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 – RNase vs DNase Single/double strand – Exonuclease vs 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 Hydrolysis of pGpGpUpApCpUpG gives: Biochemistry 1998, 37, 12121-12132

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

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

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

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

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

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

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

17. Answers 1.C 2.B 3.D 4.A 5.B