BIO 326R 1st EditionExam # 2 Study Guide Lectures: 9 -15 Lecture 9 (September 20)DNA Replication History- know what Roslind Franklin and Linus Pauling did- DNA discovery experiments were done on bacteria in 1952Steps to Replication (bacteria)- find origin— know function of OriC and DnaA- unwind DNA—know function of helicase (DnaB, uses ATP) and DnaCo unwinging at OriC—A/T richo replication bubble—single strand binding protein- assemble replisome—DNA polymerase I (RNA primer removal) and DNA polymerase III (DNA replication, bidirectional)Initiation of Synthesis- know function of RNA primase and RNA primer- continuous vs. discontinuouso leading vs. lagging Okazaki Fragments- Err rate is 1 in 10^9-10^10 bases- Can have multiple replication forksControlling Initiation- DnaA doesn’t bind to hemimethylated DNA well (old strand is methylated)o seqA= binds hemimethylated OriC and prevents DnaA binding- Cell size—understand how concentration of DnaA affects replicationLecture 10 (September 23)Proofreading- how is it so low?o Base pairing is specific and preciseo DNA polymerase I and III have 3’5’ exonuclease activityo Posy synthesis mismatch repairTerminating Replication- stop at terminating sequence- double strand break, pass through other chromosome, relegateTranscription- = RNA from DNA- Know differences between RNA and DNA- Types of RNA: mRNA, tRNA, rRNA, sRNA- 3 steps are initiation, elongation, termination- RNA polymerase (RNAP)o Enzyme that makes RNA, common antibiotic targeto 2 forms: core enzyme and holoenzyme—know the differenceo Sigma factor (from holoenzyme) helps RNAP recognize promoterInitiation of Transcription- start site at +1 (remember there’s no zero, directly upstream position is -1)- promoter sequence is upstream from a transcripto consensus sequence is recognized by the sigma factor -35 and -10o strength of the consensus sequence effects gene expressionLecture 11 (September 25)Continuation or transcription steps…Initiation- promoter binding— sigma factor recognizes promoter, RNAP binds- RNAP complex—know open versus closedElongation- sigma factor falls off the RNAP and transcription can continue by core RNAP- proofreading—RNAP has proofreading activityTermination- Rho independent vs. Rho dependentSidenotes- bacterial mRNAs are often polycistronico operons- RNA half lives varyLecture 12 (September 27)Translation- transcription and translation are coupled, most antibiotics target translation, 90% of E. coli energy production is used for translation, 60% of macromolecules are involved in translation- ribosomes: 50s and 30 s subunit make a 70s unit (s= Svedberg)o know what composes the 30s and 50s units- tRNA= transfer RNA that can have an amino acid attached covalently to 3’ end (charged when attached)o know function of amino acyl tRNA synthetaseo understand why they can “wobble”Initiation- recognize start site (start codon)—use N- formylmethionine- Shine Delgarno sequence—tells ribosome where to beo Consensus determines gene expression- 30s subunit and initiation factors find SD, tRNA with formylmethionine and GTP, ready totranslate, kick off initiation factorsElongation- binding of 2nd aminiacyl tRNA, enters bound to EF-Tu and GTP (Ef- Tu hydrolyzes GTP to GDP), helps find binding site, make a peptide bond between the 2 amino acids with 23s ribozyme, translocation- speed: ~15 amino acids/sec—matches transcription of 45 nt/secTermination- finds stop codon (no tRNA has anticodon for these), release factor interacts with stop codon, releases ribosomeLecture 13 (September 30)Cell Division- binary fission- bacteria genome differs per cell, but faster growing cells typically have larger genomeso genes around origin are present at higher levels in fast growing cellsBinary Fission- 2 equal progeny- Elongate, form septum, form cell walls, separate- Can follow proteins with GFP (fluorescent protein from jelly fish)- FtsZ (like tubuline)—makes microtubules, forms ring at septum, contracts, and pinches cell to divideo Finds the middle with MinC, MinD, MinE Oscillate back and forth to sense where mid-cell is Know other functions of MinC and MinD- MreB= shape determining factoro Know what happens-- whether cell is rod or cocci shaped- Bacteria can divide on multiple planes: one, two (tetrad), all (clusters of grapes)Lecture 14 (October 2)Terminology- Phenotype vs. genotype, definition of mutantPlasmids- autonomous replicating DNA molecules- use host machinery (DNA polymerase I and III and ligase from host)- know how to name a plasmid- host range (narrow or broad)o broad host range plasmids encode additional factors to replicate in many different bacteria- replication of plasmid can be theta (like chromosome replication) or rolling circle (conjugation of new strand to make a new plasmid)- copy number 15-20—know how this is maintainedo RNA I, with the help of Rop, binds to RNA II to prevent replication initiationo Proper ratio and more RNA II than RNA I allows cell to maintain this copy numberLecture 15 (October 4)Plasmid compatibility- incompatibility groupso same copy number or same origin= not compatible- formula for probability that all plasmids will be in one cell when it divides: p=2(1/2)^2n- improve probability with portioning systemsCloning DNA into a plasmid- know the three characteristics of a good plasmid candidate for DNA cloning- PCR= amplifying a desired piece of DNAo Know the three temperatures and what their function iso Three steps: amplify the gene, digest with restriction endonuclease, DNA