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RNA and Nucleic Acid Reactions

RNA and Nucleic Acid Reactions. C483 Spring 2013. 1. Which is not a difference between RNA and DNA? A) RNA is more prone to basic aqueous hydrolysis. B) RNA contains uracil; DNA usually does not. C) RNA cannot form helices. D) RNA is usually single-stranded; DNA is double-stranded.

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RNA and Nucleic Acid Reactions

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  1. RNA and Nucleic Acid Reactions C483 Spring 2013

  2. 1. Which is not a difference between RNA and DNA? A) RNA is more prone to basic aqueous hydrolysis. B) RNA contains uracil; DNA usually does not. C) RNA cannot form helices. D) RNA is usually single-stranded; DNA is double-stranded. 2. ________ catalyze the hydrolysis of phosphodiester linkages to release nucleotide residues from the middle of a polynucleotide chain. A) Topoisomerases B) Endonucleases C) Exonucleases D) Restriction enzymes 3. Which are the products of the RNase A-catalyzed hydrolysis of pGpApUpApApCpG? A) pG + pA + pUpA + pA + pCpG B) pGpApU + pApApC + pG C) pG + pApUpApApCpG D) pGpApUp + ApApCp + G E) two pG + three pA + pU + pC

  3. 4. Which does not occur during the hydrolysis of RNA by RNase A? A) Covalent catalysis involving a bond between a pyrimidine and a lysine of RNase A. B) Acid-base catalysis to cleave the phosphodiester bond. C) Abstraction of a proton from the 2'-hydroxyl group by histidine. D) Transition-state stabilization of a pentavalent phosphorous atom. 5. Which enzyme would be least useful for recombination experiments that introduce new fragments of DNA into an existing DNA molecule? A) EcoRI G↓AATTC. B) SmaI CCC↓GGG. C) XhoI C↓TCGAG. D) All are equally useful.

  4. RNA • Single stranded with secondary structure, including helices • Stem-loop • Ribosomal RNA (rRNA) • 80% • Transfer RNA (tRNA) • 15% • Messenger RNA (mRNA) • 3% • Small RNA • 2%

  5. RNA Structure and Stability • Structural difference of 2’ hydroxyl • H-bonding in RNA structure • Reactions of catalytic RNA (rare) • Hydrolysis • Structure dictates role difference in DNA/RNA

  6. Catalytic Hydrolysis • Enzymes can catalyze hydrolysis • Very important reactions! • Nucleases • RNasevsDNase • Single/double strand • Exonucleasevs Endonuclease • Orientation of hydrolysis

  7. RNase A • Key protein in study of protein folding • Endonuclease • Selective for pyrimidines • Cleaves ester to give free 5’ hydroxyl (like alkaline hydrolysis)

  8. Products of RNase A Biochemistry 1998, 37, 12121-12132 Hydrolysis of pGpGpUpApCpUpG gives:

  9. Mechanism of RNase A • Mechanisms of catalysis • Proximity • Acid/base • TS stabilization • also includes Lys41 • Pentavalent phosphorus Biochemistry 2001, 40, 4949-4956.

  10. Restriction Enzymes • DNases (endonuclease) that some bacteria have to “restrict” virus infection • Work by recognizing and cutting up foreign DNA • Specificity • Palindrome • Sticky ends

  11. EcoR1 • Many restriction enzymes have been isolated • Many sources • Unique specificities • Recognize base pairs in the major groove

  12. Host vs. Foreign DNA • Based on methylation (SAM) • Type 1 Restriction enzyme has both endonuclease and methylation catalysis • Methylation blocks restriction

  13. Application 1: Restriction Map • “Map” restriction sites onto a DNA sequence • Useful for locating specific genes relative to each other

  14. Application 2: DNA Fingerprints • Identifies individuals in a heterogeneous population • Not as useful until PCR was developed

  15. Application 3: Recombinant DNA • Insert a gene into a cloning vector • Vector put into microorganism • Independently replicated and expressed

  16. Answers • C • B • D • A • B

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