Detailed Notes on RNA, DNA, and Replication

RNA vs DNA - Wednesday 19

• RNA has ribose instead of deoxyribose (DNA). The only difference is one oxygen atom.

• RNA has uracil instead of thymine.

• RNA is single stranded, making it less stable but more diverse.

• RNA tends to be shorter than DNA.

• mRNA corresponds to a gene, microRNAs are smaller.

• RNA is diverse in function, including gene regulation and catalysis.

• Three Major RNA Groups:

  • messenger RNAs

  • ribosomal RNAs

  • transfer RNAs

• All RNAs are transcribed from DNA, not created de novo.

• RNAs are temporary and degraded, unlike stable DNA. Enzymes called RNAses degrade RNA.

• mRNA vaccines are advantageous because:

  • They are safer due to short lifespan in the system.

  • They are more efficient to produce.

  • They don't interact with DNA in the cell nucleus.

  • Protein is synthesized by the individual.

Types of RNA

• Ribosomal RNAs (rRNAs) are components of ribosomes and necessary for protein synthesis; some drive catalytic events.

• Messenger RNAs (mRNAs) are templates for protein synthesis, corresponding to a specific gene.

• Transfer RNAs (tRNAs) bring amino acids to the ribosome for protein synthesis.

• Other RNAs:

  • Telomerase RNAs (eukaryotic cells)

  • RNA primers

  • Small nuclear RNAs (eukaryotic cells)

  • MicroRNAs, small interfering RNAs (gene regulation in all cells)

• All RNAs are encoded by DNA, transcribed, and then degraded.

DNA Accessibility

• DNA is a double helix, making genetic information not freely accessible.

• Transcription requires the DNA double helix to unwind to access a single strand.

• DNA strands are held together by hydrogen bonds, which are weaker than covalent bonds.

• Adding energy, such as heat, can separate DNA strands.

• The longer a DNA strand, the higher the temperature needed to unwind it.

• $G-C$ content affects unwinding temperature. Three hydrogen bonds between $G$ and $C$, two between $A$ and $T$.

• Melting Temperature ($T_m$): The temperature at which DNA starts to separate.

• $T_m$ is important in PCR and DNA hybridization.

• GC content: Higher GC content requires a higher melting temperature.

Molecular Hybridization

• Used to detect specific sequences in a sample (e.g., virus or bacteria).

• A known DNA sequence (probe) is used to see if it hybridizes (attaches) to DNA from a sample.

• The probe may be labeled with a radioactive isotope, fluorescent protein, etc.

• Process:

  • Purify DNA from the sample.

  • Melt (denature) the DNA into single strands.

  • Add the labeled DNA probe.

  • Observe if hybridization occurs.

• Principle relies on complementary base pairing (e.g., A-T, C-G).

• PCR uses repeated cycles of hybridization to amplify DNA.

• Applications include identifying species or DNA presence in a sample and gene expression experiments.

Fluorescence In Situ Hybridization (FISH)

• FISH: Fluorescence in situ hybridization.

• Uses fluorescent probes to monitor hybridization.

• Can be used with RNA to study developmental genes.

• Enables tracking of gene expression in specific cell types and at specific time points.

• Requires a DNA probe complementary to the target sequence, labeled with a fluorescent protein (e.g., GFP).

Gel Electrophoresis

• Separates DNA (or RNA) based on size, using a gel matrix with pores.

• Larger molecules travel slower; smaller molecules travel faster.

• A DNA ladder (fragments of known sizes) is used for comparison.

• Can determine if a specific DNA fragment of a known size is present in a sample.

• Allows for purification of specific DNA fragments.

• Process:

  • DNA runs through an agarose gel (a porous matrix).

  • An electrical current is applied, causing charged DNA to move.

  • Smaller DNA fragments move faster through the gel.

  • Visualized using chemicals like ethidium bromide under UV light.

DNA Replication

• Semiconservative: DNA replicates by unwinding and using each strand as a template.

• Each new DNA molecule has one old and one new strand.

• If you have one strand, you have all the information you need to.

• Process:

  • DNA unwinds to form single strands.

  • Each strand serves as a template for a new strand.

  • New nucleotides are added based on complementary base pairing.

• Single-stranded DNA can be synthesized from a single strand during bacterial conjugation or transformation.

• In eukaryotes, this occurs during the S phase of interphase, creating sister chromatids.

Hypotheses of DNA Replication

• Conservative: Original DNA is a template. The new DNA molecule is completely new.

• Semiconservative: Each new molecule contains one old and one new strand.

• Dispersive: Parental strands are dispersed into new helices, forming a hybrid.

Meselson and Stahl Experiment

• Used isotopes of nitrogen ($^{15}N$ and $^{14}N$) to label DNA.

• Grew E. coli in $^{15}N$ media, then transferred to $^{14}N$ media.

• Separated DNA using a high-speed centrifuge.

• Results:

  • After one generation, DNA consisted of one strand of old and one of newly synthesized strands.

  • Eliminated the conservative replication model.

  • After another cycle, eliminated the dispersive model.

  • Confirmed semiconservative replication.

Taylor, Woods, and Hughes Experiment

• Showed that the Vikia fava DNA replication was semi conservative through the use of autoradiography.

Bacterial DNA Replication

• Bacteria usually have a single, circular, double stranded DNA chromosome.

• Replication initiates at a single origin of replication, or OriC site.

• Replication proceeds in both directions until the replication forks meet at the terminus.

• Replisomes are protein collections that synthesize the new DNA.

• About 4.5 million bases in E. coli.

Enzymes in DNA Replication

• DNA polymerases are enzymes that polymerize DNA.

• 5 different DNA polymerases in E. coli, numbered in order of discovery.

• DNA polymerases require a template strand of RNA or DNA. They can only initiate DNA polymerization using a template. They do not do it de novo.

RNA Primers

• An RNA polymerase lays down a short primer is an RNA template for DNA polymerase to attach to.

• RNA primer of RNA that tells the DNA polymerase when to attach.

• DNA polymerases can only synthesize in the 5' to 3' direction.

• DNA polymerases add one nucleotide at a time at the three prime end. (like Legos)

• Requires an exposed 3' hydroxyl group to attach the new nucleotide.

• The phosphodiester bond is formed between the phosphate and the sugar.

• The process of synthesizing a nucleotide involves 2 nucleotides. You'll have a 5’ end and a 3’ end. DNA polymerases can only attach the next nucleotide to the 3’ hydroxyl group.

• Nucleotide triphosphates are used, removing two phosphate groups to provide energy for the phosphodiester bond.

DNA Polymerase Types

• DNA polymerases 1, 2, and 3:

  • Elongate existing DNA strands using a primer.

  • Cannot initiate DNA synthesis.

  • Have exonuclease activity (3' to 5' and 5' to 3') for proofreading and removing DNA/RNA.

• DNA polymerase 1:

  • Removes RNA primers.

• DNA polymerase 3:

  • Main enzyme for new DNA synthesis.

  • Works 5' to 3'.

• In an average bacterial cell, there are 400 copies of DNA polymerase one. DNA polymerase three has only 15.

DNA Polymerase Functions

• DNA polymerase 3: main enzyme for DNA replication.

• DNA polymerase 1: removes RNA primers and fills the gaps.

• DNA polymerases 2, 4, and 5: primarily involved in DNA repair.

DNA Polymerase III Holoenzyme

• DNA polymerase III functions as a holoenzyme: many separately encoded polypeptides combine.

  • Alpha subunit: polymerizing portion (5' to 3').

Requirements of DNA Replication

1. Unwind the double helix.

2. Alleviate supercoiling.

3. RNA primer for DNA polymerase.

4. Synthesize a second strand using the old strand as a template.

5. DNA polymerase 1, removes the RNA primers.

6. DNA ligase to attach everything.

7. A high fidelity as possible, make as few of errors as possible. DNA goes through a proofreading process.

Enzymes in DNA Replication

• DNA helicase:

  • Unwinds the double helix.

  • Moves in front of DNA polymerase III.

• Topoisomerases (e.g., DNA gyrase):

  • Cut DNA.

  • Allow it to unwind to alleviate coiling/pressure.

  • Reattach the strands.

• Single-stranded binding proteins:

  • Attach to single-stranded DNA.

  • Ensure strands don't re-anneal.

• DNA Polymerases only synthesize in the five prime to three prime direction because they can't synthesize three prime and five prime.

  • Because the synthesis only happens in the five prime to three prime direction, it can't synthesize three prime and five prime, which causes a problem.

  • The problem happens because as you unwind this, you what you have here is what's known as the leading strand, and this is going to have a template on it, and it's going to have a three prime hydroxyl on this newly synthesized strand. This tells the DNA polymerase to go in this direction.

  • In the other direction its anti parallel so it can't be synthesized this way, which makes it the discontinuous or lagging strand.

• Leading Strand:

  • Continuous synthesis (5' to 3').

• Lagging Strand:

  • Anti-parallel.

  • Discontinuous synthesis.

  • Okazaki fragments: short DNA fragments synthesized backwards.

  • Requires repeated RNA primers.

• DNA polymerase one must remove RNA primers on the lagging strand.