Lecture 7A

Question 1

What is the Central Dogma of Molecular Biology?

Answer 1

DNA -> RNA -> Protein

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

Question 2

In structure of DNA, what bonds covalently link deoxyribonucleoside monophosphates, and in what direction? Which enzyme can cleave these bonds?

Answer 2

3’ -> 5’ phosphodiester bonds

Enzymatically cleaved by nucleases

Question 3

Melting Temperature Tm is defined as what?

Answer 3

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

Question 4

Steps in Prokaryotic DNA Synthesis

Answer 4

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.

Question 5

Steps in Eukaryotic DNA Synthesis

Answer 5

1.) Multiple origins of replication

Question 6

Replication fork proteins in Prokaryotic DNA synthesis and what they do

Answer 6

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)

Question 7

Cell cycle.

Answer 7

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.

Question 8

Important Eukaryotic DNA polymerases

Answer 8

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

Question 9

Telomeres

Term used to describe when cell is no longer able to divide

Answer 9

Several thousand tandem repeats of AGGGTT base.

Senescent.

Telomerase: Stem cells/cancer cells have these to maintain telomeric length.

Question 10

Reverse Transcriptase

Answer 10

RNA back to DNA

Question 11

Histone, Nucleosome, Chromosome

Answer 11

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

Question 12

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

Answer 12

Can give rise to pyrimidine dimers, specifically thymine dimers

Question 13

Base Excision Repair (BER)

Answer 13

Removes / replaces individual damaged bases

Question 14

Nucleotide Excision Repair (NER)

Answer 14

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

Recognizes the physical distortion over specific base sequences

Question 15

Mismatch Repair

Answer 15

Removes nucleotides that have incorrect matching.

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

Question 16

Ataxia Telangiectasia

Answer 16

Poor coordination
Defects in excision repair
Neurodegenerative disease

Ataxia Telangiectasia Mutated protein is what causes this.

Question 17

Xeroderma Pigmentosum

Answer 17

Pyrimidine dimers formed in skin cells exposed to UV light

Defects in excision repair due to mutant UV-specific endonuclease

Question 18

rRNA

Answer 18

Ribosomal RNA

4 species in eukaryotes: 5S, 5.8S, 18S, 28S

Comprise about 80% of RNA in cell

Question 19

tRNA

Answer 19

Transfer RNA

15% of RNA in cell

Transfers amino acids

At least one tRNA for each 20 amino acids

Question 20

mRNA

Answer 20

Messanger RNA

Carries genetic info from nuclear DNA to cytosol, used as template for protein synthesis

5’ end cap, coding region, poly-A tail

Question 21

Prokaryotic RNA Polymerase

Answer 21

One species of RNA polymerase synthesizes all the RNA except for short RNA primers needed for DNA replication.

Transcription occurs in 5’ -> 3’ direction

Question 22

Sense strand

Antisense strand

Answer 22

Sense: DNA strand with same sequence as mRNA

Antisense: DNA strand being transcribed into mRNA

Question 23

Steps in Transcription of Prokaryotic Genes

Answer 23

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.

Question 24

Transcription of Eukaryotic Genes

Chromatin structure 2 forms

Answer 24

Most acitively transcribed genes: Relaxed Chromatin = euchromatin. More loosely associated.

Most inactive segments found in Condensed form = heterochromatin. DNA interacts more tightly with histones.

Question 25

How to go from one form of DNA to next

Answer 25

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.

Question 26

All RNA polymerases in Eukaryotic Cells

Answer 26

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.

Question 27

RNA processing in prokaryotes and eukaryotes

Answer 27

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

Question 28

Eukaryotic RNA processing: What is the cap made of?

Answer 28

7-methylguanosine cap at 5’ end of mRNA

This protects 5’ end from exonucleases and promotes mRNA translation by ribosomes.

Question 29

Eukaryotic RNA processing: Function of polyA tail?

Answer 29

polyA tail helps to stabilize mRNA, facilitates mRNA exit from nucleus and aid in translation.

Question 30

Important thing for exams when reading nucleotide sequences:

Answer 30

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.

Question 31

Alterations of Genetic Code

2 types of substitution

Answer 31

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?

Question 32

The genetic code: insertion or deletion mutations called what?

Answer 32

Called frameshift mutations, and they alter reading frame.

Question 33

3 types of point mutations

Answer 33

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)

Question 34

Translation:

Important components required

Answer 34

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.

Question 35

Ribosomes:

Sedementation rates, small subunits and large subunit

Answer 35

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.

Question 36

What is nontraditional base-pairing and what is the purpose of it?

Answer 36

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.

Question 37

Enzyme in ribosome that catalyzes peptide bond formation?

Answer 37

Peptidyltransferase. Combines the amino acids via peptide bonds in large subunit of ribosome.

Question 38

Ubiquitination

Answer 38

Proteins that are misfolded / defected tagged by ubiquitin. Degraded by proteasome in cytosol.

Question 39

Gel electrophoresis

Answer 39

Separates macromolecules on basis of size / charge

Question 40

Western Blotting
Southern Blotting
Northern Blotting

Answer 40

SNOW DROP

Southern - DNA
Northern - RNA
O - O
Western - Proteins

Question 41

Polymerase Chain Reaction (PCR)

High Performance Liquid Chromatography (HPLC)

X-Ray Crystallography / Nuclear Magnetic Resonance (NMR)

Answer 41

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.

Question 42

Edman Degradation method

Mass Spectrometry

Answer 42

Edman Degradation: Determines AA sequence of peptide (label and take off one by one)

Mass Spec: Determines AA sequence of peptide.