Lecture 7A Flashcards by Alex Shin

What is the Central Dogma of Molecular Biology?

DNA -> RNA -> Protein

DNA -> DNA = Replication
DNA -> RNA = Transcription
RNA -> Protein = Translation

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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

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Melting Temperature Tm is defined as what?

When DNA is heated, the temperature where 1/2 of the helical structure is lost.

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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.

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Steps in Eukaryotic DNA Synthesis

1.) Multiple origins of replication

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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)

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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.

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Important Eukaryotic DNA polymerases

Pol (delta): Elongates Okazaki fragments of the lagging strand.
Pol (epsilon): Elongates the leading strand.

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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.

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Reverse Transcriptase

RNA back to DNA

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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

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In DNA damage, what can UV light do to your cells?

Can give rise to pyrimidine dimers, specifically thymine dimers

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Base Excision Repair (BER)

Removes / replaces individual damaged bases

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Nucleotide Excision Repair (NER)

Removes / replaces larger areas of bases (bulky, 2-30 nucleotides)

Recognizes the physical distortion over specific base sequences

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Mismatch Repair

Removes nucleotides that have incorrect matching.

Example: A-G, shud be A-T. Original (parent) strand = typically methylated.

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Ataxia Telangiectasia

Poor coordination
Defects in excision repair
Neurodegenerative disease

Ataxia Telangiectasia Mutated protein is what causes this.

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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.