DNA Structure and History
What was the popular misconception regarding DNA?
Scientists thought it was proteins.
What is the enzyme that is responsible for proofreading mismatches during replication & replacing the mismatch w/ the correct nucleotide?
DNA polymerase
List and define the types of RNA.
Messenger RNA (mRNA): encodes proteins; contains codons; used as a template to make proteins
Transfer RNA (tRNA): "translates" mRNA -> amino acid
Ribosomal RNA (rRNA): found in the ribosome; protein synthesis
Vocabulary: Define the following terms)
1. Euchromatin
2. Heterochromatin
3. Activators
4. Repressors
5. Alternative RNA Splicing
1. Loose chromatin that's actively transcribed
2. Tightly wound chromatin that cannot be transcribed
3. Transcription factor proteins that bind to DNA seq. called enhancers to initiate transcription.
4. Transcription factor proteins that bind to DNA seq. called silencers to stop transcription.
5. allows multiple different proteins to be built from one gene
Which DNA technology is used to separate DNA based on size?
What is Gel Electrophoresis.
Name the individuals involved in the discovery of DNA and list their contributions.
Watson & Crick: Described the double helix, Used Chargaff's rules, and Rosalind Franklin's X-ray
Rosalind Franklin: Used X-ray crystallography to reveal the double helix
Chargaff: Discovered the relative abundance of nitrogenous bases in DNA, which helped provide a clue to DNA's structure. (# Purines = # Pyrimidines; #As = #Ts [double h-bond]; #Gs = #Cs[triple h-bonds])
AG: Purines [2 rings]
TC: Pyrimidines [1 ring]
What is the complement of this strand of DNA?
5' - AGT CGA TTG GTA ACC TAG - 3'
Follow-up: What is the RNA complement of this strand of DNA?
3' - TCA GCT AAC CAT TGG ATC - 5'
and
3' - UCA GCU AAC CAU UGG AUC - 5'
Define the types of base substitutions.
Follow up: What's the second type of mutation? What happens?
Missense: changes the amino acid
Nonsense: changes the amino acid codon into a stop codon
Silent: changes the nucleotide seq. (AGT) that does not change the amino acid
*Don't get missense mutations confused w/ silent mutations, only missense changes the amino acid, silent does not affect the amino acid*
What is an Operon?
A series of genes that are turned on/off together as one unit to make one mRNA molecule.
Define DNA Cloning.
Uses bacteria to make millions of copies of a specific DNA frag. Includes recombinant DNA, restriction enzymes, plasmids.
What are Telomeres? Telomerase?
Telomeres are sections of repetitive, "junk" DNA at the ends of chromosomes.
Telomerase is an enzyme that extends the ends of chromosomes with "junk" DNA (telomeres).
Name all of the enzymes involved in DNA replication and their functions. Include or illustrate their locations on a strand of replicating DNA.
What is Helicase, SSB Proteins, DNA ligase, DNA polymerase, Primase, Topoisomerase
Helicase: unzips
SSB Proteins: keeps dna strands from recoiling
DNA ligase: glues Okazai frags.
DNAP: builds nucleotides
Primase: adds RNA primer
Topoisomerase: Breaks, turns, and rejoins double-stranded parental DNA to relieve strain created by unwinding/replication fork
1. Mistakes in DNA rep.
2. Mistakes in Mitosis or Meiosis
3. Mutagens like radiation & chemicals
4. Exposure to viruses
What are the functions of Methylation and Acetylation during Eukaryotic Gene Regulation?
Methyl: prevents transcription by packing DNA so tight that its inaccessible
Ace: loosens chromatin so DNA is accessible for transcription
What is used to detect COVID19?
Quantitative Reverse Transcription PCR
What are the major characteristics of DNA?
Complementary nitrogen bases, antiparallel, grow from 5' to 3', double-stranded [double helix]
List the define the steps of the leading strand. (Ex: Which way does it grow? What enzymes are being actively used?)
1. Helicase unzips DNA @ replication fork
2. DNA primase adds RNA primer to create a 3' end to which the 1st DNA nucleotide can bond
3. DNAP attaches 5' end of the 1st DNA nucleotide to 3' end of RNA primer
4. DNAP continues to add nucleotides (5' to 3') towards replication fork
Briefly explain the steps of Transcription. Intitation, elongation, and termination. (Use whiteboards and paper to write out the steps. Be ready to explain.)
There are three sites: Promoter [RNAP binds to DNA to begin transcription] [TATA box], Transcriptional unit [the DNA sequence that will be used to transcribe RNA], Terminator [DNA seq. that signals the end to transcription]
- Located in the nucleus
Initiation: Proteins bind to TATA box -> RNAP binds to Promoter on DNA template strand -> RNAP unwinds DNA and transcribes DNA seq. to RNA seq.
Elongation: The building mRNA 1 nucleotide at a time until termination
Termination: mRNA hits a termination seq. -> mRNA is processed [splicing out introns] to create a mature mRNA strand [only exons, no introns] it becomes capped w/ a 5' and ended with a 3' tail -> leaves the nucleus
Describe the structure and function of the Trp Operon? (What type of operon is it? What happens when there is excess tryptophan?)
Repressible: gene series that's usually ON, unless a "corepressor" triggers it to turn off
- Excess trp will turn the trp operon off (trp binds to repressor protein, changing its shape, and stopping transcription)
Trp + Repressor protein = trp operon OFF
What is PCR? Ingredients?
Uses PCR ingredients and thermocycler to make billions of copies of a gene. Ingredients: DNA sample, free nucleotides w/ diff. bases, primer, and DNA polymerase
Define and Illustrate the parts that make up a strand of DNA. Include all of the bonds involved in the structure.
What is Pentose sugar, Nitrogen bases, and a Phosphate group. "Sugar-phosphate backbone"
Strong Covalent bond between the 3' sugar and the phosphate group of another molecule.
Weak Hydrogen bonds in between the nitrogen base pairings
List and define the steps of the lagging strand. (Ex: Which way does it grow? What enzymes are being actively used?)
1. DNA primase adds RNA primer to create a 3' end to which the 1st DNA nucleotide can bond
2. DNAP adds nucleotides to the 3' end of the RNA primer. DNAP continues to add nucleotides in the 5' to 3' direction away from the replication fork.
3. The first Okazaki fragment has been made after replication reaches a dead end.
4. 2nd RNA primer is added to begin a new Okazaki fragment. DNAP adds a nucleotide to the primer and continues.
5. The 1st RNA primer degrades and gets replaced with nucleotides.
6. DNA ligase joins the two Okazaki fragments. Repeat.
Briefly explain the steps of Translation. Intitation, elongation, and termination. (Use whiteboards and paper to write out the steps. Be ready to explain.)
Located on ribosomes, using Large subunits (LSU) and Small subunits (SSU).
There are 3 active sites:
A: Arrival of charged tRNA molecules
P: holds tRNA that keeps the growing polyp. chain
E: Site where used tRNA exits the ribosome
tRNA sites:
Amino acid attachment site & Anticodon (mRNA attachment site)
Initiation: SSU binds to mRNA and 1st tRNA moves into the P site [codon attaches to anticodon] -> LSU joins
Elongation: (a.a's are added to the peptide chain 1 by 1) 2nd tRNA w/ matching anticodon arrives @ A site -> ribosome "clicks" and shifts positions, so now the A site is empty and the 2nd tRNA is now in the P site -> Repeat until a STOP codon arrive
Termination: hits a stop codon and complex falls apart (UGA, UAG, UAA)
Describe and illustrate the structure of the Lac Operon? (What type of operon is it? What happens when lactose is present? Absent?)
Inducible Operon: gene series that's usually turned OFF, unless an "inducer" turns it on.
Lactose Operon requires three enzymes: lac a, lac z, and lac y
1. RNAP binds to the promoter
2. RNAP runs thru the operator & transcribes genes z, y, and a at the same time
3. mRNA is translated into 3 separate proteins
Lactose Absent [Usual, repressed state]: lactose repressor binds to operator and blocks RNAP, blocking transcription of ZYA
Lactose Present [Induced state]: lactose repressor protein w/ 2nd site binds to lactose -> repressor falls off -> RNAP can transcribe ZYA
What is the process of PCR? (Hint: 4 Steps)
1. Denaturation: Uses heat to break H bonds and break open the double helix into 2 single strands.
2. Annealing: Cooling and using primers to stick to complementary places on the target DNA
3. Extension: Warmed up to copy the gene -> DNA replication begins after this point using DNA Polymerase
4. Repeat