Week 13 Notes
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This 5 page Class Notes was uploaded by Cassidy Zirko on Sunday April 24, 2016. The Class Notes belongs to BCH 110 at University of Montana taught by Scott Samuels in Spring 2016. Since its upload, it has received 9 views. For similar materials see Intro Biology for Biochemist in Biology at University of Montana.
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Date Created: 04/24/16
Mechanisms of DNA Replication 4/18/16 Three phases of replication initiation, elongation, termination Bidirectional replication o Origin of replication (Starting location for replication) o DNA double helix unwinds and replication begins o Bidirectional replication: polynucleotide chains are synthesized in both directions from origin of replication o Two replications fork from each origin 4 locations where nucleotides are being added from a single site of replication Synthesis of nucleotide chains (orientation of sugar only 5’ 3’ template strand is 3’ 5’ because it is template is antiparallel The reaction o 3’OH group at end of growing DNA chain acts a nucleophile o Adjacent phosphate to sugar is attacked, then added to growing chain o Phosphate groups: dATP, dCTP, dGTP, TTP o Deoxynucelotides o Hydrogen bonds determine type of sugar o Phosphate reacts with phosphate group which is unfavorable o Using coupling reaction to drive reaction o As extra phosphates leave drives reaction forward Semidiscontinous Replication o DNA synthesis 5’ 3’ and template read from 3’5’ (antiparallel) o Leading strand is synthesized continuously 5’ to 3’ toward the replication fork o Lagging strand is synthesized discontinuously away from replication fork as okazi fragments o Leading strand synthesized as normal (5’ 3’) o Lagging strand (Template is 5’ to 3’) so it cant just replication 5’ to 3’ must be antiparallel o Okazi fragments allow replication from 3’ 5’ not continuous but fragments will eventually become stitched together creating a strand DNA Replication Machinery o DNA polymerase enzyme that catalyzed DNA synthesis o Polymerizes nucleotides into a nucleic acid o Requirements all four deoxyribonucleoside triphosphates TTP, dATP, dCTP, dGTP and magnesium o Needs primer: Short polynucleotide strand to which to growing polynucleotide chain is covalently bonded to initiate replication DNA polymerase function o DNA polymerase I: repair and patching of DNA o DNA polymerase III: responsible for the polymerase of the newly formed DNA (lowest concentration of cell) o DNA polymerase II, IV, V: proof reads and repair enzymes Other proteins o Helicasehelix destabilizing enzyme promotes unwinding by binding at replication fork o Single stranded binding stabilizes single strand DNA o Primase catalyzes copying of short stretch of DNA template strand RNA primer sequence o DNA ligase seals nicks in DNA acts as glue DNA polymerase acts as dimer: 2 enzymes one for each strand Proofreading o DNA replication takes place only once in each generation in each cell o Errors in hydrogen bonding leads to errors in a growing DNA chain once in 10 to 10 base pairs o Can lead to mutations o Incorrect nucleotides are removed immediately after they are added to the growing DNA chain during replication o Polymerase feels for a mistake if one is present, it pops nucleotide out and puts a new one in stran d o It is possible that polymerase cant fix/find mistake Transcription 4/20/16 Genetic flow of information: Central dogma RNA is machine that hooks up amino acids in correct sequence Transcription: RNA synthesissynthesis of RNA strand, using a DNA strand as a template, complementary General features of Transcription o Requires DNA template o Catalyzed by RNA polymerase o Requires the ATP, GTP, CTP, UTP o Requires magnesium o No RNA primer is required o RNA chain is synthesized 5’ to 3’ o DNA contains signals for termination and initiation o DNA template is unchanged used again Bacterial RNA polymerase o 5 different subunits: alpha, gamma, beta, beta primer, and sigma o Core enzyme has alpha, gamma, beta, and beta primer, the beta is catalyzed formation of phosophdiester bond o Holoenzyme: alpha, gamma, beta, beta prime and sigma, this gets things started o Sigma will dissociate o Core can transcribe but cant start it o Sigma recognizes sequence in DNA for starting (promoter) o Bacteria uses different sigma factor to control different sets of genes Template: o 2 DNA strands Template or antisense strand Coding or sense strand o RNA polymerase binds to and transcribes the template strand o Coded in triplets each triplicate codes an amino acid Promoter o Sequence of DNA flags where transcription will start o Transcription start (TSS) 2 different parts 10 region and 35 region o Regions are almost 17 factors apart o Different sigma factors bacteria has housekeeping sigma factor o Consensus sequence most common sequence o The closer the promoter sequence is to TATA box the better that the promoter will work o 35 and 10 regions are upstream from TSS o Sigma factor will recognize 35 and 10 regions o Transcription start site, no zero nucleotide Mechanisms of transcription o Initiation, elongation, termination Initiation o RNA polymerase binds to promoter (recognize by a factor) o Forms a closed complex bound to double stranded DNA o DNA unwinds at promoter to form open complex o RNA polymerase begins transcribing complementary base pairing o Simpiler in transcription then in replication o Replication needs 3’OH group o Replication separating strands completely o Sigma factor is only used in transcription unwinding DNA o Only similarly between transcription and replication is unwinding of DNA Elongation o Transcription bubble of 17 base pairs o Phosphodiester bond between incoming nucleotides and RNA chain catalyzed by RNA polymerase o Sigma factor released after 10 nucleotides DNA swivel o Topoisomerase relaxes supercoil in front of and behind transcription bubble o Keeps an even supercoiling after and before the location of the RNA polymerase Intristic Termination (Rhoindependent) o Two inverted repeats, which can form a stem loop followed by a stretch of A’s which encode a strecht of U’s o After inverted repeats stretch of U’s o Terminators encode AU base pairs because they have weaker bonds Rhodependent o Rho ATP dependent RAN o DNA helicase that releases RNA from template Eukaryotic Transcription 4/22/16 Compared to bacterial o Cellular components of transcription and translated separated o RNA processing o Chromatin (nucleosomes) substrates adds regulator potential o Larger genome o Multicellular organisms are differentiate and cells have specialized function o Three RNA polymerases Gene Expression in eukaryotes o Two cellular components Transcription in nucleus Translation in cytoplasm o RNA processing 5’ capping (5’5’) linkage protects mRNA RNA splicing 3’ polyadenylation (almost all mRNA have poly A tails) In splicing introns removed, exons spliced together Splicing creates variation of proteins Eukaryotic RNA polymerase o 3 main polymerases that catlyazes transcription o RNA polymerase I: pre rRNA o RNA polymerase II: premRNA (protein coding) o RNA polymerase III: pretRNA, pre5.8s RNA, Small structural RNA’s RNA polymerase II o Transcribes premRNA, protein coding genes o Largest subunit contains essential carboxyl terminal 7 amino acid repeats Phosphorylated during initiation Tyrosine, serine, proline, threonine, serine proline, serine Eukaryotic Promoters o Determine site of transcription initiation o More complex then in bacteria o Several different sequence elements o Different for each RNA polymerase o Not as conserved as bacterial promoters Transcriptional Regulation Transcriptional regulation o Bacteria respond to changes in environment conditions such as presence of nutirents or change in temperature o Most gene regulation in bacteria occurs at the level of transcription initiation certain examples of that following regulation is transcription o Not many genes are transcribed Principles of transcription o DNA protein interactions transcription factors (repressors and activator) DNA binding proteins Protein protein interactions transcription factors interact with each other an or RNA polymerase RNARNA interactions small regulatory RNAs regions of mRNA’s regulate expression Response to environment nutrients in unicellular organisms, o Hormones multicellular organisms
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