Anaphy 12H Unit 2

DNA

Deoxyribose Nucleic Acid.

Substance of inheritance. Hereditary information (comes from parents) is encoded here and reproduced in all cells of the body. Directs development of biochemical (what proteins we need, how many proteins we need), anatomical, physiological, and behavioral traits.

Rosalind Franklin

1953, discovered structure of DNA through the technique called x-ray crystallography, take DNA, put it in a solvent and turn it into crystals, freezing it and shock x-rays at it. Produced a picture, photo 51, that helped Watson determine the double helix structure. Photo 51 was stolen by Maurice Wilkons and given to Watson and Crick

Bacteria/viruses

Used to study DNA since they replicate quickly and efficiently with a small scale.

Frederick Griffith

1928, discovered genetic role of DNA. Worked with two strains of a bacterium, one that could infect, and the other was harmless. Mixed heat killed remains of infecting strain with living harmless strain, and some of those harmless strains became infective. This phenomenon was called transformation.

Transformation

Change in genotype and phenotype.

Bacteriphages/Phages

Viruses that infect bacteria. Used in molecular genetics research to gain more evidence about DNA being the genetic material

Alfred Hershey and Martha Chase

1965, Performed an experiment to prove that DNA was a genetic material. Grew some viruses in a medium that contained radioactive phosphorus (radioactive DNA), and grew other viruses in another medium that contained radioactive Sulphur (radioactive protein). Both were allowed to infect bacteria separately. After they infected bacteria, the viral codes were removed by the bacteria by agitation. The viral particles were separated from the bacteria through a center fuge. Bacteria were tested for the presence of radioactivity. Bacteria infected by radioactive phosphorus contained radioactive DNA that was passed on. Bacteria that were infected by the radioactive Sulphur contained no radioactive protein. Confirmed that genetic material is DNA.

Radioactive tagging

Taking elements and using the isotopes that give off a signal. Track where the molecules go.

Chargaff’s rule

In any species, there is an equal number of adenine and thymine bases and an equal number of guanine and cytosine bases.

DNA replication

2 complimentary DNA strands splits apart and each strand is used as a template to build a new complimentary strand producing 2 DNA molecules and a replication bubble. The base pairings would be the same, but the information would not be the same.

DNA replication (in the cell)

Untwist the double helix. There is a 3’→5’ end and a 5’→3’ end orientation. The two strands run in opposite directions. Starts at specific sites called origins of replication. Proteins attach here and separate DNA strands, forming a replication bubble that grows in both directions. Enzymes called DNA polymerases move along DNA template strands and catalyze the elongation of new strands. Can only assemble new DNA in the 5’→3’ direction. Only one strand can synthesize in a continuous piece, whilst the other strand is synthesized in short pieces. These short pieces are synthesized together by the enzyme DNA ligates. Eventually all the replication bubbles merge, showing 2 new identical DNA molecules.

Semi-conservative model

Semi = half

Conservative = conserving the original

One new DNA molecule would have one half of the parent molecule, and the other half as the offspring molecule.

Conservative model

Original parent molecule stays the same and an entire new daughter molecule is synthesized.

Dispersive model

Each new strand is a mixture of old and new DNA segments from parent and daughter molecules.

Semi-conservative advantages

More accurate, efficient, and faster. It’s easier to copy something than to make a whole new thing.

Meselson-Stahl experiment

Determine which of the three DNA replication models was correct. Labeled nucleotides that the old strands of the heavy isotope of nitrogen. The new strands were lighter isotopes of nitrogen.

Predictions of Meselson-Stahl experiment

Conservative: after 1 round, would have 2 bands, 1 heavy and 1 light. After 2 rounds, there would still be 2 bands.

Semi-conservative: after 1 round, would have 1 intermediate band (half heavy, half light). After 2 rounds, would have 2 bands, 1 intermediate and one light.

Dispersive: After 1 round, 1 intermediate band (a mix). After 2 rounds, would have 1 slightly lighter intermediate band (all molecules a mix of old and new).

Results of Meselson-Stahl

After one round, a single band of intermediate density, meaning that the conservative model could not be a candidate anymore.

After 2 rounds, two bands were observed, one intermediate and one light band, confirming that the semi-conservative model was how DNA replicates

Origin of replication

Specific site of DNA sequences where replication begins. Proteins attach and separate DNA strands, that forms replication bubbles.

Prokaryotes

Typically have one origin due to small, circular DNA.

Eukaryotes

Have hundreds to thousands off origins to quickly replicate their massive linear chromosomes.

Replication forks

Y-shaped regions at the ends of replication bubbles where new DNA strands are elongated.

Key enzymes and proteins

1. Helicase - Unwinds and untwists the double helix, separating the two DNA strands.

2. Single-Strand Binding Proteins (SSBPs) - Bind to and stabilize the separated single strands, preventing them from re-annealing

3. Topoisomerase - Relieves the overwinding tension ahead of the replication for by breaking, swiveling, and rejoining DNA strands

4. Primase - Synthesizes short RNA primers, which provide the necessary 3’ end for DNA polymerase to start adding nucleotides.

5. DNA Polymerase - Catalyzes the elongation of new DNA strands by adding nucleotides complementary to the template. It has 2 limitations.

6. DNA Ligase - Joins together the short DNA fragments, also known as Okazaki fragments, on the lagging strand

DNA Polymerase limitations

1. Can only synthesize DNA in the 5’→3’ direction, meaning only put in base pairings in the 5’→3’ direction.

2. Cannot initiate synthesis without a pre-existing 3’ hydroxyl group (end).

Antiparallel Nature

DNA strands run in opposite directions (one 3’→5’, the other 5’→3’).

Leading strand

Synthesizes DNA continuously in the 5’→3’ direction, moving towards the replication fork. Only needs one primer at the origin.

Lagging strand

Synthesizes DNA repeatedly in short segments known as Okazaki fragments. Synthesizes in the 5’→3’ direction, but away from the replication fork. Needs multiple RNA primers, one for EACH Okazaki fragment. After synthesis, DNA polymerase 1 removes the RNA primers and replaces them with DNA nucleotides. DNA ligase then joins the Okazaki fragments to form a continuous strand.

DNA damage and repair

The DNA in one cell is damaged tens of thousands times per day, which equates to about a quintillion DNA errors daily across the body’s trillion cells. Damage to DNA causes serious problems, like cancer

Types of errors

• Damaged nucleotides (DNA’s building blocks)

• Incorrectly matched nucleotides, causing mutations

• Nicks (breaks) in one or both strands, which interfere with replication or cause DNA sections to get mixed up.

Repair Mechanisms

Cells have ways of fixing most problems, usually relying on specialized enzymes. There are specific repair pathways.

Mismatch Repair

The enzyme, DNA polymerase, makes an error about once every 100,000 additions during DNA replication. The DNA polymerase usually catches and corrects most mistakes immediately by cutting off and replacing nucleotides. A second set of proteins performs mismatch repair, checking for missed errors, cutting out the incorrect nucleotide, and replacing it. Together, these systems reduce base mismatch errors to about 1 in 1 billion.

Base excision repair

Used when just one base is damaged (often by chemical changes from environmental exposure). One enzyme snips out the damaged base, and other enzymes trim the site and replace the nucleotides.

Nucleotide Excision Repair

Used for harder-to-fix damage, such as when UV light causes two adjacent nucleotides to stick together, distorting the double helix shape. A team of proteins removes a long strand, about 24 nucleotides, and replaces them with fresh ones. The process involves a nuclease, cut out sections, DNA polymerase 1 fills the gap, and DNA ligase links the ends.

Double Strand Breaks

Caused by very high-frequency radiation (gamma rays and x-rays) and are the most dangerous type of damage; even one break can cause cell death.

Homologous Recombination

Uses and undamaged section of similar DNA as a template to repair the break, filling in missing gaps

Non-homologous end joining

Does not rely on a template. Series of proteins trims off a few nucleotides and fuses the broken ends back together. This method is less accurate and can cause genes to get mixed up/moved around, but is useful when sister DNA is unavailable.

Mutagens

Falls into three types

• Physical: Radiation, such as X-rays and UV light.

• Chemical: Molecules like acetone, cleaning products, or compounds in cigarette smoke.

• Biological: Viral or bacterial infections

Mutations

Often harmful, but beneficial mutations can allow a species to evolve. Defects in DNA repair are associated with premature aging and many kinds of cancer.

DNA shortening

Usual DNA replication machinery cannot complete the 5’ ends of linear eukaryotic chromosomes after the primer is removed. DNA polymerase can only add nucleotides to a 3’ end, not a 5’ end.

Repeated rounds of replication result in DNA molecules becoming shorter by 5-10 nucleotides each round. If this shortening occurs in “junk DNA” (sequences that do not code for a particular trait), there is no immediate consequence. However, if the shortening progresses downstream to an important gene, it will affect the type of protein made.

Telomeres

Eukaryotic chromosomes have nucleotide sequences called telomeres at their ends. Extra bits of DNA that postpone the shortening and the erosion of essential genes. These shortenings are proposed to be connected to aging. As DNA shortens, essential genes may lose information necessary to make functional proteins.

Germ cells

To ensure essential genes are not lost form gametes (sperm and egg cells, which are germ cells, the enzyme telomerase catalyzes the lengthening of telomeres in these cells.

Eukaryotic chromosomes

Linear DNA molecules associated with a large amount of protein.

Bacterial chromosomes

Double-stranded circular DNA associated with a small amount of protein, supercoiled, and located in a region called the nucleoid.

Histones

Proteins responsible for the first level of DNA packing in chromatin.

Chromatin

The complex mixture of DNA wrapped around histone proteins, often visualized as a “string of beads.” The purpose of wrapping is to protect the DNA and prevent access to genes until they are needed. Chromosomes are only visible during cell division, appearing during prophase and lasting through telophase.

Naked DNA

Pure, unwrapped DNA, only visible when a gene needs to be expressed, only visible when a gene needs to be expressed. DNA exists as chromatin or naked DNA about 99% of the time.

Euchromatin

Loosely packed chromatin.

Heterochromatin

Highly condensed chromatin. Dense packing makes it difficult for the cell to express genetic information in those regions.

Gene expression

Also known as protein synthesis, is the process by which DNA dictates the synthesis of proteins, leading to specific traits (phenotypes). Information content of DNA is in the form of specific nucleotide sequences. The gene specifies what kind of protein to make. Involves transcription and translation, are central to life, representing the flow of genetic information from DNA to mRNA protein.

Gene expression cheer

ONE GENE = ONE PROTEIN

Many proteins are composed of several polypeptides, each with its own gene. Most accurately stated as the one gene, one polypeptide hypothesis.

Transcription

The process of making a copy of the required gene into messenger RNA (mRNA). The mRna carries the instructions out of the nucleus.

Translation

the process of translating the mRNA message into a polypeptide (protein)

Eukaryotes vs. Bacteria

In eukaryotic cells, the nuclear envelope separates transcription from translation. Transcription happens in the nucleus, and translation happens outside in the cytoplasm. In bacteria, both occur in the cytoplasm.

Primary Transcript (pre-mRNA)

The initial RNA transcript, which needs to be processed or edited before it becomes mature.

Ribosomes

The machinery that translates the mRNA. Ribosomes are made up of proteins and rRNA. They are produced in the nucleolus. Ribosomes consist of a large subunit and a small subunit and have binding sites for mRNA, as well as E, P, and A sites.

Triplet Code

The flow of information. Every three nucleotides codes for an amino acid. This allows the four nucleotide bases to specify the 20 amino acids.

Codon

The name for a base triplet (three nucleotides) on the mRNA. One codon codes for one amino acid. 61 codons code for amino acids.

Transcription process

• Only one strand of DNA is used as a template: the template strand.

• RNA polymerase - creates the mRNA transcript

• RNA polymerase reads the template strand in the 3’ to 5’ direction.

• mRNA transcript is synthesized in the 5’ to 3’ direction.

• Read up, write down

• Base pairing - During transcription, Adenine in DNA pairs with Uracil in RNA instead of Thymine

Translation process

• mature mRNA leaves the nucleus and into the cytoplasm, where ribosomes are located, floating in cytoplasm or attached to rough endoplasmic reticulum.

• The ribosome reads the codons along the mRNA in the 5’ to 3’ direction.

• The sequence of three nucleotides dictates which of the 20 amino acids is placed at the corresponding position on the polypeptide.

• Reading must be done sequentially, left to right; reading in reverse will yield the wrong amino acid sequence.

• Resulting amino acid sequence is the polypeptide, which presents the primary structure of the protein.

Gene

Codes for a specific protein. The resulting proteins perform the tasks necessary for living things to function.

Non-template strand (trick)

Goes 5’ to 3’. Resulting mRNA will be identical to the non-template strand, but with all Thymine replaced with Uracil.

Properties of the Code

Redundant but not ambiguous.

Redundancy - There is more than one codon for a specific amino acid, acting as a backup plan, increasing the chance that a mutation still results in the correct amino acid.

Not ambiguous - No codon specifies more than one amino acid. The codes are specific, meaning a particular codon codes for one specific amino acid.

Reading Frame

Determines how the nucleotides are grouped into three-letter codons. Ribosome finds the correct one by locating the start codon. If no codon is specified, assume that it begins with the first three nucleotides.

Types of mutations

• Point mutation - a mutation involving a single base change (a deletion of one base)

• Frame shift - Occurs when a base is deleted/added, causing all subsequent nucleotides to shift over, fundamentally changing the reading frame.

• Nonsense - Cuts the protein short by causing premature stopping codons. Results in an incomplete/non-functional protein.

• Missense - Results in the use of the wrong type of amino acid.

Amino Acid sequence

Determined by matching the mRNA codons to a codon table

Genetic Code

Nearly universal, shared by organisms from the simplest bacteria to the most complex animals. Genes Can be transplanted and still be transcribed and translated in the new species.

Transcription unit

Segment of DNA transcribed into an RNA molecule

Promoter

Area on the DNA located prior to the gene. Promotes transcription by encouraging/helping the RNA polymerase to bind the DNA strand

RNA polymerase function

1. unwinds the two DNA strands

2. picks the template strand to read (3’-5’)

3. synthesizes an RNA molecule

Initiation

Start of transcription. Promoter signal this of RNA synthesis

Transcription Factors

Other proteins that mediate the binding of RNA polymerase and the initiation of transcription. Coded by other genes, possibly in regions previously considered “junk DNA”

TATA box

Crucial promoter sequence in eukaryotes. TFs bind to this, which creates a specific 3D space/shape that acts as an adapter, making it easier for RNA polymerase to bind to the gene site.

Transcription Initiation Complex

Formed when the necessary transcription factors and RNA polymerase 2 are bound to the promoter.

Signal Transduction Connection

Production of transcription factors can be triggered by external signals or hormones that initiate a chemical cascade leading to gene expression

Elongation

Process of synthesizing the RNA transcript by adding RNA nucleotides using complementary base pairing.

Terminator

A sequence on the DNA found after the gene. Tells the RNA polymerase to stop transcribing, unclamp, and fall off the DNA. Ensures the polymerase only transcribes the necessary gene and prevents transcription of adjacent genes. Mechanisms of this differs slightly between bacteria and eukaryotes..

RNA processing

Modifications made to the primary RNA transcript (premature) inside the nucleus before its ready.

End modifications

Both ends of the primary transcript are altered.

1. 5’ Cap - A modified nucleotide added to the 5’ end

2. Poly-A Tail - A large number of Adenine bases added to the 3’ end.

Functions of Cap and Tail

Help facilitate the export of the mRNA from the nucleus, protect the mRNA from being recycled or digested by hydrolytic enzymes, and help ribosomes attach to the 5’ end.

RNA splicing

Interior parts of the molecule are cut out, and others are added together. Resulting in mature mRNA to be shorter than the primary transcript

Introns

Regions within the gene that are cut out or ripped because they are not needed. Thought to be either inherited, non-useful genes or regions that protect the actual gene from damage/mutations

Exons

Regions that are kept and expressed because they code for the protein. The exons are glued together after introns are removed.

Splicing Machinery

Spliceosomes (enzymes/proteins) and ribozymes (RNA molecules that function as enzymes) are responsible for cutting out introns and gluing exons.

Alternative RNA splicing

Exons can be rearranged in different ways after splicing to create different proteins from the same gene

tRNA (Transfer RNA)

Transfers the appropriate amino acid. Its codons are NOT the amino acid. folded RNA molecule that carries a specific amino acid on one end and an anti-codon (three nucleotides) on the other end.

rRNA (Ribosomal RNA)

A component of ribosomes

tRNA vs. mRNA

The anti-codon's job is only to match up with the complementary codon on the mRNA; it acts as a check to ensure the correct package (amino acid) is delivered. The amino acid sequence is dictated by the mRNA codon table, not the tRNA anti-codon.

Cell Structure

The cell is the basic unit of all living tissue. The nucleus contains the genome.

tRNA Charging

An enzyme called aminoacyl tRNA synthetase (or acetyl tRNA) is responsible for adding the correct amino acid to the tRNA molecule. Cells must assemble these charged tRNAs ahead of time.

Ribosomal Sites (EPA)

The ribosome contains three sites where translation occurs:

    ◦ A site (Aminoacyl): Holds the tRNA that carries the next amino acid to be added to the chain (the incoming amino acid).

    ◦ P site (Peptidyl): Holds the tRNA that carries the growing polypeptide chain.

    ◦ E site (Exit): The exit site where the empty or discharged tRNA is ejected.

Release factor

Stop codon is in a site. Inserts a water molecule instead of an amino acid. This reaction (hydrolysis) frees the polypeptide chain, and the translation machinery comes apart.

Post-Translational Modifications

Polypeptide chains are often modified after translation. Modifications may occur in the Endoplasmic Reticulum (ER) and the Golgi body. Some proteins are inactive initially and require cutting or folding under special conditions to become active (e.g., blood clotting enzymes).

Targeting Secreted Proteins

◦ Polypeptides destined for the ER or secretion possess a signal peptide.

◦ A signal recognition particle (SRP) binds to this signal peptide.

◦ The SRP brings the ribosome complex to an SRP receptor on the ER membrane, opening up a channel.

◦ The growing polypeptide is fed through this channel into the lumen (inside space) of the ER. The signal peptide is removed.

◦ The protein then moves to the Golgi body for further modification, repackaging, and eventual export from the cell via a vesicle (exocytosis).

Table of Mutations

Mutation Type	Mechanism	Effect on Protein/Amino Acids	End Result Classification	Frame Shift?
Substitution	One nucleotide is replaced by a different one.			No
Silent Mutation	A substitution occurs, but due to redundancy in the genetic code, the resulting codon codes for the same amino acid.	No change to the amino acid sequence.	Silent mutation (or neutral).	No
Missense Mutation	A substitution results in a codon coding for a different amino acid.	The same number of amino acids are produced, but one or more are incorrect. (Example: Sickle cell anemia).	Missense mutation.	No
Nonsense Mutation	A substitution changes an amino acid codon into a stop codon prematurely.	Results in a truncated (chopped off) or incomplete, non-functional polypeptide.	Nonsense mutation.	No
Insertion	An extra nucleotide is added.	Shifts the reading frame for all subsequent codons.	Usually disastrous effects. Often leads to a nonsense mutation immediately.	Yes
Deletion	A nucleotide is removed.	Shifts the reading frame for all subsequent codons.	Usually disastrous effects. Often leads to extensive missense mutations or premature nonsense.	Yes
Codon Deletion/Insertion	A whole codon (three nucleotides) is removed or added.	Results in the loss or gain of one amino acid without affecting the reading frame.	Considered a missense (if the remaining sequence is correct) or nonsense (if the resulting chain is too short).	N

Metabolism

refers to the totality of all chemical reactions that happen in the body. Anytime a process involves/requires energy

Includes:

    ◦ Breaking down food and converting it into energy.

    ◦ Creating hormones.

    ◦ Making proteins (protein synthesis).

    ◦ DNA replication.

    ◦ Active transport and endo/exocytosis.

Cellular Factory

The living cell functions as a miniature chemical factory where thousands of reactions occur, extracting and applying energy to work.

Emergent Property

Metabolism is this of life that arises from the interactions between molecules in the cell.

Metabolic pathway

Unlike simple chemistry reactions, chemical reactions in living organisms are often split into multiple steps. Begins with a specific starting molecule (reactant) and ends with a final product, involving multiple steps in between.

Enzymes

Each step in a biological pathway is catalyzed by this. Special proteins that help catalyze chemical reactions. Is like a Catalyst. Regulates/facilitates chemical reactions. Highly specific. Only seed up one reaction. Names usually end in the suffix -ase (e.g., sucrase, maltase, lipase, DNA polymerase) or sometimes -sin (e.g., trypsin, pepsin).

Catabolic Pathways

  ◦ Release energy by breaking down complex molecules into simpler ones.

    ◦ Examples: Cellular respiration (breaking complex glucose into simpler CO2 and water), or digestion (breaking down proteins into amino acids or starch/glycogen into glucose).