Chapter 6: Nucleotides, Nucleic Acids, and Genetic Information

purine

pyrimidine

Adenosine Monophosphate (& ADP & ATP)

nucleotides most commonly contain 1-3 phosphate groups

ATP: energy transfer agent

ATP formation (from ADP and Phosphate)

condensation ADP and phosphate

endergonic (nonspontaneous)

process of breakdown of metabolic fuels provides energy for this

catalyzed by ATP synthase (complex V of mitochondria)

Chemical PE of ATP is made available when

it transfers 1 of its 2 phosphate groups to another molecule

hydrolysis of ATP —→ ADP

ADP-Glucose

starch synthesis in plants proceeds by repeated additions of glucose units donated by ADP-glucose

group attached to ADP is usually linked to the nucleotide via mono- or diphosphate groups

(deoxy)guanosine

(deoxy)cytidine

(deoxy)thymidine

uridine

nucleobase

planar, aromatic, heterocyclic structural derivative of…

1. purine (adenine, guanine)

2. pyrimidine (cytosine, thymine/uracil)

nucleoside

nucleobase N + pentose

nucleotide

nucleobase + sugar + one or more phosphate groups

Purines: ribose at N_{_}

N₉

Pyrimidines: ribose at N_{_}

N₁

functions of nucleotides

information storage and transfer

free nucleotides and derivatives perform an enormous variety of metabolic functions

phosphate esters can form 3’ or 5’

nucleic acids = chain of nucleotides

• phosphate groups bridge the 3’ and 5’-positions of neighboring ribose units

• phosphates of polynucleotides = acidic

• 
  

at physiological pH (7.4), nucleic acids are

polyanions

information encoded in sequence (Chargaff’s rules)

complementary base pairing in dsDNA

G% = C%

A% = T% (or U%)

Dominant tautomer form of guanine and thymine

keto form

Double-Helical Model of DNA

Watson-crick-franklin

• R-handed helix

• Keto tautomer dominates

• Chargaff’s rules

• crystal structure

dsDNA (B-DNA) is antiparallel

complementary base pairing with H-bonding between b.p.

Number of H bonds between G:C and A:T?

3 between G:C

2 between A:T

Higher G:C content means a _____ T is required to melt/separate strands

higher T is required

Ring stacking

base stacking is enthalpically driven (greater thermal stability)

stacking interactions are a form of van der Waals (London dispersion/instantaneous dipoles)

interactions between G&C is greater due to greater thermal stability

How does Na⁺ help stabilize nucleic acids

cations shield the negative charges of the backbone

DNA is stabilized by Na ions b/c ions electrostatically shield the anionic phosphate group

DNA Melting curve

melting temp = temp at midpoint — gives useful info for G:C content

hyperchromic effect

DNA’s UV absorbance (due to aromatic bases) increases on denaturation as a consequence of electronic interactions among neighboring bases

UV absorbance increases as dsDNA—>ssDNA

Base orientations in B-DNA structures

Purines can be syn or anti conformation

Pyrimidines are stable in anti (syn configuration introduces steric interference)

Biologically most common form of DNA

B-DNA

In most double helical nucleic acids, all bases are in ____ conformation except in Z-DNA

most in anti conformation

Z-DNA is alternating

What conditions take B-DNA to A-DNA?

dehydrating conditions

A-DNA

right handed

major groove=narrow and deep

minor groove=wide and shallow

B-DNA

right handed

major groove: wide and deep

minor groove: narrow and shallow

standard

solid core

Z-DNA

left handed

solid core

RNA-RNA Double helix

the genetic material of certain viruses (synthesized only as a single strand)

RNA can form an A-form on double helix

RNA stem-loop

single strand of RNA folds back on itself to form a double-helical "stem" paired with an unpaired "loop" at the end

Yeast tRNA^Phe Structure

• Highly compact and L-shaped, with each leg ~60Å long.

• covalently modified bases (such as pseudouridine) and unusual base pairs

  • RNA exhibits greater structural/chemical variety than DNA because of these modifications.

• Tertiary Stabilization: Relies on unique hydrogen-bonding networks—including base triples (associations involving three bases)—to maintain its tightly folded, compact structure.

16s rRNA structure

• 16S = sedimentation coefficient of ribosome

• rRNA = ribosomal RNA

• Divided into distinct structural regions, including the 5'-domain, central domain, and 3'-domain.

• Characterized by numerous internal loops and hairpin turns that create binding sites for ribosomal proteins.

16S rRNA has unusual base-base hydrogen bonding interactions in 16S rRNA

Non-Watson-Crick Interactions: rRNA relies on complex hydrogen-bonding networks outside of normal base-pairing

These unusual interactions help stabilize the intricate 3D tertiary architecture necessary for ribosomal function.

Why is DNA more stable than RNA?

RNA is highly susceptible to base-catalyzed hydrolysis

this makes DNA the dominant molecule of heredity

Step-by-Step Mechanism:

1. Deprotonation: A base induces deprotonation of the 2'-OH group.

2. Nucleophilic Attack: The activated 2'-oxygen attacks the adjacent phosphorus atom in the backbone.

3. Cleavage: This cleaves the RNA backbone, forming a 2',3'-cyclic nucleotide intermediate before yielding a 2'- or 3'-nucleotide.

Ribozymes: Catalytic RNA molecules that can accelerate reactions and cleave RNA strands.

Hammerhead Ribozyme

Features three distinct helical regions (Stem I, Stem II, and Stem III) connected by conserved sequence motifs and a characteristic uridine turn.

The 3D fold positions key residues (such as G-8, C-1.1, A-9, and G-12) precisely around the scissile bond to execute site-specific self-cleavage.

Semiconservative DNA Replication

Each resulting DNA molecule consists of one parental (old) strand and one progeny (new) strand.

DNA Polymerase: Synthesizes the new strand using the template strand.

Helicase: Unwinds the double-stranded DNA parental helix.

Complementarity: Base-pairing rules ensure transmission of genetic info

flow of genetic info

transcription and translation

1 strand of DNA directs mRNA synthesis

base sequence of transcribed RNA is complementary to DNA strand

message=translated when tRNA molecules align with mRNA by codons

**each tRNA carries a specific amino acid

Polymerase Chain Reaction (PCR)

apparatus = thermocycler

1. denature DNA with heat (sep. by melting)

2. Annealing with primers for the gene of interest while cool

3. Add dNTPs and Taq polymerase for synth of new strands using primer as starting point

4. repeat cycles

Gel Electrophoresis

DNA is negatively charged (b/c phosphate backbone), so it moves neg—>pos on gel

gel = usually agarose (for DNA)

Sanger sequencing (Chain termination sequencing)

1. denature DNA with heat

2. annealing with primers for gene of interest while cool

3. add dNTPs and Taq polymerase AND fluorescently-labeled dideoxynucleotides

4. repeat cycles — but not sometimes the ddNTPs are incorporated instead of dNTPs

5. If ddNTPs are tagged with fluorescent molecules, sequence can be determined following DNA strand separation based on size (using a gel)

ddNTPs in Sanger Sequencing

stop the chain from growing b/c no 3’ OH

very important to keep dNTPs » ddNTPs

Illumina sequencing (reversible terminator sequencing)

1. DNA is fragmented to a few hundred b.p./ fragment

2. DNA is bound to specific adapters of known sequence and immobilized on a flow cell

3. in place, some Illumina preps now do PCR to form “clusters” of identical DNA strands at a particular location

4. Add primer specific adapter of known sequence, DNA polymerase, and all modified dNTPs

5. in between each bonding, the fluorescent label and blocking group are removed with washing, allowing the next modified nucleotide to bind

   1. all of these clusters are sequenced simultaneously

difference in modified dNTP role between Sanger sequencing and illumina

modified dNTPs are chain terminators

modified dNTPs are reversibly chain terminators

why is a flow cell helpful in illumina sequencing

solid support: anchor ssDNA fragments

primary rxn chamber: houses the physical space needed for bridge amplification/exclusion amplification