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.

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

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.

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%

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

Catalytic Hydrolysis

• Enzymes can catalyze hydrolysis

• Very important reactions!

• Nucleases– RNase vs DNase

• Single/double strand– Exonuclease vs

Endonuclease– Orientation of hydrolysis

RNase A• Key protein in study of

protein folding• Endonuclease• Selective for

pyrimidines• Cleaves ester to give

free 5’ hydroxyl (like alkaline hydrolysis)

Products of RNase A

Hydrolysis of pGpGpUpApCpUpG gives:

Biochemistry 1998, 37, 12121-12132

Mechanism of RNase A

• Mechanisms of catalysis– Proximity – Acid/base– TS stabilization

• also includes Lys41• Pentavalent phosphorus

Biochemistry 2001, 40, 4949-4956.

Restriction Enzymes

• DNases (endonuclease) that some bacteria have to “restrict” virus infection

• Work by recognizing and cutting up foreign DNA

• Specificity• Palindrome• Sticky ends

EcoR1

• Many restriction enzymes have been isolated

• Many sources• Unique

specificities• Recognize base

pairs in the major groove

Host vs. Foreign DNA

• Based on methylation (SAM)

• Type 1 Restriction enzyme has both endonuclease and methylation catalysis

• Methylation blocks restriction

Application 1: Restriction Map

• “Map” restriction sites onto a DNA sequence

• Useful for locating specific genes relative to each other

Application 2: DNA Fingerprints

• Identifies individuals in a heterogeneous population

• Not as useful until PCR was developed

Application 3: Recombinant DNA

• Insert a gene into a cloning vector

• Vector put into microorganism

• Independently replicated and expressed

Answers

1. C2. B3. D4. A5. B