Biochemistry Chapter 2; Nucleic Acids

Nucleic Acids and PCr

What is the structure of ribonucleotides?

Ribonucleotides contain a nitrogenous base (A, G, U, C), a carbohydrate sugar, and a phosphate.

• The 5 carbon sugar contains an OH group.

What is the structure of deoxyribonucleotides?

Deoxyribonucleotides contain a nitrogenous base (A, G, C, T), a carbohydrate sugar, and a phosphate.

• The 5 carbon sugar only contains H, no OH.

What are the properties of nitrogenous bases?

1. They are heterocyclic; containing 1 or 2 rings (pyrimidine:purine).

2. They contain ketone, amine, or methyl groups to participate in H-bonding).

3. They’re never found in an ionized state - are nonpolar.

4. They’re aromatic and have resonance; can absorb UV.

What are the purines?

Heterocyclic n-bases with 2 rings;

• Adenine

  • Has an amino bonded to a ring C.

• Guanine

  • Has an amino and double bonded oxygen bonded to ring C’s.

*Used in DNA, RNA, metabolism, signalling.

What are the pyrimidines?

Heterocyclic n-bases with 1 ring;

• Cytosine

  • Has an amino group bonded to a ring C, and a double bonded oxygen.

  • Bonds to Guanine.

• Thymine

  • Has two double bonded oxygens and a methyl group.

  • Bonds to Adenine (DNA).

• Uracil

  • Has two double bonded oxygens.

  • Bonds to Adenine (RNA).

What is tautomerism?

When nitrogenous bases migrate protons, which results in manipulated functional groups in the enol form.

• NH2 → C=NH

  • Amino → Imino

• C=O → C — OH

  • Ketone → Hydroxyl

Why is tautomerism bad?

It can modify hydrogen bonding properties, and alter catalytic centers.

DNA vs deoxyribonucleotide?

DNA is made of multiple deoxyribonucleotides.

What is a nucleoside?

A 5 carbon sugar linked to a nitrogenous base through a glycosidic bond at the 1’C. 

• DNA; A, G, C, or T glycosidically bonded to the 1st carbon of the sugar ring.

• RNA; A, G, C, or U glycosidically bonded to the 1st carbon of the sugar ring.

What is a nucleotide?

A nucleoside bound to 1 phosphate group through a phosphoester link to the OH group on the 5’C.

• Nucleoside monophosphate.

Additional phosphate groups can be added through phosphoanhydride links.

• Nucleoside di/tri=phosphates.

Characteristics of nucleotides?

1. The phosphate group produces a negative charge.

2. Triphosphates can donate 3 protons - polyprotic; ATP.

   1. They can link with divalent metal ions.

   2. They hydrolyze with negative ΔG; they can be used in coupling to drive non-spontaneous reactions.

How do nucleotides come together to form DNA or RNA? What type of bond and where?

A phosphodiester bond is created between the phosphate of one nucleotide (at the 5’ C), and the hydroxyl on the 3’C of another.

• Water is released as a byproduct.

Where is the code of life stored, and transferred, according to the central dogma?

In purine and pyrimidine base matching, through hydrogen bonds.

What base pairs are harder to denature?

G-C base pairs, because they have 3 sites of H-bonding, increasing the melting point. Therefore they are harder to break compared to A-T/A-U.

• G-C dense areas are harder to denature.

DNA vs RNA?

DNA:

• Two antiparallel strands of deoxyribonucleotides, twisted into a double helix.  

  • T pyrimidine.

  • Deoxy.

RNA:

• A single strand of ribonucleotides bound together by phosphodiester bonds, folded into complex figures.

  • U pyrimidine.

  • Ribo.

How do proteins interact with DNA?

Through the major and minor grooves formed by the double helix.

What forces stabilize the DNA double helix?

Stacking of n-bases in the hydrophobic core of the helix.

1. Triggers the hydrophobic effect, allows water to be less ordered and have higher entropy.

2. Has dipole-dipole, pi-pi, and London dispersion forces.

Charge-charge repulsion of phosphate groups.

• Coulombic effect.

Three types of DNA helixes? Most common one in cells?

A, B, and Z DNA.

B DNA is most common cells; wider major groove and narrower minor groove.

A DNA is common in labs.

Z DNA is a left-handed twist common in complexes.

Ratios of purines:pyrimidines and their pairs?

The ratio of purine:pyrimidine pairing is 1:1 in any strand; if 30% of my DNA strand contains A, then 30% of it must also contain T (its base pair).

• 40% is left, split between G:C; so 20% each.

What forces fold the RNA strand?

RNA forms double stranded sections of 3-12 nucleotides using a single strand.

• Hydrophobic effect.

• Base stacking; dipole-dipole, h-bond, london dispersion.

Why does replication occur from 5’-3’?

Because the OH on the 3’C is capable of nucleophilic attack, allowing the formation of a new phosphodiester bond with another nucleotide (via their phosphate group). Replicating from 5’-3’ leaves this site open for new polymerization.

• Binding at the phosphate end (3’-5’) would make proofreading impossible due to the loss of energy needed for coupling.

Structural features of DNA replication? What keeps it at a low error rate?

DNA is organized into chromosomes, containing all the genetic material of the cell.

• The antiparallel double strand formation allows for two template strands.

  • A daughter cell receives one parent and one synthesized — semi-conservative.

• Replication forks make leading and lagging strands; okazaki fragments have to be ligated on the lagging strand.

  • DNA pol can only synthesize 5’-3’.

What is the rate-determining step in eukaryotic cell division?

DNA replication.

What is bidirectional replication and why is it helpful?

It refers to the idea that DNA pol can only add nucleotides to the 3’ end OH of the strand. This means that in a replication fork both a leading and lagging strand are synthesized, bidirectionally and outwards..

• Allows for multiple replication forks to be working on a chromosome simultaneously.

What is the pathway to synthesizing proteins?

DNA is first transcribed into mature mRNA, which is translated into protein by ribosomes and tRNA.

Key features of transcription?

1. Eukaryotic genes coded in DNA are separated into introns (non-coding) and exons (coding).

2. Transcription factors bind to the 5’ end, promoter region, to elicit RNA pol to make a premature mRNA.

3. Introns are spliced out to form a mature mRNA.

4. A 7-meth.guan cap and poly-A-tail are placed on the 5’ and 3’ end to prevent degradation and stabilize.

Key features of transcription?

1. Ribosomes catalyze the reaction.

2. tRNA reads the mRNA code in 3’s, and transfers a specific amino acid to the chain. 

   1. There is 1 code for 1 AA.

How can we regulate gene expression ?

1. Transcription factors can up or downregulate gene expression and mRNA synthesis.

2. mRNA riboswitches fold it into a complex shape, disabling translation.

3. RNAi forms a dsmRNA.

Virus vs Retrovirus?

A virus contains DNA or RNA as they genetic material.

A retrovirus has RNA genes, but uses a DNA intermediate at some point.

Both infiltrate host cells to survive and replicate.

How do mutations affect the central dogma?

Chemical degradation via UV light or heating can denature or mutate DNA

• Chemical damage can invoke crosslinks in bases, altering the transcription pathway.

What is the central dogma?

The flow of genetic material; how information is expressed in the genome.

• DNA→RNA→Protein.

Name the three steps of PCr?

1. Denature; heat to break h-bonds and separate strands (90c).

2. Anneal; to create forward and reverse sites through primers (cool temp).

3. Extension; Heat to synthesize via taq (72c).

Amplify and repeat.

What are the properties of PCr? What are the 5 components?

• In vitro.

• Amplifies specific DNA fragments.

• Only replicates selected pieces.

• Creates an unmodified DNA copy.

1. DNA primers to target each end.

2. Taq polymerase to bind and add.

3. dNTP’s to create new strand.

4. Buffers

5. Target DNA

What amount/ratio does PCr amplify?

It doubles the number of double-stranded pairs of DNA each cycle.

• Quadruples the number of single DNA strands.

• Amplified 10^6 times after 25 cycles.

What enzymes can manipulate DNA?

1. Restriction enzymes; cut sequences.

2. Ligase; joins fragments.

3. Polymerase; generates a copy.

4. Topoisomerase; unwinds DNA.

5. Reverse transcriptase; converts RNA→DNA.

How does DNA cloning occur?

Occurs in vivo (a living organism), using plasmids, target DNA, and a carrier host.

• Restriction enzymes cut a fragment of target DNA and a plasmid.

  • Plasmids (short bacterial loops, cloning vectors) are capable of inserting and replicating in a host.

• The target DNA is ligated to the plasmid, forming recombinant DNA.

• The recombinant DNA is inserted into a bacterial host through transformation, transfection, or infection.

• Host cells replicate the recombinant DNA.

What does cloning mean?

To generate copies of DNA by inserting a fragment into another living host, and propagating it through growth and replication.

How can we verify successful cloning transformations?