MCB 250 EXAM III STUDY GUIDE
MCB 250 EXAM III STUDY GUIDE MCB 250
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This 17 page Study Guide was uploaded by Jessica Logner on Sunday May 15, 2016. The Study Guide belongs to MCB 250 at University of Illinois at Urbana-Champaign taught by Kirchner, N in Spring 2016. Since its upload, it has received 4 views.
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Date Created: 05/15/16
MCB 250 EXAM III central dogma DNA, RNA, Protein Mendel used purebreeding lines, used brush to transfer pollen, two clearly distinguishable forms of peas monohybrid cross two traits; round peas and wrinkled peas F2 generation, monohybrid cross 3:1 ratio Archibald Garrod first to propose specific disease could result of a heritable deficiency homologous chromosomes chromosome from mom and dad, humans has 23 distinct pairs alleles multiple forms of genes, RR or rr mitosis sister chromatids break apart, 2n dihybrid cross 9:3:3:1, both traits dominant, one trait dominant other recessive, both recessive tratis independent assortment occurs in meiosis, when the maternal and paternal copies of each chromosome homologous recombination when chromosomes will cross over eachother and switch parts of the genetic information herman muller discovered exposing living organisms to x rays have high rate of heritable mutations forward genetics new way of studying genes, mutant phenotype, find the gene, sequence DNA mutagenesis inducing mutations in organisms homologous recombination DNA repair, nearly identical, meiosis in eukaryotes, double strand break model break occurs, processed to create 3' tails, 3' strand invades homologous chromosome and is replicated by PolI to create holiday junctions collapsed replication fork repaired by homologous recombination, uses 5' end processing REC BCD helicase/nuclease that binds to a double stranded break helicase unwinds and nuclease degrades both strands, creates single strand 3' end helps load Rec A, once hits a chi site, will stop degrading the molecule RecA forms filament on a single strand of DNA, mediates strand invasion and is NECESSARY for all pathways, form a 5' to 3' filament, RecA and ATP necessary to change the conformation of Rec A to leave the reaction RecA is required for all homologous recombination mutants do not do homologous recombination, conserved through evolution, requires at least 50bp of homology RuvAB responsible for migration of holliday junctions, RuvA recognizes and binds to holliday junction, RuvB is an ATPdependent helicase RuvC cuts the holliday junction randomly, determining a orientation of if there is a crossover site specific recombination short sequences in both donor and recipient and special enzymes can direct recombination with little to no homogology transposition uses transposons to cut out one piece of DNA for another, without requiring homology illegitimate recombination very low frequency recombination between nonhomologous sequences nonhomologous end joining can repair DSBs without needing an intact copy, cannot repair a break perfectly , be used in nonproliferating cells nonhomologous end joining process Ku recognizes the DSB, DNAPKcs binds to recuir Artemis to protect ends to potentially be joined back together DNA cloning DNA molecule of interest moved into DNA vector, transformed into bacteria, propogated at will DNA library mixture of DNA sequences from a common source are cloned into a large # of bacterial cells, used to reconstruct basepair sequence of the entire genome, introns still present reverse genetics genome first sequenced, gene is found, phenotype is discovered open reading frame where the sequence will be translated into proteins cDNA DNA molecule synthesized by the reverse traanscription of RNA, isolate total processed mRNA from the cell, then convert back to DNA with reverse transcriptase reverse transcriptase DNA polymerase that uses RNA as the template cDNA library contains DNA products without the introns that can be cloned into plasmid or other DNA vectors, contains only sequences to make that particular tissue isoforms multiple forms after RNA splicing grind and find grind up a tissue sample, then homogenate and probe for the prescence of RNAs or proteins northern blot mixed RNAs bound to physical substrate and allowed to hybridize with a labeled nucleic acid probe DNA microarrray need cloned DNA library, spots of DNA in an orderly pattern on a glass slide, use cDNAs with labels to see where they will glow on the microarray transcriptome the entire set of RNAs being synthesized by a cell or tissue at a given time in situ hybridization place labeled nucleic acid prove to hybridize with a certain sequence in a cell, wash out to visualize the labeled cells antisense probe the complementary sequence that will be created to bind to the DNA in in situ hybridization, preferred to be RNA immunohistochemistry using proteins labeled with antibodies constitutive expression genes produced at essentially a constant level under all conditions facultative gene expression expression of many genes is actively regulated basal transcription the RNA polymerase can act by itself to recognize promoters and initiate transcription transcription factors sequencespecific DNAbinding proteins that regulate transcription core promoter the specific sequence that RNA polymerase will bind to promoter the entire regulatory region of the gene 3 themes in the function of DNA binding proteins sequencespecific binding (specificity), noncovalent bonds, chemical affinity for strength purposes electrophoretic mobilityshirt assay (EMSA) bound proteins on DNA vs. unbound DNA on a gel, if unbound will travel the normal length as that DNA proteins will connect to the major groove of DNA, but will not disrupt the hydrogen bonds holding the bases together DNAbinding protein vs. DNAbinding site the amount of binding protein will always be changing where the binding site is fairly constant, the more protein, the more likely binding site will have a protein activated level of transcription there is an activator molecule to help the RNA polymerase bind to the core promoter most efficient for growth in E coli. glucose, a simple sugar that does not need to be broken down further lactose permease membrane transport protein that allows lactose to enter the cell, lacY beta galactosidase enzyme that cleaves the disaccharide lactose into glucose and galactose, lacZ thiogalactose transacetylase enzyme that inactivates certain toxic sugars that can enter the cell through lactose permease, lacA lacI gene is not part of the lac operon, constitutively active, the lacI protein binds to lacO and acts as a repressor, helixturnhelix structural motif how lacI protein binds uses two helixturnhelix molecules, look like a mirror image of one another (opposite orientations), with the recognition helix in the major groove, creates an inverted repeat inverted repeat when two lacI protein are bound to lacO, create two symmetric halfsites Jacob and Monod model when lactose is present, an inducer molecule binds to the lacI repressor protein and prevent it from binding to DNA allostery protein function can be turned on or off by a ligand, ligand binding site is separate from the active site of the protein, can reduce or influence chemical functions allolactose when lactose is passed through the membrane, some is converted to the isomer, allolactose, binds to the lacI protein lac operon promoter weak promoter, needs help to have the RNA polymerase bind catabolite activator protein CAP when cap protein is attached to the cap site upstream of the promoter, the alpha subunit can recognize and attach there, increases the affinity of the RNA polymerase for the promoter CAP vs. glucose expressed constitutively, the affinity for CAP binding to the site is controlled by glucose concentration( lack of glucose) cAMP with CAP will bind to CAP and the dimer willl undergo a conformation change that rotates its recognition by 90 degrees, as glucose levels decrease, amount of cAMP will increase, recognition helices must be parallel to one another both lactose and glucose present neither lacI or CAP binds, the operon experiences basal transcription in cis must be on the same DNA molecule to interact in trans two elements can interact when located on the same or different DNA molecules, CAP lacI is a tetramer it can bind two different operator sites simultaneously, lac operon has three operator sites 500 bp away from one another, fewer operators, less transcription two means of regulation in bacteria modifying the structure of RNA polymerase, placement of proteins on binding sites and their functions heat shock proteins proteins created when there is a heat shock, molecular chaperones sigma 32 is used when the temperature is raised to 42 degress celsius, the protein is degraded less quickly, determined by posttranscriptional regulation, usually at a lower temperature the secondary structure will block translation of sigma 32, but the heat erases those structures and allows it to be translated cro key gene for lysis cI key gene for lysogeny Pr will be the first promoter expressed, leads to expression of cro gene, if not stopped, the cell will go into the lytic cycle, also expresses CII gene Pre gene will be expressed if cII is created, then will begin producing cI which will translate cI, which will lead to lysogeny Cro protein has most affinity for... the OR3 operator, which will block the production of cI, RNA has to transcribe the opposite away, creating more of the cro protein cI protein has the highest affinity for... OR1 (dimer), and then can bind to OR2 (tetramer), increasing the affinity for RNA polymerase to bind and create more cI cI repressor protein structure nterminal region to bind to DNA and activating region, and a cterminal region that has domains for tetramerization and dimerization autoregulation when cI recruits RNA polymerase to influence its own synthesis FtsH when an E. coli cell is healthy, this is created to degrade the cII protein and gives Cro the upperhand favoring lysis no FtsH when the bacterium is infected there is relatively little of this protein, so this favors lysogeny lambda integrase used to place phage DNA into the bacterial chromosome, lysogeny conservative sitespecific recombination aligns the two recognition sequences on phage DNA and bacteria DNA, enzyme then recombines two sequences at crossover region, both break and recombine into a single chromosome, can occur ANYWHERE in two regions that share similarity when lambda is integrated into the bacterial chromosome... the phage is silenced except for the cI gene, maintains lysogeny and the Cro from being expressed SOS response in lysogenic cycle uses RecA to excise lambda from the bacterial chromosome and begin lysogeny, recA destroys the cI protein excess cI protein can bind to pL, OL1 and OL2, and can connect to OR1 and OR2 and then OL3 and OR3 can bind together to create a tetramer and cease translation of cI, and Cro is still ceased as well general transcription factor needed in eukaryotes to begin transcription with RNA polymerase (TATA box) enhancers eukaryotes, proteins can bind here to enhance transcription rates, one enhancer usually has binding sits for multiple transcription factor proteins mediator complex eukaryotes, decide whether transcription should occur based on regulartory proteins present, signal to pol II to escape the promoter and elongate, transfers signals to pol II DNAbinding domain on a transcription factor and binds to a DNA sequence activation domain on a transcription factor to interact with mediator domain to communicate the RNA polymerase fusion gene?? ... homeodomain protein alpha helix, version of helixturnhelix but in eukaryotes, can bind in monomers or dimers basic helixloophelix protein eukaryotic transcription factor, bind as dimers, alpha helix basic leucine zipper protein backbone is a dimerized leucine zipper, alpha helix that inserts into the major groove of DNA, eukaryotic transcription factor zinc finger protein eukaryotic transcription factor, uses Zn2+ to read the major groove, binds as monomers or dimers chromatin remodeling complex will displace the nucleosome away from the promoter histone acetyltransferase will add a acetyl group to a lysine sidechain on the Nterminal histone tail, will take away the positive charge, causes diassembly of the histone, increasing accessibility of DNA methylation of histone tails will cause the histones to coil more maternal gene products RNA and proteins contained within the egg produced by the mother's genes Macho1 a mother's gene in a sea squirt embryo, cells will have different concentrations of this gene (encodes for a zinc finger), 3' UTR will connect to motor proteins to move around the cell cell to cell interactions contain a signal and receptor, ligand which will connect into receptor signal transduction pathway ligand is detected on outside of the cell, activates sequence of biochemical event on the inside of the cell to begin transcription somewhere JAKSTAT pathway cytokine moves into receptor that dimerizes and is bound to JAK, it will phosphorylate to serve as binding sites for STAT (which will be phosphorylated), STAT functions as a transcription factor receptor autophosphorylation when the protein can phosphorylate the other protein and the other receptor subunit on a ligand receptor eukaryotic chromosomes can loop to.. bring transcription factors bound at distant enhancers close to the transcription units they regulate insulator sequences stops one enhancer to being used on another promoter, without other genes can be transcribed that aren't meant to be Homologous Recombination (Meiosis) Homologous recombination. Crossing over between maternal and paternal chromosomes to increase genetic diversity Homologous Recombination (DSBs) Homologous recombination. Repairs double stranded breaks in chromosomal DNA RecB Homologous recombination. Has 3'>5' helicase and nuclease activity and aids in generating a 3' overhang for strand invasion. Helicase unwinds the DNA, nuclease degrades both strands, but the 3' end is shortened more slowly RecD Homologous recombination. Has 5'>3' helicase activity and aids in generating a 3' overhang for strand invasion. Helicase unwinds the DNA RecC Homologous recombination. Recognizes the Chi site Chi Site Homologous recombination. When RecBCD encounters the Chi site, it stops degrading the 3' strand altogether and becomes more active in degrading the 5' strand RecA Homologous recombination. DNAdependent ATPase, initiates strand invasion and duplex formation by using ATP hydrolysis to stretch DNA and enhance recognition and strand exchange RuvAB Homologous recombination. Responsible for branch migration of Holliday Junctions RuvA Homologous recombination. Recognizes and binds to the Holliday Junction RuvB Homologous recombination. ATPdependent helicase unwinds the DNA purebreeding If a purebreeding individual is mated to itself, all of its descendants will share the same trait homozygous RR, rr heterozygous Rr recombinant DNA Cloning. A DNA sequence of interest is covalently ligated into a DNA vector PCR (primers) Cloning...sort of. Experimenters synthesize a DNA oligonucleotide that base pairs with the template strand and can act as a primer for strand elongation cDNA production Purify RNA from tissue. Anneal primers. Reverse transcribe expression vectors require... A bacterial promoter, a ribosomebinding site, and a transcriptional terminator Northern blot Molecular analysis of gene expression. RNA. RNA is physically separated by gel electrophoresis, blotted onto paper, hybridization by a complementary nucleic acid probe allows for detection DNA microarray Molecular analysis of gene expression. RNA. A spotted glass slide, each spot containing copies of ssDNA. Labeled cDNA probes from mRNA in specific tissue samples bind spots at different levels, showing different levels of gene transcription in the sample in situ hybridization Molecular analysis of gene expression. RNA. Labeled probes are inserted into fixed and permeabilized cells. Unhybridized probes are washed out constitutive gene expression Typical of housekeeping genes essential for survival facultative gene expression Typical of gene products only required under specific conditions basal transcription Recognition of promoter DNA and initiation of transcription by RNA polymerase alone EMSA Electrophoretic mobilityshift assay. Measures protein:DNA binding experimentally. Gel electrophoresis shows bound protein:DNA as two bands (because not all DNA will be bound), unbound protein and DNA as one lactose permease Lac operon. Membrane transport protein that allows lactose to enter the cell Betagalactosidase Lac operon. Enzyme that cleaves dissacharide lactose thiogalactoside transacetylase Lac operon. Enzyme that inactivates certain toxic sugars that can enter the cell through lactose permease lacZ Lac operon. Gene that ultimately leads to Betagalactosidase lacY Lac operon. Gene that ultimately leads to lactose permease lacA Lac operon. Gene that ultimately leads to thiogalactoside transacetylase lacI Not part of the lac operon. Has its own promoter. Ultimately leads to the lacI protein lacI protein Lac repressor. Binds to lacO and represses transcription of the lac operon lacO Lac operator adjacent to the lac promoter. Binding by lacI blocks transcription allostery Protein function is turned on or off by the binding of a particular ligand molecule allolactose Isomer of lactose. Acts as inducer by binding lacI protein CAP Catabolite activator protein. Helixturnhelix transcription factor that allows glucose to repress lac operon transcription CAP site Upstream of the lac promoter. Binding by CAP increases the affinity of RNAP cAMP Cyclic AMP. Intracellular ligand that binds CAP dimer. Concentration is inversely proportional to glucose levels heat shock proteins Molecular chaperones that help other proteins refold properly upon disruption of covalent bonds by elevated temperatures sigma 32 Sigma factor that directs RNAP to genes that encode heat shock proteins at higher temperatures lytic cycle DNA is replicated repeatedly. Genes are used to synthesize large quantities of viral proteins. DNA and protein assemble into roughly 100 new viral particles which are released by cell lysis lysogenic cycle DNA is integrated into the bacterial chromosome and is replicated with each subsequent cell divison cro promotes... lysis cI promotes... lysogeny strong promoters (lambda phage) PL (cIII) and PR (cro, cII) weak promoters (lambda phage) PRM, PRE (cI, cIII) cro protein Transcription factor expressed from the PR promoter which can bind to the already weak PRM promoter, further repressing the cI gene cII protein Transcription factor expressed from the PR promoter that can bind next to the weak PRE promoter, activating it and leading to cI transcription OR3 Cro protein has a high affinity for OR3. When bound, cro blocks the PRM and represses cI cI protein Lambda repressor. High affinity for OR1. When bound, cI blocks the PR and represses cro cI protein affinity High affinity for OR1. Tenfold lower affinity for OR2 and OR3 cooperative binding Simultaneous binding increases the probability that both binding sites will be occupied. Found in cI autoregulation Protein influencing its own synthesis FtsH Protease found in healthy and actively dividing E. coli that degrades cII protein lambda integrase Enzyme expressed from the phage genome that inserts the phage DNA into the bacterial chromosome during lysogeny conservative sitespecific recombination Integrase unites the phage and bacterial chromosome through this mechanism autocleavage Protein selfdestruction. CI protein undergoes autocleavage in response to contact with activated RecA enhancers Functional units of multiple transcription factors mediator Large protein complex that signals RNAP to initiate transcription if an appropriate set of transcription factors is bound to gene's regulatory region insulator sequences Flank eukaryotic genes and bind a protein that acts as a barrier to prevent inappropriate enhancer:promoter interactions CTCF Protein that acts as a barrier to prevent inappropriate enhancer:promoter interactions chromatin remodeling Dynamic modifications of chromatin architecture that facilitate transcription RSC Chromatin remodeling complex. Ensures that enhancer DNA is associated with a partially unwound nucleosome SWI/snf Chromatin remodeling complex. Displaces the nucleosome that covers the core promoter and the +1 start site of target gene, exposing the DNA for assembly of general tfs acetylation of histone tails Promotes transcription of neighboring genes methylation of histone tails Inhibits transcription of the neighboring genes HAT Histone acetyltransferase. Can covalently bond an acetyl group to a lysine R group in a histone protein HDAC Histone deacetylase. Can remove acetyl group from histone protein CpG Cytosinephosphodiester bondguanine. Target for DNA methylation in mammals gene silencing Occurs when multiple sites of a CpG island are methylated epigenetic regulation Persistence of a regulatory state long after the initiating signal has vanished enhancers Functional units of multiple transcription factor binding sites grouped together
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