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MBB 407/511 Lecture 17: Structures of DNA and RNA (Part II)

Nov. 5, 2004. MBB 407/511 Lecture 17: Structures of DNA and RNA (Part II). 6Å. 3.4Å. 34Å. DNA is:1) double stranded 2) a right-handed helix. Helix vs Spiral. Merriam Webster’s Collegiate Dictionary. Helix ( n )—a. “Something spiral in form.”

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MBB 407/511 Lecture 17: Structures of DNA and RNA (Part II)

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  1. Nov. 5, 2004 MBB 407/511 Lecture 17: Structures of DNA and RNA (Part II)

  2. 6Å 3.4Å 34Å DNA is:1) double stranded 2) a right-handed helix Helix vs Spiral Merriam Webster’s Collegiate Dictionary Helix (n)—a. “Something spiral in form.” b. “A coil formed by winding wire around a uniform tube.” c. “A curve traced on a cylinder…”

  3. Examples of helices

  4. Hydrogen Bonds Hold the Two DNA Strands Together What are hydrogen bonds? Notice that: A-T bp have 2 H bonds G-C bp have 3 H bonds G-C bp are more stable Helix has constant diameter Why? 1) Purines always pair with pyrimidines 2) The H bonding distance between the A-T and G-C bp is the same

  5. DNA Strands are Antiparallel Why? 1. Strands must run anti-parallel in order to get H bonding Phosphodiester bonds link adjacent nucleotides Phosphomonoester bond

  6. The Double Helix Has Alternating Major and Minor Grooves Why? Unequal offsetting of the two strands

  7. How is DNA Synthesized? Requires dNTPs Why?

  8. DNA can exist in 3 different 3-dimensional forms B-DNA Base pair spacing = 3.4Å/bp 10.5 bp/helix repeat 36 bp Helix diameter ~ 20Å

  9. How to Detect DNA and RNA Nucleic acids absorb UV light Why? A spectrophotometer Determine [nucleic acid] For dsDNA: 1 A260 unit = 50 mg/ml of DNA

  10. Double stranded DNA denatures at high temperature UV light absorbance increases as DNA goes from double-stranded to single-stranded “Hyperchromic shift” Melting temperature (Tm) What is the basis of hyperchromic shift? Stacked bases absorb less UV light

  11. Melting temperature Tm increases with G:C content

  12. Cot Curve Analysis of DNA Samples

  13. Visualizing DNA by Ethidium Bromide Staining

  14. Nucleic Acids Can Be Precipitated 1. Salt (0.3M NaOAc, 0.5M NH4OAc, 0.4 M LiCl) Monovalent cations neutralize the repulsive effects of the phosphate groups 2. Alcohol (70% Ethanol or 50% isopropanol) To concentrate DNA/RNA by exluding H20

  15. The Effects of Alkali on DNA and RNA Alkali hydrolyzes RNA Alkali denatures dsDNA The 2’OH groups are susceptible to nucleophilic attack At high pH, deprotonation of bases disrupts hydrogen bonding

  16. Q: Does RNA have a secondary structure? RNA is single stranded and is most stable when it Engages in intrastrand base pairing A tRNA 16S rRNA

  17. How about the 3-D structure of RNAs?

  18. Roles of DNAs and RNAs DNA RNA Messenger RNAs (mRNAs) Transfer RNAs (tRNAs) Ribosomal RNAs (rRNAs) Small nuclear RNAs (snRNAs) Catalytic RNAs (“Ribozymes”) — self-splicing (e.g., group I and group II introns) — cleavage of other RNAs (e.g., RNA component of RNaseP cleaves tRNAs) — “hammerhead RNAs” (

  19. Examples of catalytic RNAs Group II intron (self-splicing) RNaseP cleavage of tRNA 5’ ends

  20. Hammerhead ribozyme Ribozymes catalyze: 1. Cleavage reactions 2. Transesterification reactions

  21. How come only RNAs be catalytic? They are single-stranded —can adopt complex 3-D structures They have 2’ OH groups —highly reactive Newest ribozymes: the snRNAs in the spliceosme & the 23S rRNA in ribosomes What are the implications?

  22. DNA and RNA are Degraded by Nucleases (phosphodiesterases) Exonucleases —5’3’ exo and 3’ 5’ exo Endonucleases —cut internally

  23. DNAs are more stable than RNAs DNA is very stable: it stores the genetic information There aren’t many DNases in the cell. DNA is protected. RNA is very unstable: its turnover is important for regulating gene expression Lots of RNases in the cell. RNAs generally not protected. Some RNAs more stable than others: rRNAs and tRNA aka “stable RNAs mRNAs have half-lives from minutes to hours

  24. Answer to last lecture’s question: “Why is thymine found only in DNA, uracil only in RNA?” Deamination of cytosine in DNA is very common in the cell, but can lead to mutations after DNA replication. deamination C:G U:G DNA replication DNA replication C:G U:T & C:G There is a repair system that looks for uracil attached to deoxyribose sugars and excises the base then replaces the uracil with a cytosine. If uracil was a normal base in DNA, the cell might not detect the U:G bps and there would be too many C:G to U:T mutations.

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