What are genes?
- Elements that store specific information.
- They are found on Chromosomes which contain both DNA and Proteins
What part of the cell contains genetic information?
The Molecule that carries the heritable information is DNA
What is DNA made of?
- A double stranded Helix
- Sugar Phosphate backbone
- Non-Covalent Hydrogen bonding of base pairs: A=T (Double bond); G=C (Triple bond)
- Phosphodiester bond
In what direction is DNA read?
ALWAYS 5' to 3'
Describe DNA base pairing.
- DNA base pairing is complementary & antiparallel (they form "steps")
- Purines: Adenine, Guanine
- Pyrimidines: Thymine, Cytosine
- A binds to T through a double bond
- G binds to C through a triple bond
In DNA how many Bases/ repeat?
10 base pairs / 360* turn
What makes the DNA Helix Stabilization?
- Base stacking (Pi Electrons of stacked bases)
DNA is the genetic information for essentially all living organisms
- Each species' genetic instructions are coded in the sequence of the four nucleotides making up DNA
- Every species has at least slightly different sequence of nucleotides in their genes
Describe the structure of Chromosomes in Eukaryotic*/Prokaryotic
- -Eukaryotic: may have linear pieces of DNA; Packages by protein in Chromatin; Create visible chromosomes during Mitosis
- -Prokaryotic: (bacteria) circular genomes
How many Chromosomes to Humans have?
- Total = 23
- 22 pairs of somatic chromosomes
- 1 pair of sex chromosomes
How can you tell the difference between Homologus chromosomes?
- 1 pair Homologus: 1 Paternal, 1 Maternal
- The same gene exists on the DNA of each chromosome in the same order
- But the actual DNA sequence is usually different
Why do chromosomes need to be Condensed in mitosis?
Because a chromosome is a descrete UNIT of genetic info that needs to pull apart into daughter cells
In human chromosomes, how do you tell if something is "wrong"?
- Trisomy 21*- (Down syndrome in female has 3 instead of 2)
- ID by banding pattern
- If its longer on one side than the other
What happens to Chromatin in Interphase?
- "Between Mitosis"
- Can't see individual DNA or Chromosomes
Describe the chromosome organization within a nucleus
- You can pick out different chromosomes in a nucleus
- DNA of each chromosome is attached continuously to internal protein "scaffolds" to keep coming back
- Decondensed but Localized
How is DNA packaged in chromatin?
- Histones1. start with 1 DNA molecule with 2 'strands'
- 2. The DNA has (2) Telomere- "END" sequence
- 3. DNA has hundreds of replication origins along it
- 4. 1 centromere
- Starts with Interphase.
- Goes through Mitosis/ Cell division
- then Interphase again (Ending with duplicated chromosomes in 2 separate daughter cells- IDENTICAL)
How many DNA must be packed in one nuclues?
- Humans have 23 mitotic chromosomes (Haploid) ~20,000 genes in ~1m DNA(3.2 billoin bp)
- Humans are Diploid with 23 Materal & 23 Paternal chromosomes (Thus, 23 Homologus chrom.)
- Mitotic chromosomes contain 2 chromatids (each is Identical because DNA duplication, NOT homologus)
- A human mitotic cell contains 92 DNA molecules ( [23 x 2 diploid] x 2 mitotic = 92) This is 4 m DNA packaged into a nuclues of 10 micrometers
- Daughter cells have 46 DNA molecules
DNA Organization occurs at two levels:
- 1. The protein "scaffold" with periodic DNA attachments
- 2. The packaging of DNA by basic histones into Chromatin :
- Add linker histone + engage histone tails 30nm chromatin fiber - Can't transbribe (OFF), (Roll up into a helix)
- Add core histones (beads on a string)- 4 (+) pos. basic charged proteins bind to the (-) charge of DNA strands and compact into "beads" called nucleosomes
Chromatin = DNA [acidic with many P (-) grps] + small basic (+) proteins named "HISTONES"
- "beads on a string": Nucleosomes with 8 core histone on DNA - Can transcribe (ON)
- 30nm chromatin fiber of compacted nucleosomes- Can't transcribe (OFF)
First Level of organization of chromatin: Nucleosomes
- Histones = Proteins of nucleosomes at the core of each 'bead'
- 1. Start with linker DNA and core histones of nucleosomes
- 2. Nuclease digests "cuts" the linker DNA
- 3. Have a released nucleosome core particle
- 4. Dissociation with High concentration of salt- gets 146 nucleotide pair & octameric histone core
- 5. Dissociate the histone core particles
Why is it important that DNA is wrapped around the outside of th histone core?
DNA outside makes it easier for proteins to recognize it and get to it to perform work
Second level of organization of chromatin: 30 nm chromatin fiber
- (35 folds)
- 1. Last levels of packing: short region of DNA double helix
- 2. Chromosome "scaffold": "beads on a strong" form of chromatin
- 3. Euchromatin (Good chromatin): 30nm chromatin fibers of packed nucleosomes
- 4. Heterochromatin: Section of chromatin in extended form
- 5. condensed reaction of chromosomes
- 6. entire mitotic chromosome
- Net Result: each DNA molecule has been packaged into a mitotic chromosome that is 10,000 folds shorter than its extended length
DNA structures in interphase cells
- Heterochromatin: up to 90% of DNA
- Euchromatin: ~10-20% in most cells
What is the current hypothesis reguarding RNA polymerase, Euchromatin, & Heterochromatin?
- RNA polymerase cannot function in dense, heterochromatin
- Euchromatin still has nucleosomes: (11nm-30nm) RNA polymerase with help, can read through nucleosomes
- Histones must be fully displaced for DNA duplication
Regulation of Chromosome structure
- 1. Chromatin remodeling complexes are ATP driven machines that push/pull nucleosomes out of the way of moving RNA Polymerase
- - Move Nucleosomes
- - partially unfold nucleosomes
- - displace some nucleosomes
- - destabilize 30nm fiber
- 2. 30nm chromatin is repressed
- Histones are unacetylated: bind (-)neg. DNA
- Reversible modification of histone tails: Acetylation, phosphorylation, methylation..
- Histones are highly acetylated
What does the Histone code Hypothesis "Epigenetics" propose?
Specific combinations of modifications help determine chromatin configuration and influence transcription
- Each change:
- -affects higher-order packing of chromatin
- -brings in transcription factors
- -brings in more modification enzymes
- -brings in chromatin remodelers
- -brings in RNA polymerase, etc..
- (or the reverse!!!) And Thus changes Gene expression
What enables DNA Replication?
- Complementary base pairing synthesizes new strands correctly
- Building blocks are dNTPs- deoxyribo-nucleotide tri-phosphates
Each replicated double helix has:
- one original strand +
- one new strand
In what direction is DNA synthesized?
*All DNA is synthesized in the 5' to 3' direction! *
RNA synthesis at origins
- Step 1: Synthesize RNA primers
- 1. RNA polymeras ("primase") enters the Origin bubble
- 2. RNA adds to the first NTP 3'OH a second nucleotide
- 3. The nucleotide choice is based on the template base
- 4. after adding ~10 nucleotides, the stable double-stranded structure exists
- 5. Now DNA polymerase can add nucleotides to 3'OH end using dNTPs and the template strand.
- *ALWAYS: 5'-->3'*
first strand DNA synthesis at origins
- Step 2: DNA strand extention
- 1st strand = "leading strand"
- synthesis: continuous
- DNA ploymerase requires:
- -a 3'OH end to add to: a RNA primer or a 3' DNA terminus
- -a template DNA strand
- *DNA polymerase only extends a new strand in the 5' to 3' direction*
second strand DNA synthesis at origins
- Step 3: Okazaki pieces
- "discontinuous" lagging strand synthesis
- 1. take old primer & 2. Add Okazaki piece to new RNA primer synthesis by DNA primase
DNA synthesis at origins: Joining Okazaki pieces
- Step 4: Ligation of Okazaki pieces
- 1. DNA polymerase fills in the gap until it reaches the RNA primer
- 2. 5'-->3' exo-nuclease activity in DNA polymerase complex removes RNA nucleotides until primer is gone
- 3. DNA polymerase fills the last gap
- 4. the 'nick' (missing link between 3'OH of previous Okazaki piece & 5' phosphate of completed piece) is Ligated by DNA ligase to extend the growing, disconinuously, synthesized DNA chain 'backwards'
Many proteins control DNA replication. How can you ensure that DNA polymerase doesn't fall off?
Answer: hook it onto a sliding clamp
- At site of RNA primer:
- 1. Clamp loader makes ring form from clamp pieces
- 2. Load a DNA polymerase
- 3. Start DNA strand elongation
- 4. Only let DNA polymerase go when DNA ligase is ready to seal the nick.
Name the proteins required for DNA replication
- 1. Helicase: opens DNA double helix
- 2. ssDNA-binding protein: coats ssDNA on lagging strand
- 3. Primase: makes RNA primers
- 4. DNA polymerase: DNA synthesis
- 5. Sliding clamp & loader: makes ploymerase processive
- 6. Exo-nuclease: removes RNA primers
- 7. Repair DNA polymerase: fills in primer gaps
- 8. DNA ligase: joins Okazaki fragments, DNA from other origins
- Many more.....
How can a chomosome replicate all the way to the end of the chromosome if it needs to start with an RNA primer that is not stable and readily destroyed?
- Telemorase (with built in RNA primer)
- If you have insufficient Telemorase (aging) your chromosomesbecome shorter, genes get lost, and cancer and death follow.
- *Use telemorase- Doesn't shorten!*
How do you replicate the ends of chromosomes?
- Telemorase adds additional repeats to template strand
- 1. Telemorase enzyme comes in with the primer
- 2. It then extends the primer on the 3'OH end
- 3. Completion of lagging strand by DNA polymerase
- (This type of reverse transcriptase synthesizes DNA on an RNA template)
What does the cell have to do in order to repair DNA?
- 1. Recognize DNA damage or mismatches
- 2. Identify the strand with the correct sequence*
- 3. Execute a repair
- *Repairing the proper strand is crucial; the copied DNA should be flawless!!*
What is the Mechanism for DNA Repair?
- 1. Proof-read exo-nuclease activity of DNA polymerase
- 2. Replicative mismatched pair (only repair the new strand, use old strand as template*)Remove the newly syntesized DNA strand and repair the gap by DNA polymerase and Ligase!
- - If the mismatch is NOT repaired: one daughter cell will be fine, the other daughter cell & its progeny will carry a mutation (50% progeny have mutation)
- -if the mismatch IS repaired: All progeny are OK or All progeny are Mutated3. Post-Replicative Patch Repair:
- - Nucleases excise the damaged DNA & leave gaps
- - Repair DNA polymerase binds to the 3'OH end of the cut strand
- - Repair DNA polymerase fills the gap using the opposite strand as a template
- - DNA Ligase catalyzes formaton of the final phosphodiester bond and seals the nick
What does UV radiation do to Thymine?
- If you have two Thymine next to eachother, with UV radiation it becomes a Thymine Dimer
- *A Thymine Dimer requires excision repair*
What protein is required for ALL DNA Repair?
How do you repair UV damage?
- T=T dimer formation
- Patch excision
- DNA polymerase
- DNA Ligase-seals
- No Mutation
In Chemical damage to DNA, what is Depurination? Demination?
- Depurination: Guanine being lost
- Demination: C turns into U
How do you repair double-strand breaks? (caused by ionizing radiation, or mechanical stress)
- Broken end strand is processed by Nuclease
- Non-homologus end-joining: end joining by DNA ligase ( *Nucleotides are usually lost at the repair site*)
- Net Result: Double-Strand break repaired with deletion of nucleotides at repair site
How do we repair double-strand breaks in Diploid organisms?
- Use homologus DNA from other parent as a tmplate
- If the Maternal copy of the chromosome is broken:
- 1. line it up with the Paternal DNA strand
- 2. Open up the Paternal DNA double helix at the right location
- 3. Use the Correct single strand Paternal DNA as the template
- 4. After synthesis of the other Maternal DNA strand
- 5. Untwist the mess
- 6. Repackage the DNA back into Chromatin
What is the Central Dogma?
- DNA is Transcribed into RNA
- RNA is Translated into Protein
- *ALWAYS 5' to 3'*
- *ALWAYS N to C*
RNA vs DNA
- RNA: Sugar- 2' hyroxyl on ribose; Base- Uracil; single stranded
- DNA: Sugar-Deoxyribose; Base- Thymine; double stranded
Can RNA base pair since it is single stranded?
What are the steps in Bacterial Transcription?
- 1. Unwind DNA- separate into 2 strands
- 2. RNA polyermase use DNA as a template- open *(RNA polymerase does NOT Proofread!)
- 3. New Ribonucleotide added
- 4. RNA is displaced and folds, DNA rewinds (RNA lets go and end of process)
- *(Transcription can start without a primer)
What are the 3 types of RNA?
- Messenger RNA (mRNA): code for proteins (need ribosomes to get code)
- Ribosomal RNA (rRNA): Structural RNA in ribosomes (Catalytic functions- "ribozyme" defines structure)
- Transfer RNA (tRNA): Structural RNA for Tranlation (*Amino acids, catalyticall active)
What is RNA without protein coding information?
What are the 3 main phases of RNA synthesis?
- Initiation: RNA polymerase must find a place to start RNA synthesis
- - sequence recognition of the 'Promoter sequence'(interaction to regulate and enhance transcription, sometimes represses transcription) Loading of RNA polymerase
- - separate the DNA strands
- - base-pair the first NTP and creates the first phosphdiester bond
- Elongation: continued addition of NTPs using DNA template strand code
- Termination: Stop Polymerization
- - Realease RNA "primary RNA transcript"
- - Release RNA polymerase for re-use
Describe Bacterial Transcription: Start (Initiation)
- 1. RNA poymerase (with bound Sigma factor) binds DNA
- 2. Scan DNA for Promoter Sequence
- 3. Open TATA sequence
- 4. Start RNA synthesis 1 DNA turn Downstream
Describe Bacterial Transscription: Continue (Elongation)
- 1. Bacterial promoter typically require Sigma Factor (TATA) to open the bubble
- 2. Sigma Factor is released (for re-use)
- 3. RNA polymerase moves along the template strand while synthesizing the RNA chain
When will a growing RNA chain in Bacteria bind ribosomes to start translation?
Immediatly! As soon as the ribosome can bind!
Describe Bacterial Transcription: Stop (Termination)
- 1. RNA polymerase recognizes terminator sequence
- 2. RNA is released
- 3. RNA polymerase releases
- 4. Sigma Factor binds RNA polymerase to allow initiation
What are the differences with Prokaryotic transcription?
- Eukaryotic genes are packaged in chromatin
- Eukaryotic cells have more variations of RNA polymerase
- Eukaryotic cells have more complex protein complexes for initiation and elongation
- Eukaryotic cells regulate gene expression in more complex ways
- Eukaryotic genes contain non-coding 'introns' that must be removed to make mRNA
- Nuclear transcription is separated from cytoplasmic translation
Describe Eukaryotic Transcription: Initiation (1)
- *(TATA-binding protein (TBP): OPEN the TATA box!)*
- 1. Bind to TATA sequence
- 2. Twist the DNA helix
- 3. Ease the (weak A=T) strands apart
- (Only have H-bonds & use less force to open)
Describe Eukaryotic Transcription: Initiation (2)
- TBP is part of a complex named TFIID with other 'general' Transcription Factor proteins that work with RNA polymerase II
- TBP forms a loading platform to add additional Transcription initiation factors
- Sigma-like factor TFIIE creates the bubble
- Ultimately, RNA polymerase II (with additional Transcription Factors) binds 'downstream' of the TATA box
Describe Eukaryotic Transcription: Initiation (3)
- TFIIH phosphorylates the CTD (C-terminal domain) "tail" of RNA polymerase
- This releases the brake!
- Transcription can start!
- Produce the "primary transcript" RNA
- (load polyermase and release by phosphorylation)
Eukaryotic RNA Processing comprises 3 distinct processes. What are they?
- 1. RNA Splicing: to remove non-protein coding RNAs
- 2. RNA Capping: To create a ribosome binding site
- 3. polyA addition: to measure the age of a mRNA
What are the 3 binding sites that Ribosomes have for tRNA?
- A-site: entry site
- P-site: make peptide bond
- E-site: exit site
*tRNAs move from A to P to E*
Transcription & Translation Overview
- 1. Tag with Ubiquitin
- 2. into proteasome
- 3. Digestion
- 4. result: small peptides
What is the Principle of Membrane Transport?
All cells maintain an internal ion concentration that is different from the extracellular fluid
What types of Molecules can cross directly through a lipid bilayer?
- Can Cross:
- - Small Nonpolar molecules
- - Small Uncharged polar molecules
- (Rate depends on Size)
- - Steroids
- Can't Cross:
- - Ions
- - Larger polar molecules
- - Charged molecules
- (Sugars, amino acids, nucleotides, etc. must use proteins to get across the membrane)
Membrane Transport Proteins fall into what 2 classes?
- Channels: (door) are selective based on size and charge of molecule
- - hydrophilic pore
- - many are gated
- - molecules cross at high rate when channel is open
- Transporters: (Turnstile) "Carriers" are selective based on specific binding to molecule
- - undergoes conformationa changes to tranfer molecule
- - Transport can be saturated under normal physiological conditions
What are the 3 types of movement across a cell membrane?
- Simple Diffusion: molecules move down their concentration gradients
- - Pass through the lipid bilayer
- Passive Transport: molecules move down their concentration gradients
- - Uses a channel or transport protein
- Active Transport: moles move against thier concentration gradient
- - Uses a Transport protein
- - Requires Energy input*
Passive Transport can move molecules in either direction across cell membrane
Active Transport has 3 main forms that move solutes against their electrochemical gradient.
- 1. Coupled Transport
- 2. ATP Driven Pumps
- 3. Light Driven Pumps
Active Transport requires energy input into a system so as to move solutes against their electrochemical and concentration gradients. Which of the following is not one of the common ways to perform active transport?
- A) Na+-coupled
- B) ATP-driven
- C) Light-driven
- D) NADPH-driven
What is the correct order of events occuring at the end of the axon in the presynaptic neuron (the side of the synapse which releases neurotransmitters)? KNOW THIS
- 1. Action potential generated in presynaptic neuron
- 2. Voltage-gated Ca++ Channels open
- 3. Ca++ Flows into the cell
- 4. Neurotransmitter released
Which channel(s) is/are open during the falling phase of the action potential, when the membrane potential is becoming less positive?
- The voltage-gated K+ channel
What happens during Hyperpolarization?
Little K+ rush through channels that arn't completely closed (Underneath the Resting Membrane Potential)
Both Excitatory and Inhibitory neurons form junctions with muscles. By what meschanism do Inhibitory neurotransmitters prevent the postsynaptic cell from firing an action potential?
- By Opening Cl- Channels
How does a cell Make ATP?
- 1. Oxidative Phosphorylation
- 2. Mitochondria
What happens if an axon membrane potential depolarizes beyond the threshold?
Inactivated gates of voltage-gated Na+ channels close and cannot be reopened
An example of an electron carrier which moves in the lipid bilayer to transport e- from one proton-pumping complex to the next, but is not part of a proton-pumping complex, is
- Ubiquinone- Carries 1 or 2 electrons from one complex to the next.
- Resides in lipid bilayer but not bound to protein
Which component of the Electron transport chain is required to combine the pair of electrons with molecular oxygen?
Cytochrome oxidase complex
Which is not an electron carrier that participates in the electron-transport chain?