BIO 319Exam # 2 Study GuideExam # 2 Study Guide• Know Dr. Hollingsworth’s lecture, 2 questions will be on the exam• Know the experiment done to isolate DNA• Definitions• Know what is template or non-template• Be able to do okazaki fragments and also label what is 5’  3’ or 3’  5’I. General Features of ReplicationRead Experiment 11A pages 263-265 Meselson/Stahl Experiment A. Semi-Conservative• The double-stranded DNA contains one parental and one daughter strand following replication• Conservative Model: Both parental strands stay together after DNA replication• Dispersive model: Parental and daughter DNA are interspersed in both strands following replication B. Starts at Origin C. Bidirectional D. Semi-Discontinuous II. Identifying Proteins and Enzymes of ReplicationA. Combine Genetics and Biochemistry1. Genetic Approach: Obtain Mutants that are defective in Replicationa. Such mutations are Lethal!b. Conditional lethalc. Temperature sensitive (ts) lethal2. Method:• mutagenize cells• plate the cells on agar plates and grow at 30C• replica plate and grow at 37C3. Identifying which ts lethal mutants have defects in Replication a. pick ts colonies from 30oC plate and grow them in liquid medium at 30oC. b. shift them to 37oC c. add Bromodeoxyuridine (BrdU) and continue growth for a short time at 37oC d. remove the BrdU and irradiate the cells with UV light 1) If BrdU is incorporated into the DNA the UV light will kill the cells e. Return the cells to 30 oC f. the cells that revive and continue to grow did not incorporate BrdU because they have a defect in Replication!!! g. Those ts cells that had other defects continued to replicate their DNA and incorporate BrdU, and hence the UV light killed them.III. Detailed Examination of the Mechanism of ReplicationA. InitiationSummary of Initiation after DnaA binding• DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them• This generates positive supercoiling ahead of each replication fork– DNA gyrase (Topoisomerase) travels ahead of the helicase and alleviates these supercoils • Single-strand binding proteins bind to the separated DNA strands to keep them apartProperties of DNA Polymerase III1) Synthesizes DNA in the 5’ to 3’ direction2) Can’t initiate a new strand: Requires a primer3) Highly processive (Beta subunit clamp)4) Proofreading 3’ to 5’ exonucleaseIf this problem is not solved1) The linear chromosome becomes progressively shorter with each round of DNA replicationThe cell solves this problem by adding DNA sequences to the ends of chromosome: telomeres2) Small repeated sequences (100-1000’s)Catalyzed by the enzyme telomeraseTelomerase contains protein and RNA3) The RNA functions as the template 4) complementary to the DNA sequence found in the telomeric repeat1) This allows the telomerase to bind to the 3’ overhang B. Priming C. Elongation D. Proofreading and TerminationBacteria Replication• DNA synthesis begins at a site termed the origin of replication• Each bacterial chromosome has only one (OriC)• Eukaryotic chromosomes have many origins• Synthesis of DNA proceeds bidirectionally around the bacterial chromosome– eventually meeting at the opposite side of the bacterial chromosome• Where replication ends• Not as simple with eukaryotic linear chromosomes• Replication is Semi-Discontinuous – Know the Fig. 11.11 and how to do it.TRANSCRIPTIONI. Transcription in BacteriaA. General FeaturesB. RNA PolymeraseC. InitiationI. The promoter functions as a recognition site for initiation D. ElongationI. RNA polymerase slides along the DNA in to synthesize the RNA transcript in the 5’ to 3’ directionE. TerminationI. A termination signal is reached that causes RNA polymerase to dissociated from the DNA Important Definitions –You need to know this!!!• The DNA strand that is copied is termed the template strand (non coding)• The opposite DNA strand is called the coding strand or the sense strand– The base sequence is identical to the RNA transcript• Except for the substitution of uracil in RNA for thymine in DNA• RNA polymerase is the enzyme that catalyzes the synthesis of RNA• RNA polymerase– Core enzyme• Four subunits = a2bb’ (may include δ and ω) • Capable of transcribing DNA into RNA but randomly• Does not recognize promoters– Holoenzyme add Sigma factor• One s subunit• Specificity subunit allows RNA polymerase to recognize promoters • Promoters are DNA sequences that “promote” gene expression– More precisely, they direct the exact location for the initiation of transcription by telling RNA polymerase where to bind on the DNA.• What does a promoter look like?• What are the steps of transcription initiation at a promoter? • The position in DNA where the RNA chain starts is called +1. DNA 5’ to the start site is called “upstream” and is given negative values (-10). DNA to the 3’-side is called “downstream” and is given positive values. • Transcription Initiation takes place in several steps• The binding of the RNA polymerase to the promoter forms the closed complex• Then, the open complex is formed when ~17 bp of DNA in the vicinity of the TATAAT box is unwound• Abortive Initiation: Many short RNAs 2-10 nts are made within the open complex without any movement of RNA polymerase• Promoter clearance: Eventually RNA polymerase moves away from the promoter 5’ to 3’ – The sigma factor is released at this point• This marks the end of initiationTERMINATION• Two different mechanisms for termination– 1. Intrinsic termination• Only requires sequences in the DNA-àRNA• Factor independent• Rho independent – 2. rho-dependent termination• Requires a protein known as rho (r) TRANSLATION tRNAs Share Common Structural Features• The secondary structure of tRNAs exhibits a cloverleaf pattern– It contains• Three stem-loop structures; Variable region• An acceptor stem and 3’ single strand region• The actual three-dimensional or tertiary structure involves additional folding • In addition to the normal A, U, G and C nucleotides, tRNAs commonly contain modified nucleotidesMore than 60 of these can occurAminoacylating tRNAs• The enzymes that attach amino acids to tRNAs are known as aminoacyl-tRNA synthetases – There are 20 types • One for each amino acid•