RNA and Nucleic Acid Reactions C483 Spring 2013
Dec 14, 2015
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)
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