Genetics Unit Review

DNA structure

• double helix

• backbone outside (sugar and phosphate)

• nitrogenous bases inside (A, C, T, G)

  • complementary base pairing (A-T, C-G)

  • hydrogen bonding (A-T = 2 bonds, C-G = 3 bonds)

  • purines, double rings (A and G)

  • pyrimidines, single rings (T and C)

• anti-parallel strands (3’🡪5’ and 5’🡪3’)

• 0.34 nm between bases, one complete turn every 10 bases/3.4 nm

• strong structure

  • hydrophobic interior, hydrophilic exterior

  • hydrogen bonds

  • even spacing between strands

  • phosphate bridges (link sugars)

Organization of genetic material

• prokaryotes

  • nucleoid (DNA packed into a loop structure)

  • plasmid (carry non-essential DNA)

• eukaryotes

  • nucleus

• Prokaryotic DNA coiling:

  • protein scaffolds, radial loops form

  • supercoil

• Eukaryotic DNA coiling:

  • DNA wraps around a histone (group of 8 proteins) 1.65x to form nucleosomes

  • nucleosomes are connected by linker DNA

  • H1 histone protein interacts with nucleosomes to form a 30 nm fiber

  • protein scaffolds help to form radial loops and create a 300 nm fiber

  • supercoil

Turning Genes “Off”

• DNA methylation (the attachment of methyl to DNA)

  • DNA tightly wraps around histones

  • methyl “turns off” the gene because the gene cannot be read and a protein cannot be made

• Acetyl

  • DNA loosely wraps around histones

  • acetyl “turns on” the gene because the gene can be read

• Epigenetics

  • the study of how cells control gene activity (on/off) without changing the DNA sequence
    

DNA Replication

• Semi-conservative model (each new DNA molecule contains one strand of the parent molecule)

• Stages

  • Initiation

    • Helicase (unwinds DNA)

    • SSB’s (prevent H bonds between nitrogenous bases from reforming)

    • Gyrase (cuts one DNA backbone, allows the DNA to swivel, relieves tension)

  • Elongation

    • RNA Primase (places down RNA primers onto DNA strand)

    • DNA Polymerase III (binds to RNA primer and adds complementary bases in a 5’ to 3’ direction)

    • Leading strand (3’ to 5’ direction - DNA Polymerase III adds from end to replication fork)

    • Lagging strand (5’ to 3’ direction - DNA Polymerase III adds from replication fork to end, creating Okazaki fragments)

  • Termination

    • DNA Polymerase I (replaces RNA primers with DNA)

    • DNA ligase (joins Okazaki fragments together)

    • DNA Polymerase II (checks DNA - exonuclease activity (cut out incorrect DNA and replace it))

• Prokaryotic vs. eukaryotic DNA replication Similarities

  • require origins of replication

  • elongation occurs in 5’ to 3’ direction

  • leading and lagging strand

  • use primers

  • Okazaki fragments

  • use DNA polymerase enzymes

• Prokaryotic vs. eukaryotic DNA replication Differences

  • eukaryotic DNA replication takes longer

  • DNA polymerase enzymes differ

  • prokaryotic DNA may have one origin of replication, eukaryotic DNA may have thousands

  • linear structure of eukaryotic DNA causes loss of DNA during replication

    • telomeres (repetitive sequences) are lost instead of important DNA

  • telomerase (enzyme that creates telomeres)

History

• Mendel

  • pea plant experiments

  • “factors” played a role in the transmission of traits

  • each parent contributes a possibility for each trait that makes up the organism

  • dominant and recessive alleles

• Garrod

  • illness in family members

  • each gene provided the information to produce one enzyme

  • an error in one of the enzymes could create the illness, “inborn errors of metabolism”

  • enzymes had to be under the control of the genetic material and therefore, an error in the genetic material led to an error in the enzyme

• Beadle and Tatum

  • Garrod’s ideas were further confirmed by Beadle and Tatum who looked at inborn errors of metabolism in mutant strains of a bread mould (Neurospora crassa)

  • determined the metabolic pathway responsible for synthesizing one of the amino acids

  • an error in one enzyme shuts down the pathway at that point since the product of that particular enzyme was not available for the next step in the pathway

• Ingram

  • sickle cell anemia

  • one amino acid change from the normal protein was responsible for the disease

  • now, each gene codes for one polypeptide chain that may by itself make up a protein or it can combine with other chains to form larger proteins

  • current hypothesis is one gene codes for one polypeptide

Protein Synthesis and Gene Expression

• Transcription

  • creation of mRNA from RNA

  • Stages

    • Initiation

      • RNA polymerase binds to the promoter (area rich in As and Ts) on DNA

    • Elongation

      • RNA polymerase (adds complementary bases in a 5’ to 3’ direction)

      • DNA antisense strand, template strand (3’ to 5’)

      • mRNA strand

    • Termination

      • RNA polymerase reaches a terminator sequence at the end of the gene

      • RNA releases from DNA

    • Post-transcriptional modification 

      • 5’ cap (7-methyl guanosine) added to prevent mRNA from being digested by enzymes in cytoplasm

      • poly-A tail (200 As) added to 3’ end

      • only in eukaryotes

        • exons (coding regions)

        • introns (non-coding regions)

        • splicing (spliceosomes cut out introns and connect exons)

          • the protein will not code properly without it because there are introns

      • RNA can leave the nucleus because it is smaller (one strand), DNA cannot leave the nucleus because it is big (two strands)

• Translation

  • decoding mRNA into a polypeptide chain

  • large subunit of ribosome has three sites

    • A (acceptor site, tRNA enters the ribosome)

    • P (peptidyl site, holds the growing polypeptide chain)

    • E (exit site, tRNA is released from)

  • tRNA (3 nucleotides (anti-codon) that are complementary to the codon in

    mRNA)

  • mRNA codon (codes for a different amino acid)

  • Stages

    • Initiation

      • large ribosomal subunit binds to the 5’ cap, causes the small ribosomal subunit to bind

      • Ribosome reads mRNA in 5’ 🡪 3’ direction until AUG (start) is in the A site

    • Elongation

      • the ribosome reads the codons on the mRNA to determine the correct amino acid

      • tRNA (which has the anticodon) binds the amino acid to the acceptor stem (amino acid is joined to the tRNA using the enzyme aminoacyl tRNA synthetase), and is attracted to its complementary codon on the mRNA transcript and binds to the A site

      • A peptide bond forms between the amino acids

      • the ribosome shifts over one codon

      • The growing polypeptide chain moves to the P site

      • tRNA brings a new amino acid into the A site (based on the mRNA codon)

      • tRNA is released from the E site

      • process repeats until the entire amino acid chain is creates and the stop codon is reached

    • Termination

      • the ribosome reads a stop codon (UAA, UAG or

        UGA)

      • A release factor moves into the A site (not an amino

        acid) and the chain is released

      • The polypeptide chain must fold into its final shape before it is functional

      • Once the polypeptide is released, the ribosomal subunits release the mRNA

Regulating Gene Expression

• Prokaryotes - Transcriptional methods

  • operon (cluster of genes under the control of one promoter that produces a

    protein)

  • promoter (where the RNA polymerase complex binds to begin transcription)

  • operator (DNA sequence that a protein binds to start transcription (found after the promoter))

  • repressor (protein that binds to the operator)

  • lac operon

    • required to make enzymes to breakdown lactose

    • coding region - three enzymes that are required for the breakdown of lactose

    • regulatory region - promoter, operator, activator protein

    • If no lactose is present, the lac repressor protein binds to the operator, preventing RNA polymerase from binding to the

      promoter

    • If lactose is present, allolactose is produced and binds to the repressor. The result is transcription of the genes to produce lactase.

  • trp operon

    • coding region - 5 genes for enzymes required for the synthesis of tryptophan (amino acid)

    • regulatory region - promoter and operator

    • tryptophan must be synthesized, so the repressor does not bind to the operator, and transcription occurs

    • When tryptophan levels are high, a repressor protein binds to the operator, reducing transcription

• Eukaryotes - Control mechanisms

  • pre-transcriptional (cell controls how much DNA is exposed to the transcription enzymes, condensed DNA cannot undergo transcription)

  • transcriptional (cell is able to control whether the exposed DNA is transcribed, transcription factors (helper proteins) need to bind to the promoter before the RNA polymerase)

  • post-transcriptional (cell controls the rate at which the unmodified mRNA transcript is modified into finished mRNA, the addition of the 5′ cap or 3’ poly-A tail may

    not occur)

  • translational (cell is able to control how often and how quickly the mRNA gets translated, regulatory proteins in the cytoplasm can bind to the 5′ cap and prevent/slow down mRNA from binding to the ribosome)

  • post-translational (if the protein needs additional processing before it

    becomes functional, this processing can be slowed down or eliminated)

Genetic Mutations

• Mutation (a permanent change in the genetic material)

• Point mutations

  • missense (the substituted base changes the codon and codes for a similar amino acid)

  • nonsense (the substituted base changes the codon to a stop codon)

  • silent (the substituted base changes the codon but it still codes for the same

    amino acid)

• Frameshift (changes entire reading frame)

  • insertion/duplication (adding a base)

  • deletion (removing a base)

  • inversion (codon is cut out and put in backwards)

  • translocation (codon is cut out and places elsewhere)

• Mutations can happen naturally or by mutagens (physical or chemical agents that cause mutations). Mutations that happen due to outside sources are referred to as induced

Transposon

• the movement of specific DNA sequences within and between chromosomes

• not mutations, occurs naturally

Genetic Technologies and Ethics

• Biotechnology (The use of a biological system to make a product or process)

• DNA technology (The sequencing, analysis, and cutting-and-pasting of DNA)

• Polymerase chain reaction (PCR) (make many copies of small amounts of DNA)

• Gel electrophoresis - (separate DNA fragments according to their size, allows us to visualize DNA and note similarities in DNA samples)

• Recombinant DNA - DNA that is assembled out of fragments from multiple sources

• DNA cloning (makes many identical copies of a piece of DNA)

• DNA sequencing (determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA)

• Bioethics (the ethical debate surrounding biotechnology (e.g. privacy, accessibility, discrimination, ethical dilemmas))