BCHM 4116 1st EditionExam # 1 Study Guide Lectures: 1 - 12Lecture 1 (January 21) & Lecture 2 (January 23) I. DNA vs. RNA a. Pyrimidine : Cytosine, Thymine, Uracil (Uracil replaces Thymine in RNA) i. One ring ii. 6-membered aromatic ringb. Purine : Adenine, Guaninei. Two ringsii. 5-membered imidazole ring fused with pyrimidine c. N-glycosidic bond : links base to sugar in DNA & RNAd. Nucleoside = sugar + basee. Nucleotide = phosphate + sugar + basef. Phosphodiester bond : links a nucleotide to another nucleotide II. Keto-enol tautomeric shift DNA RNABases: A C G T Bases: A C G U3’ hydroxyl group 2’ hydroxyl groupGenetic information Genetic information mRNA, tRNA, rRNA, snRNADouble stranded Single strandedRichly adorned w/ proteins, a class of arginine and lysine rich basicproteins called histonesMore chemically stable b/c: 1. RNA’s 2’OH group makes the 3’phosphodiester bond susceptible tonucleophilic cleavage2. cytosine deaminates to form uracil and b/c DNA does not have uracilthis change would be seen as a mistake and repaired; whereas, U isnormal in RNA and this conversion from C U would not be seen as amistake and therefore go un-repaired.a. To quantify DNA/RNA use bases because bases are aromatic and have a strong absorption at 260 nm b. Keto : C= Oc. Enol : C – OH , C = C Lecture 3 (January 26) I. Structural transitions of DNAa. Denaturation and Renaturation i. Double helix is held together not by covalent bondsii. Heat doesn’t happen in cell so instead denature of the double helix occurs via enzyme called HelicaseII. Implication for nucleic acid hybridization a. Hyperchromic shirft: increase in the absorbance of DNA upon denaturationb. Tm: melting temperaturei. High Tm = more stableii. Low Tm = less stable iii. Guanine/Cytosine (GC) content, pH, salt, and solutes affect Tm1. High GC = high Tma. Because GC has more stacking energy than AT and therefore is more stable 2. High salt = high Tma. Because salt neutralizes base and therefore makes it more stable3. Forms H-bond = lowers Tma. Formamide steals base-to-base attraction to make formamide-base bond and this makes it less stable Lecture 4 (January 28) I. Tertiary Structure in DNAa. Spiral around an imaginary toroidb. DNA interwinds and wraps about itselfc. Long linear DNA loops, ends restrained i. DNA attached to nuclearmatrixii. 1 end fixed so forms supercoilsiii. Make sure as you transcribe release stress/tensioniv. Enzyme topoisomerase I dues this by cutting 1 line to release pressure and seals back when finished v. Enzyme topoisomerase II does this by cutting both strands (And will seal when done) vi. DNA gyrase is a topoisomerase that introduces negative supercoil into DNAII. L = T + W a. L = linking number i. Number of times two strands are intertwined b. T = twisti. Number of helical turnsii. HOW MANY DARKER SHADE COLOR THERE IS ON DNAc. W = writhe i. Number of supercoils ii. HOW MANY OVERLAPS ARE THERE III. Supercoil a. Negative supercoil: more relaxed between the two twisted strandb. Positive supercoil: more tight IV. DNAa. DNA is negatively charged b. Histone is positively charged i. This is why DNA wraps around histone c. “histone code” V. The secondary and tertiary structures of RNAsa. Pseudouridine: N- glycosidic which would have linked base to sugar via N but in this case base is linked to sugar via C VI. DNA attached to nuclearmatrix  1 end fixed so forms supercoil  make sure as you transcribe releases stress/ tension a. Release tension via topoisomerasei. Enzyme topoisomerase I does this by cutting 1 line to release pressure and seals back when finishedii. Enzyme topoisomerase II does this by cutting both strands and will seal when done iii. DNA gyrase is a topoisomerase that introduces negative supercoils in DNALecture 5 (January 30) I. Denaturation and Renaturationa. Increase in absorbance = denaturation  Tmi. Because when DNA is denatured there are more bases exposedb. Tm: Melting Temperaturei. High Tm = Stableii. Low Tm = unstablec. Factors that affect Tmi. High G-C content in DNA = high Tm = STABLE1. G-C pairs have higher base stacking energies than A-T ii. High salt concentration = high TM = STABLEiii. H-bond = low Tm = UNSTABLE1. Formamide steals base-to-base attraction to make formamide-to-base bond so less stable II. Single stranded DNA can renature to form DNA duplexesa. Denatured DNA can renature to reform duplex if denaturing conditions are removedb. Renaturation requires reassociation of DNA strands into double helix  reannealing c. Renaturation is dependent on both DNA concentration and time d. Process can be sped up via warm temp. to promote diffusion of large DNA molecules but not too warm where can cause melting Lecture 6 (February 2) I. Basic Manipulation of DNAa. DNA ---trancription mRNA ----translation--- proteins b. transcription begins with 5’ untranslated regions (UTR) and ends with the 3’ UTRc. translation begins with the start codon (ATG) and ends with stop codon (TAA, TAG, or TGA) d. promoters are DNA sequences at the 5’ region adjacent to the transcriptional start site / before the 5’ UTR i. promoter sequences = “TATA” box (TATAAA) e. RNA polymerase (transcriptionfactors) bind to promoter andtranscribe DNA to mRNA f. Exons: coding regionsg. Introns: noncoding regions i. Transcription = entire gene is copied into pre-mRNA which includes exons & introns ii. RNA splicing = introns are removed & exons join to form a contiguous coding sequence1. This is known as mature mRNA & is ready for translationh. Poly A tailsi. A residues at the end of mRNAii. Poly a tails are not encoded in DNAiii. They are added to pre mRNA after transcription 1. End of pre-mRNA is cut by specific enzyme and poly A tail is addedto mRNA strand 2. Signal sequence (yellow) in 3’UTR guides the addition of poly A tailII. Basic Manipulation of DNAa. Isolation of DNAi. Cell lysis – remove cell debris – remove protein (phenol; protease; ion-exchange column) – remove RNA (RNase) – [recipitation of DNA (ethanol) b. Gel electrophoresis i. Separation of DNA fragments III. Hydrolysis of Nucleic Acid a. Nucleases = hydrolyze nucleic acids b. Nucleases are phosphodiesterases b/c they catalyze the cleavage of phosphodiester bonds by h2o c. Nucleases that act only on DNA  DNased. Nucleases that act only on RNA  RNase IV. Restriction Endonucleasea. Act only at a particular nucleotide sequence 4 – 8 nucleotides in length b. Nucleases that cleave