Lecture 7A Flashcards
What is the Central Dogma of Molecular Biology?
DNA -> RNA -> Protein
DNA -> DNA = Replication
DNA -> RNA = Transcription
RNA -> Protein = Translation
In structure of DNA, what bonds covalently link deoxyribonucleoside monophosphates, and in what direction? Which enzyme can cleave these bonds?
3’ -> 5’ phosphodiester bonds
Enzymatically cleaved by nucleases
Melting Temperature Tm is defined as what?
When DNA is heated, the temperature where 1/2 of the helical structure is lost.
Steps in Prokaryotic DNA Synthesis
1.) Begins at origin of replication (Lots of A-Ts, bc must melt)
2.) Formation of replication fork
3.) Direction of DNA replication, synthesis 5’->3’ bc DNA polymerases read 3’->5’
4.) RNA primer required for DNA polymerase via primase. Free OH on the 3’ end. Later on removed.
5.) Chain elongation, catalyzed by DNA pol III. goes from 5’->3’. DNA pol III has 3’->5’ exonuclease activity to correct mismatch bases. (Goes backwards)
6.) Excision of RNA primers/replacement by DNA. DNA pol I does this.
7.) DNA Ligase: Final phosphodiester linkage.
Steps in Eukaryotic DNA Synthesis
1.) Multiple origins of replication
Replication fork proteins in Prokaryotic DNA synthesis and what they do
DnaA protein: binds to origin, causes AT rich regions to melt
DNA helicases: unwinds double helix
SsDNA-binding proteins: Strands stay apart to protect DNA from nucleases that degrade ssDNA
Topoisomerase I: Cut / rejoin one strand of double helix. Removes supercoils.
Topoisomerase II: Cuts / rejoins both strands (DNA Gyrase)
Cell cycle.
Mitosis: PMAT: prophase, metaphase, anaphase, telophase.
Interphase: G1, S, G2.
G1: Cell prepares DNA synthesis
S: DNA content doubled. (DNA Synthesis)
G2: Biosynthesis for mitosis to occur.
G0: Cells exiting cell cycle. (Nerve cells) Withdrawing Growth factors send cells back to G0. Adding back GFs go to G1.
Important Eukaryotic DNA polymerases
Pol (delta): Elongates Okazaki fragments of the lagging strand.
Pol (epsilon): Elongates the leading strand.
Telomeres
Term used to describe when cell is no longer able to divide
Several thousand tandem repeats of AGGGTT base.
Senescent.
Telomerase: Stem cells/cancer cells have these to maintain telomeric length.
Reverse Transcriptase
RNA back to DNA
Histone, Nucleosome, Chromosome
Histone: Eukaryotic DNA associated with tightly bonded basic proteins
Nucleosomes: Order the DNA in structural units
Chromosomes: nucleosomes further arranged to this.
Goes from Naked DNA -> Histone H1 -> Nucleosomes -> nucleofilaments -> chromosome
In DNA damage, what can UV light do to your cells?
Can give rise to pyrimidine dimers, specifically thymine dimers
Base Excision Repair (BER)
Removes / replaces individual damaged bases
Nucleotide Excision Repair (NER)
Removes / replaces larger areas of bases (bulky, 2-30 nucleotides)
Recognizes the physical distortion over specific base sequences
Mismatch Repair
Removes nucleotides that have incorrect matching.
Example: A-G, shud be A-T. Original (parent) strand = typically methylated.
Ataxia Telangiectasia
Poor coordination
Defects in excision repair
Neurodegenerative disease
Ataxia Telangiectasia Mutated protein is what causes this.
Xeroderma Pigmentosum
Pyrimidine dimers formed in skin cells exposed to UV light
Defects in excision repair due to mutant UV-specific endonuclease
rRNA
Ribosomal RNA
4 species in eukaryotes: 5S, 5.8S, 18S, 28S
Comprise about 80% of RNA in cell
tRNA
Transfer RNA
15% of RNA in cell
Transfers amino acids
At least one tRNA for each 20 amino acids
mRNA
Messanger RNA
Carries genetic info from nuclear DNA to cytosol, used as template for protein synthesis
5’ end cap, coding region, poly-A tail
Prokaryotic RNA Polymerase
One species of RNA polymerase synthesizes all the RNA except for short RNA primers needed for DNA replication.
Transcription occurs in 5’ -> 3’ direction
Sense strand
Antisense strand
Sense: DNA strand with same sequence as mRNA
Antisense: DNA strand being transcribed into mRNA
Steps in Transcription of Prokaryotic Genes
1.) Initiation: Binding of RNA polymerase to promoter region.
2.) Elongation: To make RNA using RNA polymerase, do NOT need a primer. Proceeds along DNA anti-sense strand, RNA grows from 5’->3’ direction.
3.) Termination:
a.) Rho-independent-RNA folds forms hairpin loop. Separates RNA from DNA. (more common)
b.) Rho-dependent: Uses ATP-dependent helicase activity to separate RNA from DNA.
Transcription of Eukaryotic Genes
Chromatin structure 2 forms
Most acitively transcribed genes: Relaxed Chromatin = euchromatin. More loosely associated.
Most inactive segments found in Condensed form = heterochromatin. DNA interacts more tightly with histones.
How to go from one form of DNA to next
Histone acetyltransferases: Catalyze acetylation
Histone deacetylases: Catalyze deacetylation
Acetylation causes DNA to unwind more easily from protein. Gets rid of positive charge on lysine residue.
All RNA polymerases in Eukaryotic Cells
RNA polymerase I: Synthesize precursor of 5.8S, 18S, and 28S rRNA in nucleolus
RNA polymerase II: Synthesize mRNA
RNA polymerase III: Synthesize tRNA and 5S rRNA
Mitochondrial RNA polymerase: Transcribes RNA from all mitochondrial genes.
RNA processing in prokaryotes and eukaryotes
In prokaryotes: RNA used as unaltered primary transcript as soon as it is made.
In eukaryotes: Capping at 5’ end, addition of polyA tail at 3’ end, removal of introns
Eukaryotic RNA processing: What is the cap made of?
7-methylguanosine cap at 5’ end of mRNA
This protects 5’ end from exonucleases and promotes mRNA translation by ribosomes.
Eukaryotic RNA processing: Function of polyA tail?
polyA tail helps to stabilize mRNA, facilitates mRNA exit from nucleus and aid in translation.
Important thing for exams when reading nucleotide sequences:
Mclean said that if you see a sequence but not the direction, always assume it is 5’ -> 3’.
THERE IS A QUESTION ON EXAM ABT THIS.
Alterations of Genetic Code
2 types of substitution
Transition: replace a purine with a purine or replace pyrimidine with a pyrimidine.
Transversion: replace a purine with a pyrimidine or vice versa.
Classic Question: What is the difference between transition and transversion substitution?
The genetic code: insertion or deletion mutations called what?
Called frameshift mutations, and they alter reading frame.
3 types of point mutations
Silent mutation: changed codon codes the same amino acid (no effect)
Missense mutation: changed codon codes for different amino acid (small effect)
Nonsense mutation: changed codon codes for terminal codon (big effect)
Translation:
Important components required
AAs: for finished proteins
tRNA: to transfer AAs
Aminoacyl-tRNA synthetases: catalyze attachment of AA to tRNA
mRNA: template for protein synthesis
Ribosomes: organelle where protein synthesis occurs.
NOTE*: When tRNA is covalently attached to AA, it is said to be ACTIVATED / CHARGED.
Ribosomes:
Sedementation rates, small subunits and large subunit
50S + 30S -> 70S (prokaryotic ribosome)
60S + 40S -> 80S (eukaryotic ribosome)
^^^^^^^^^^^^^^
Large subunit + small subunit = 70S / 80S.
Small subunits bind to mRNA.
Large subunits catalyze peptide bond formation.
What is nontraditional base-pairing and what is the purpose of it?
Nontraditional base-pairing is when the 3rd codon of mRNA can bind to something else (for example, U binds with G when U shud bind with A). (This is also the first base in the anticodon, the tRNA).
This allows more flexibility in binding, allowing the tRNA to not be very specific. You only have so much tRNA so it allows flexibility.
This is the wobble hypothesis. tRNAs can recognize more than one codon for a specific amino acid.
Enzyme in ribosome that catalyzes peptide bond formation?
Peptidyltransferase. Combines the amino acids via peptide bonds in large subunit of ribosome.
Ubiquitination
Proteins that are misfolded / defected tagged by ubiquitin. Degraded by proteasome in cytosol.
Gel electrophoresis
Separates macromolecules on basis of size / charge
Western Blotting
Southern Blotting
Northern Blotting
SNOW DROP
Southern - DNA
Northern - RNA
O - O
Western - Proteins
Polymerase Chain Reaction (PCR)
High Performance Liquid Chromatography (HPLC)
X-Ray Crystallography / Nuclear Magnetic Resonance (NMR)
PCR - amplify small samples of DNA. DNA multiplication.
RT-PCR : use of RNA to make c-DNA (complimentary DNA)
HPLC: Separate components via column
X-Ray/NMR: Determine 3D structure.
Edman Degradation method
Mass Spectrometry
Edman Degradation: Determines AA sequence of peptide (label and take off one by one)
Mass Spec: Determines AA sequence of peptide.
