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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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Nov. 5, 2004

MBB 407/511

Lecture 17:

Structures of DNA and RNA

(Part II)


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…”


Examples of helices


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


DNA Strands are Antiparallel

Why?

1. Strands must run anti-parallel

in order to get H bonding

Phosphodiester bonds link

adjacent nucleotides

Phosphomonoester bond


The Double Helix Has Alternating Major and Minor Grooves

Why?

Unequal offsetting of the two strands


How is DNA Synthesized?

Requires dNTPs

Why?


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


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


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


Melting temperature Tm increases with G:C content


Cot Curve Analysis of DNA Samples


Visualizing DNA by Ethidium Bromide Staining


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


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


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


How about the 3-D structure of RNAs?


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” (


Examples of catalytic RNAs

Group II intron

(self-splicing)

RNaseP cleavage

of tRNA 5’ ends


Hammerhead ribozyme

Ribozymes catalyze:

1. Cleavage reactions

2. Transesterification reactions


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?


DNA and RNA are Degraded by Nucleases

(phosphodiesterases)

Exonucleases

—5’3’ exo and 3’ 5’ exo

Endonucleases

—cut internally


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


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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