BCHM 4116 1st Edition Exam 1 Study Guide Lectures 1 12 Lecture 1 January 21 Lecture 2 January 23 DNA RNA Bases A C G T Bases A C G U 3 hydroxyl group 2 hydroxyl group Genetic information Genetic information mRNA tRNA rRNA snRNA Double stranded Single stranded Richly adorned w proteins a class of arginine and lysine rich basic proteins called histones More chemically stable b c 1 RNA s 2 OH group makes the 3 phosphodiester bond susceptible to nucleophilic cleavage 2 cytosine deaminates to form uracil and b c DNA does not have uracil this change would be seen as a mistake and repaired whereas U is normal in RNA and this conversion from C U would not be seen as a mistake and therefore go un repaired I DNA vs RNA II a Pyrimidine Cytosine Thymine Uracil Uracil replaces Thymine in RNA i One ring ii 6 membered aromatic ring b Purine Adenine Guanine i Two rings ii 5 membered imidazole ring fused with pyrimidine c N glycosidic bond links base to sugar in DNA RNA d Nucleoside sugar base e Nucleotide phosphate sugar base f Phosphodiester bond links a nucleotide to another nucleotide Keto enol tautomeric shift a To quantify DNA RNA use bases because bases are aromatic and have a strong absorption at 260 nm b Keto C O c Enol C OH C C Lecture 3 January 26 I Structural transitions of DNA a Denaturation and Renaturation i Double helix is held together not by covalent bonds ii Heat doesn t happen in cell so instead denature of the double helix occurs via enzyme called Helicase II Implication for nucleic acid hybridization a Hyperchromic shirft increase in the absorbance of DNA upon denaturation b Tm melting temperature i High Tm more stable ii Low Tm less stable iii Guanine Cytosine GC content pH salt and solutes affect Tm 1 High GC high Tm a Because GC has more stacking energy than AT and therefore is more stable 2 High salt high Tm a Because salt neutralizes base and therefore makes it more stable 3 Forms H bond lowers Tm a 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 DNA a Spiral around an imaginary toroid b DNA interwinds and wraps about itself c Long linear DNA loops ends restrained i DNA attached to nuclearmatrix ii 1 end fixed so forms supercoils iii Make sure as you transcribe release stress tension iv 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 DNA II L T W a L linking number i Number of times two strands are intertwined b T twist i Number of helical turns ii HOW MANY DARKER SHADE COLOR THERE IS ON DNA c W writhe i Number of supercoils ii HOW MANY OVERLAPS ARE THERE III Supercoil a Negative supercoil more relaxed between the two twisted strand b Positive supercoil more tight IV DNA a 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 RNAs a 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 topoisomerase i Enzyme topoisomerase I does this by cutting 1 line to release pressure and seals back when finished ii 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 DNA Lecture 5 January 30 I Denaturation and Renaturation a Increase in absorbance denaturation Tm i Because when DNA is denatured there are more bases exposed b Tm Melting Temperature i High Tm Stable ii Low Tm unstable c Factors that affect Tm i High G C content in DNA high Tm STABLE 1 G C pairs have higher base stacking energies than A T ii High salt concentration high TM STABLE iii H bond low Tm UNSTABLE 1 Formamide steals base to base attraction to make formamide tobase bond so less stable II Single stranded DNA can renature to form DNA duplexes a Denatured DNA can renature to reform duplex if denaturing conditions are removed b 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 DNA a DNA trancription mRNA translation proteins b transcription begins with 5 untranslated regions UTR and ends with the 3 UTR c 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 transcription factors bind to promoter and transcribe DNA to mRNA f Exons coding regions g 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 sequence 1 This is known as mature mRNA is ready for translation h Poly A tails i A residues at the end of mRNA ii Poly a tails are not encoded in DNA iii They are added to pre mRNA after transcription 1 End of pre mRNA is cut by specific enzyme and poly A tail is added to mRNA strand 2 Signal sequence yellow in 3 UTR guides the addition of poly A tail II Basic Manipulation of DNA a Isolation of DNA i Cell lysis remove cell debris remove protein phenol protease ionexchange 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 DNase d Nucleases that act only on RNA RNase IV Restriction Endonuclease a Act only at a particular nucleotide sequence 4 8 nucleotides in length b Nucleases that cleave double stranded DNA molecules c Classified into 3 types I II III i Type I and III require ATP to hydrolyze DNA 1 catalyze chemical modification of DNA via addition of methyl groups ii Type I cleaves DNA randomly iii Type III recognize specific nucleotide sequences w in dsDNA and cut DNA at or near those sites iv Type II