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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
Biochemistry 1998, 37, 12121-12132
Hydrolysis of pGpGpUpApCpUpG gives:
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.
2.
3.
4.
5.
C
B
D
A
B