BIO 172 1st Edition Lecture 6 Outline of Last Lecture I. MacromoleculesII. DNA ReplicationOutline of Current Lecture I. DNA replicationII. TranscriptionCurrent LectureProofreading and Repairing DNA:Accuracy: in the absence of polymerase proofreading, replication would make an error once every 100,000 base pairs. Therefore, DNA polymerase has an important job- proofreading!DNA polymerase increases accuracy when reading for chromosomal replication, by checking each nucleotide for the proper Base-Pairing. It looks at the fit of the Hydrogen Bonds, between C and G, and A and T.If the polymerase misses a mistake, then some enzymes might go back and fix the error. SYNTHESIS: 5’ to 3’ direction.Exo-nuclease (proofreading) done by DNA polymerase 3 and 1, from 3’ to 5’. Elongation Phase (of linear DNA replication): LINEAR: multiple origins of replication. Bidirectional fork movement, each way along the linear strand. Leading strand and lagging strand replication, where it is difficult on the lagging strand to replicate all the way to the end of the DNA. Telomerase builds on an extension, and then the lagging strand goes all the way to the end of DNA but excludes the extension!Telomeres: repetitive, about 6 Base Pairs repeated many times.Telomerase: becomes active once it binds to the 3’ end of the G-rich extension. Telomerase is special because it does not need a Template.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Telomerase uses base pairs to build extension. Repeats multiple extensions, once completed it can prime and go in the opposite direction so that it makes sure to copy the end ofthe DNA (chromosome)!- RNA primer taken out- we don’t want RNA in the DNA that’s replicated.- Then there’s a gap at the end of the chromosome. - No DNA could be copied there… because it needs the 3’ O-H since the RNA primer is gone.- The RNA primer was what originally provided the 3’ OH, but it was taken out so that the DNA was available.- Therefore, at the end of the chromosome (DNA), there’s no 3’ OH. The above points are why Telomeres and Telomerase is needed!With Telomeres and Telomerase, DNA polymerase has all it needs- a template, a 3’ OH (to extend the 3’ end), and the extension. DNA polymerase can get the whole chromosome! The cell loses the repeated extension made of telomeres.Human Application: We go through telomere shortening (or our cells do!). When they age, cells die and get flushed out of our system. Having old cells it bad- it means a greater likelihood of mutations or cancer.For many cancers, they have an increased expression of telomerase. This makes the cancer cells keep living because they continue to be replicated.Transcription:Supercoiling allows DNA strands to fit into tiny cells. Topoisomerases are the enzyme that makesDNA supercoiled. Topoisomerase induce supercoiling, they take long DNA strands and make them compact. For bacteria, topoisomerase has a major role! It also acts at the replication fork so it stays uncoiled.Eukaryotic Cells: nucleosomes… composed of histone proteins. Wrapping helps compact the DNA- bacteria doesn’t have this way of doing it- some proteins bind and hold the wrapped nucleosomes together.Basic features of Transcription:Use DNA as a template for RNAsynthesis.Catalyzed by RNA polymerase.Occurs in specific portions of DNA. Bacteria or Archaea: chromosomalDNA, RNA polymerase recognizes apromoter, copies DNA to mRNA, thena ribosome can begin to make theprotein.To Initiate Transcription:RNA polymerase binds to a promoter.Then it transcribes the DNA (gene).A Terminator sequence causes transcription to end the process.Major differences between DNA Polymerase and RNA polymerase:DNA polymerase needs to extend from a 3’ O-H. RNA does not need the 3’ O-H.DNA polymerase has Thymine as a base pair. RNA polymerase has Uracil.Deoxyribose (H on the 2’ Carbon). O-H on the 2’ Carbon.Dark Blue Strand: 3’ to 5’ from Left to Right.Light Blue Strand: 5’ to 3’ (L to R).Red strand (RNA): 5’ to 3’ (L to R).Transcription factors: bind to TATA box. They serve as a marker and recruitment mechanism for RNA polymerase. Then RNA polymerase 2 can bind so transcription can begin!At a Eukaryotic Promoter: ELONGATION:After the promoter, RNAPolymerase unwinds the DNA.RNA polymerase addsnucleotides.Only one DNA strand is thetemplate for base-pairing betweenDNA and RNA nucleotides.Direction of synthesis is 5’ to 3’RNA nucleotides are added tothe 3’ end.Uracil in RNA pairs to anyAdenine in the DNA strand.Unwinds DNA double helix ~ 10-20 basesThe template strand is the reverse complement of the mRNA message; the non-template strandis identical to the mRNA (except T instead of U in the DNA template).60 nucleotides per second in eukaryotes.Single gene can be transcribed simultaneously by several RNA polymerases (following like a convoy)TRANSCRIIPTION bubble does not have helicase- and moves with RNA polymerase at the same time.Template Strand:3’ H-O binds to energy-rich phosphate bonds. Polymerization for RNA and DNA are basically thesame- except for Uracil rather than thymine and that RNA has an OH and DNA just has H on the 2’ carbon.Termination of Transcription:RNA polymerase stops adding ribonucleotides (rNTPs).RNA chain is released.There is a specific site, or termination sequence, for each gene.Termination is often triggered by the formation of a RNA hairpin loop in Prokaryotes.Prokaryotes have a sigma factor protein that binds to the promoter and unwinds the DNA, threading it into the active site of the attached RNA polymerase.Transcription: Initiation, elongation, termination are the three stages! Promoter sequence. Transcription unit. Point for termination. Termination causes the formation of a hairpin and stops