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