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RNA and Nucleic Acid Reactions

This content explores the fundamental differences between RNA and DNA, focusing on their structural characteristics and the enzymes involved in RNA hydrolysis. We discuss the implications of RNA's single-stranded nature and its susceptibility to hydrolysis, highlighting the role of RNase A in catalyzing nucleic acid reactions. Additionally, we touch on various nucleases and restriction enzymes that are critical in genetic engineering and molecular biology applications. This information is vital for understanding the biochemical basis of genetic information.

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RNA and Nucleic Acid Reactions

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  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 • RNasevsDNase • Single/double strand • Exonucleasevs 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 Biochemistry 1998, 37, 12121-12132 Hydrolysis of pGpGpUpApCpUpG gives:

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

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

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

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

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

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

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

  16. Answers • C • B • D • A • B

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