Many times as the helix unwinds resulting in many

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many times as the helix unwinds, resulting in many short fragments called “Okazaki fragments.” DNA ligase joins the Okazaki fragments together into a single DNAmolecule.Helicaseopens up the DNA at the replication fork.Single-strand binding proteinscoat the DNA around the replication fork to prevent rewinding of the DNA.Topoisomeraseworks at the region ahead of the replication fork to prevent supercoiling.Primasesynthesizes RNA primers complementary to the DNA strand.DNA polymerase IIIextends the primers, adding on to the 3' end, to make the bulk of the new DNA.RNA primers are removed and replaced with DNA byDNA polymerase I.The gaps between DNA fragments are sealed byDNA ligase.DNA replication in eukaryotesThe basics of DNA replication are similar between bacteria and eukaryotes such as humans, but there are also some differences:Eukaryotes usually have multiple linear chromosomes, each with multiple origins of replication. Humans can have up to 100,100,100,comma000000000 origins of replication^55start superscript, 5, end superscript!Most of theE. colienzymes have counterparts in eukaryotic DNA replication, but a single enzyme inE. colimay be represented by multiple enzymes ineukaryotes. For instance, there are five human DNA polymerases with important roles in replication^55start superscript, 5, end superscript.Most eukaryotic chromosomes are linear. Because of the way the lagging strand is made, some DNA is lost from the ends of linear chromosomes(thetelomeres) in each round of replication.1)DNA replication is semi-conservative and template-based.2)DNA replication requires a DNA polymerase and amechanism to initiate the start of replication.DNA helicase– unwinds DNA to make it single-stranded.Primase– a DNA-dependent RNA polymerase thatmakes “primer” needed to start DNA polymerase.DNA polymerase– makes new DNA strands.Exonuclease– removes RNA primer put in by primase.DNA ligase– reseals the gaps left in the phosphate “backbone”caused by exonuclease3)DNA polymerases are always unidirectional and mayinclude “proofreading” capabilities.
4) Short stretches of DNA can be synthesized chemically.5) PCR is DNA replication in a test tube.Key Point of today’s lecture1)DNA makes RNA makes proteinGenes are transcribed at different rates making different amounts of proteinsRNA can be self-complementary, fold up, and have 3-D structureInformation in mRNA critically depends on reading frame (3 in one codon at a time)Ribosomes are complicated RNA/protein nanomachinesTransfer RNA “decodes” codons into amino acidstRNA synthetase – enzyme that “charges” tRNA with the appropriate amino acidDesign, synthesis, and testing a 57-codon genomeThe plan: use the “degeneracy” of the genetic code
to get rid of 7 natural codonsNew LectureArticle: Biocontainment of genetically modified organisms by synthetic protein design321 UAG stop codons inE. coligenome -> converted to UAAUAG now “blank” codonintroduced bipA tRNA (with AUC anti-codon) and bipA tRNA synthetaseedited essential genes (put in UAG codons) so they incorporated bipA into essential proteinsdemomonstrated engineredE. colicould only grow if fed bipAKey Points1)The primary sequence of a protein determines its 3D structure

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Term
Fall
Professor
Beck

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