BIOL 434 1st editionExam #3 Study GuideLecture 1-What is the difference between RNA vs. DNA?oHas U instead of ToSingle strandedoHas a 2' hydroxyl groupoDoes intramolecular hydrogen bonding-Describe the structure of a protein.oAmino acids determine structure-Primary-Secondary - alpha helices and beta sheets-Tertiary - R group interactions; 3D space; conformation; covalent interactions-Quaternary - multiple chains coming together; ex. HemoglobinoCan change-pH-Lac repressor - two conformations-Phosphorylation, Methylation, etc.-Covalent modificationsoDNA binding proteins-DNA binding domain - region with particular functionUnstructured when unboundBinding causes to form a structureLecture 2- Compare and contrast southern and northern blot. oSouthern blot - DNADenaturing is needed to make it shorter/single stranded by restriction enzyme.Sizes of restriction fragmentsParticular genesoNorthern blot - RNADenaturing is not needed because it is also simple.oBoth run through a gel (DNA or RNA) to separate by size.oBoth transfer to a positively charged membrane. oBoth use hybridization probe after transfer.- Compare and contrast southern or northern blot and DNA microarray.oBlots look for one particular fragment (sequence).oMicroarrays look for everything.All of the RNAs, all of the genes, which are expressed.oBoth use hybridization to get complementary sequences to anneal to each other.oMicroarray comparing two tissues.Yellow is both.Red is one.oMicroarray of one color.Intensity of color. - Compare and contrast DNA cloning and PCR.oCloningEx. Plasmid vectorsHost cellOne cycleCan amplify really large fragmentsYou don't necessarily have to know the sequence.oPCRIn vitroMultiple cyclesCan amplify small fragmentsMore mistakes b/c it doesn't take long. You need to know the sequence to make the primers.- Compare and contrast cDNA library and genomic DNA library.oCollections of clones.oThe cDNA libraryMade from RNAOnly transcribed mRNAoThe Genomic DNA libraryDigested genomesEntire thing is included- Compare and contrast dBoth synthesize a DNA molecule.oHigh throughput sequencingoDideoxynucleotide sequencing- Compare and contrast cDNA microarray and tiling microarray.ocDNA starts with RNA to create cDNA through reverse transcriptaseNo introns.Only mRNA sequences are present.oTiling can tell the difference between introns and exons.Probes can detect transcripts from every gene. Has everything- Explain the purpose of using restriction enzymes during cloning experiments. oMake it shorter and easier to clone.oNot all plasmids can take up long fragments.oThe ends can be connected to a vector. o How many fragments after Ma/I cuts?oSticky ends are better at annealing than blunt ends. oBlunt ends cuts both strands at one position.oTwo sites, three fragments. - Acrylamide gel or agarose gel?oAgarose can separate larger pieces, but less resolving power.oAcrylamide gel can separate smaller pieces, higher resolution power.oConditionstRNA from total RNA -> Acrylamide to separate small piecesSeparating inserted DNA fragment from vector -> Agarose can separate big fragments.Analyzing PCR products -> Agarose because PCR products are big.- Describe how you would visualize mRNA in a northern blot, giving at least two examples of probe types.ocDNA probe; DNA complementary to RNA you want.oProbeLabeled with radioactivity or fluorescent dyeLabeled at the end (5') or internallyConsist of either DNA or RNA sequencesLecture 3- Describe ion exchange chromatography.oIf beads are more negative charged, positive proteins are retained.oNegative charge would get pushed out b/c of charge interactions.oSalt added.Weak charge would need a low salt concentration.Strong charge would need high salt concentration.oEnzymatic assay and then purification.- Describe gel filtration chromatography.oLarge proteins can only occupy spaces devoid of obstacles.oVoid volume = available space.oSmall molecules enter aqueous spaces within beads.oKeep adding buffer.oIt will take longer for smallest.oLarge molecules cannot enter beadsoOver time, you'll get smaller and smaller proteins. -What is an antibody? Polyclonal vs. monoclonal?oProtein produced by B cells during immune responseoTwo heavy chains and two light chains held by disulfide bondsoVariable region and constant regionoPolyclonal - a lot of different antibodies bind to one antigenoMonoclonal - one antibody; same antigen; more specificLecture 4-What are the methods used to examine protein-DNA and protein-RNA interactions?oEMSA-Purify binding protein-Incubate protein with DNA-Incubate DNA fragment-Load both samples onto agarose or acrylamide gel-Native gel; if you denature the protein, it won't bindoDNase footprinting-Nuclease protection footprinting-The protein can cleave up to x number of nucleotides from the end --> easier to ID-If the cleavages were happening randomly, it'd be hard to determine how far from the end the binding site is-Internally labeling shows all the fragments, rather than showing the one that is acertain distance from the label- Describe ChIP.oAntibodies can be generated to recognize specific proteinoNucleosomes on DNAoIf RNA polymerase is associated with DNA.-Describe SELEX.oInitial DNA pooloHighest affinity site-What is the SELEX Sequence Logo.oIndicates the sequence generatedoTall letter - strong preferenceoWhat's present at each positionoConsensus binding sites helps determine how close this is to the actual binding siteoTend to bind to really short sequencesLecture 5-What occurs during transcription initiation?oRNA Polymerase will bind in a closed complex to DNA on specific sequencesoClosed complex means that DNA is double strandedoDNA is melting into open complex before transcription occursoRNA polymerase will start transcribing in initial transcribing complex (still part of initiation b/c polymerase hasn't lost contact with promoter)oLong RNA elongationoRNA fragments released-What are the variations of promoters?oSome lack -35 boxoSome have extended -10-What are some termination factors?-Rho factorDepending on rho proteinBinds to RNA so that when RNA polymerase reaches termination, it slows downIt breaks hydrogen bond between DNA template and mRNASome form of disruption to RNA helicaseLess common -No factorRho independent terminator Hairpin sequence and U sequenceHairpin forms spontaneously (similar to RNA secondary