) duplex is created from mRNA by using reverse transcriptase to synthesize a cDNA strand. 1) cDNA does not contain introns because mRNA, which has no introns, is used as a template. 2) cDNA also lacks the regulatory regions of a gene, as those sequences (promoter, promoter-proximal elements, and enhancers) are not transcribed into mRNA. Chemical synthesis of DNA - Machine can synthesize - Way to understand and study covid 19 virus - After covid 19 sequence published, 14 companies requested the viral genomes - Less than 3 weeks, 12 types of covid segments received - Able to synthesize virus RNA - Covid genome size is small - SARS CoV2 encodes 29 proteins o Spike protein is very important o Attacks human cells Dna cloning Cloning vectors: used to accommodate the DNA of interest 1. Plasmids - most commonly used vector to clone DNA 2. Bacteriophage lambda 3. Yeast artificial chromosomes 4. Mammalian viruses - All vectors carry antibiotic resistance genes - All vectors have at least one restriction enzyme sites Allow insertion of gene of interest into vector -Autonomously replicate within a host cell Amplification Circular, double-stranded DNA containing antibiotic resistance genes is cleaved by a restriction Enzyme (for example, EcoR I) - Need plasmid vector and fragment of DNA of interest (SARS-cov-2, spike) - Cut plasmid and covid with restriction enzyme (EcoR I) - Ligate relinks and inserts to plasmid vector makes chimeric plasmid with insert of DNA interest fragment - Transforms plasmid to bacterial cells - Grows bacterial cells in medium that contains antibiotics, plasmid carrying bacteria will amplify and grow To obtain DNA - Can isolate plasmids - Cut plasmid DNA - Makes cloned DNA To obtain Protein - isolate protein by bacteria to get large sars cov 2 spike protein - can use as an antigen to create antibodies 2. DNA to be amplified is cleaved by a restriction enzyme (the same enzyme as that used on the plasmid, thus producing complementary sticky ends) 3. DNA ligase covalently joins DNA fragments of plasmid and DNA ofinterest, producing a recombinant DNA molecule (hybrid plasmid) 4. Recombinant DNA can be introduced into the host by transformation or by viral infection 5. Bacteria are grown in the presence of antibiotics, thus selecting for cells containing the hybrid plasmids, which provide antibiotic resistance
Understand the difference between alkylating reagents and intercalating agents for their abilities to damage DNA
Several hydrophobic molecules containing flat aromatic and fused heterocyclic rings can insert between the stacked base pairs of DNA. These molecules called intercalating agents. Intercalating agents are potential Cancer-inducing reagents. Alkylating modifies N containing base Ethidium bromide, Acridine orange, Actinomycin D (cancer causing reagents) - Use hydrophobicity of molecules, stacking of double helix Intercalating Agents are different from Alkylating reagents - Both bind to DNA, cure cancer cells, blocks DNA replication Bind with different mechanisms
Describe how the antibiotics streptomycin, tetracycline, chloramphenicol, and erythromycin inhibit prokaryotic protein synthesis and describe ways in which resistance to these antibiotics can arise.
Streptomycin - interferes with binding of fMet-tRNA Tetracyclines: interact with small ribosomal subunits, blocking access of the aminoacyl-tRNA to the mRNA-ribosome complex (prokaryotes) Chloramphenicol: inhibits prokaryotic peptidyltransferas. High level: Mitochondria protein synthesis. Diphtheria Toxin: inactivates translocation by inhibiting eEF-2 (eukaryotes) Ricin: inactivates ribosomes by depurination of 28S rRNA (peptidyltransferase) in 60S ribosomal subunit Antibiotic resistance Three mechanisms of resistance to chloramphenicol are known: • Reduced membrane permeability • Mutation of the 50S ribosomal subunit • Expression of chloramphenicol acetyltransferase, which inactivates chloramphenicol by acetylating it. Chloramphenicol reduce intake of drug, antibiotic resistance The primary means of bacterial resistance to macrolides (Erythromycin) occurs by post-transcriptional methylation of the 23S bacterial ribosomal RNA. Clindamycin and erythromycin: Bind to a site on the 50s subunit of the bacterial ribosome, thus inhibiting translocation (prokaryotes) Erythromycin binds to the 50s subunit of the bacterial rRNA complex, preventing the transfer of the tRNA bound at the A site of the rRNA complex to the P site of the rRNA complex. Can reduce permeability, increase efflux pumps, modify antibiotic target cell, inactivates antibiotics, antibiotic modification
How does the iron level regulate the stability of mRNA of transferring receptor and the translation of ferritin?
TfR is receptor to transferrin protein (REGULATES STABILITY OF mRNA), Ferritin is regulates on translation Know 2 types of proteins synthesis Use it to absorb iron... when iron level is low, IRE binds to 3' end and stabilizes for transferrin receptor up to absorb more iron 3' end maintains stability of end... IRE located here Due to mRNA stability Ferritin is a protein that stores iron, releasing it when the body needs it. (storage) (relates to translation) IRE is located at 5', removes IRE-BP IRE conserved as template for protein synthesis to help synthesize more ferritin Production of the transferrin receptor (TfR) and ferritin is regulated at the level of mRNA by iron regulatory proteins (IRPs), which bind to iron response elements (IREs) on the 3'- (TfR) and 5'- (ferritin) untranslated regions of their respective mRNAs. Binding of IRPs to the IRE of TfR on the 3' end of mRNA stabilizes the mRNA, facilitating the translation. - Binding of IRPs to the IRE of ferritin on the 5' end of mRNA inhibits the translation. Due to protein synthesis Production of the transferrin receptor (TfR) and ferritin is regulated at the level of mRNA by iron regulatory proteins (IRPs), which bind to iron response elements (IREs) on the 3'- (TfR) and 5'- (ferritin) untranslated regions of their respective mRNAs.
Explain how some codons are recognized by more than one anticodon. List the base-pairing interactions allowed according to the wobble hypothesis.
The anticodon loop contains a sequence complimentary to the corresponding codon in a mRNA; some tRNAs can read more than one codon as first proposed by the "Wobble Hypothesis". - UAC paired with AUG - tRNA starting from 5' - >3' in anticodon, first base in anticodon (right side) pairs with third base in codon (left side) - Wobble - if first base of anticodon is C, pairs with G, A with U, U with A or G, G with U or C, I with U C or A - Why multiple genetic codes - tRNA carries amino acid to site of ribosome and follows pairing - pairing of first base in anticodon, pairing of third base in codon of mRNA, wobble comes from this pairing - don't really follow AT GC pairing Generalizations of the codon-anticodon interactions are: Codons that differ in either of the first two nucleotides must be recognized by different tRNA. 2. The first base of the anticodon determines the degree of wobble. If the first base is inosine, the anticodon can recognize three codons
Outline the role of RNA secondary structure in the regulation of iron metabolism in animals. Describe the roles of transferrin, transferrin receptor, ferritin, the iron response element (IRE), and the IRE-binding protein
Transferrin - regulation of mRNA stability Ferritin - regulation of initiation of translation (binds to 5'end of mRNA)
Diagram the structure of a "typical" human protein-coding gene, including upstream sequences, promoter, 5'UTR, transcription start site, coding sequences, introns, 3'UTR, polyadenylation site.
UTR: untranslated region 1) promoter 4) determines termination of transcription... tells wheres RNA pol synthesis should stop 6) exon coding sequence for RNA 7) intron cannot be used as template, not coding sequence Primary RNA transcript contains starting site 5'UTR, exon, intron, 3'UTR Goes through RNA processing (post-transcription modification) removes intron RNA -> mature mRNA which serves as template for protein synthesis, adds poly-A tail and 5' cap Enhancer either upstream or downstream Need post transcriptional modification 5' capping 3' poly a tail and removal of intron - tRNA modification is removal of 16 nucleotide on 5' endby RNase P
Relate the 3′ → 5′ nuclease activity of DNA polymerases to the fidelity of DNA replication
fidelity of a DNA polymerase refers to its ability to accurately replicate a template DNA polymerase III has two activities: 1) 5' 3' polymerase activity, DNA synthesis 2) 3' 5' exonuclease activity, proofreading
Understand how ubiquitination leads to protein degradation
mechanism to deliver proteins to 26S proteasome for degradation - Important in neurodegenerative diseases (proteasome) o If proteins cannot degrade properly it causes § Alzheimers § Parkinsons § Huntingtons
Explain how topoisomerase/gyrase inhibitors can act as antibacterial agents (Quinolones, ciprofloxacin) and anticancer (Camptothecins, indenoisoquinolines) agents/side effects.
o DNA topoisomerase (type 1 (strand), type 2 (loop) remove supercoil of DNA double strand) § bond covalently to the DNA phosphate as they break the phosphodiester linkage between neighboring nucleotides, storing the energy in that bond to use in the process of resealing the bond. § Antimicrobial targets specific · Ciprofloxacin: DNA gyrase (topoisomerase II) · Quinolones: topoisomerase II
Describe the principles and applications of Northern blot, Southern blot, Western blot.
o Southern blot § Use DNA probe § Single DNA § Detect DNA o Northern blot § Separate RNA § Single DNA § Transfer to membrane § Use DNA probe § Detect RNA of interest o Western blot § Use antibody probe § Detect protein of interest § Need to transfer the proteins into membrane § Common to test proteins in cells
Describe the cis-acting elements and trans-acting factors involved in eukaryotic transcription. Understand the difference between promoter and enhancer.
promoter - upstream of transcription start site (transcription factors) enhancer - upstream or downstream of promoter (cis-acting) Cis acting element - dna nucleotide sequence Trans - protein transcription factors - Important regulation is associated with promoter and enhancer (transcription factors) - General transcription factor (promoter) responsible for positioning of RNA polymerase on promoter - Trans activator associated with enhancer helps for larges complex - Regulatory DNA binding proteins and basal transcription complex are transcription factors o Oncogenes - RARa, c-myc, Fos, jun (activates genes in cell proliferation and cell survival) o Tumor suppressors - p53, RUNX3 Cis-acting elements - Promotor is always upstream of transcription starting site - Enhancer located up or downstream Both recognized by transcription factor (proteins) - Bind to promotor - basal transcription complex - Bind to enhancer - specific transcription factors
How does DNA methylation contribute to gene silencing and cancer?
tumor suppressor is inactivated by methylation C->T (most common) and hyper methylation on promoter region leads to development of cancer DNA methylation can cause long-term inactivation of genes in cellular differentiation. - If tumor suppressors are silenced, then cell loses tumor suppressor activity and cell converted to cancer cells - 2 mechanisms associated with inactivation of tumor suppressors and linked with development of cancer - 1. meCpG (methylated C followed by G) (C->T) - deamination - base alteration - becomes CpG->TpG mutation - causes tumor suppressor inactivation - 2. hyper activation of promoter - promoter silencing - causes tumor suppressor inactive - Methylation represses gene activation? Methylation specifically occurs on CYTOSINE Cytosine is followed by guanine 2 mechanisms - deamination (C->T mutation), 1) DNA methylation occurs mainly at the C5 position of CpG dinucleotides 2) DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species. 3) DNA methylation can cause long-term inactivation of genes in cellular differentiation. DNMT: DNA methyltransferase
Explain how Xeroderma pigmentosum is an autosomal recessive genetic disorder of DNA repair in which nucleotide excision repair is deficient and causes extreme sensitivity to ultraviolet (UV) light
• A rare autosomal recessive disease due to the deficiency of excinuclease or DNA polymerase n • Associated with thymine dimer damage
What is the difference between pro-carcinogen and carcinogen? How to identify carcinogenic agents (Ames test)?
• Carcinogens: agents that can cause cancer directly (e.g., radiation, chemicals) • Ability to react with DNA which cause mutations in DNA • Procarcinogen is a precursor of carcinogen. Turn into carcinogen in the body. • Can be converted to carcinogen which changes DNA • 80% of cancer cases caused by environmental & food carcinogens • Usually damage DNA, likely to induce SOS response (cell cycle arrest) • High correlation between mutagenesis and carcinogenesis (carcinogens act as mutagens). Ames Test-assays for carcinogenicity • Salmonella his-- strain: must be grown in the presence of Histidine, and the mutant allele is susceptible to back-mutation to a wild-type allele. • Many non-carcinogens are converted to carcinogens in liver. • Small amount of rat liver is added to Ames test to mimic mammalian metabolism. • Add rat liver lysates to provide cytochrome p450 to create alkylating reagents to see cancer - more colonies -> carcinogen
What is a ribozyme? Can you give an example for its function?
• Ribozymes are catalytic RNAs that function as enzymes and do not require proteins for catalysis • Binds to rna and uses activity to break down phosphodiester bonds • In nature ribozymes occur mostly within self-splicing introns and RNA encoded parasites - satellites and viroids • The catalyzed reactions in naturally occuring ribozymes are limited to cleavage and ligation of RNA
Contrast ρ-dependent and ρ-independent transcription termination. Outline the mechanisms of the ρ protein and explain the role of ATP hydrolysis in its function.
• Termination: The RNA polymerase stops moving on the DNA template. The RNA transcript falls off from the transcription complex • 1) Rho-independent (intrinsic termination): RNA transcript forms a stable hairpin turn (stem-loop); RNA transcripts contain a string of U's • Determined by structure of newly synthesized RNA • 2) Rho-dependent: p factor binds to a C-rich region (~70 nt); contains helicase activity (energy from ATP hydrolysis) unwind the 3'-end of the transcript from template; displace the DNA template strand, • Recognize certain sequence in newly synthesized RNA • Breaks H bonds since it has helicase activity to separate rna from dna template and stops replication (hydrolysis provides ATP to occur)
Describe how siRNAs are being used to easily perform "gene knockout" experiments
• microRNA: single-strand RNA some matched, more than one target non-coding region (3'-UTR). • A mechanism for regulation of gene expression. • siRNA: double-strand RNA perfectly matched, only targets at mRNA (coding region) • a natural antiviral defense in plants, fungi, and invertebrates let-7 is miRNA, inhibits expression of oncogene... cancer treatment HIV Huntington's disease Covid-19
