Lecture 1 Flashcards
What is the difference between a prokaryotic and eukaryotic cell?
- prokaryotic: single celled, lacks a membrane bound nucleus, genetic material is stored in a form of a nucleoid, outer membrane/cell wall/inner membrane
- eukaryotic: has other membrane bound organelles; mostly in multicellular organisms but not all the time
What is the Central Dogma of Molecular Biology?
DNA transcription RNA translation PROTEIN
Gene expression
In 1941, who did experiments on red bread mold Neurospora and what did they find?
Edward Tatum and George Beadle: linked genes and enzymes
- Neurospora grows well on minimal medium. Minimal midium consists of a few simple sugars, inorganic salts, and biotin
- But in order for them to grow, they must be able to synthesize other essential nutrients such as amino acids and vitamins
- Are the enzymes that are a part of these essential biosynthetic pathways needed to make these essential nutrients controlled by genes?
Exp:
1. Irradiated Neurospora spores with x-rays to induce genetic mutations. If these enzymes were really controlled by genes, then the X-ray exposure should stop their growth.
2. They selected for mutants. They placed samples on both complete medium and minimal medium. They found growth on complete medium only. So nutritional mutants were not able to grow in MM.
3. Then they had to figure out what kind of nutrition was need for these mutants to grow. Was it vitamins or aa? So they placed samples on MM+vitamins and MM+aa. They found that mutant strains grew on MM+aa.
4. Now, which aa was necessary for mutant growth? They tested the sample in multiple tubes of MM+ (an aa). The tube with MM+arginine was the only one where mutants grew. So, Arginine was being blocked from synthesis.
Conclusions:
They isolated many mutants each with a single mutated metabolic pathway enzyme and proposed a direct link between genes and enzymatic reactions.
ONE GENE- ONE ENZYME HYPOTHESIS: GENES WORK BY CONTROLLING THE SYNTHESIS OF SPECIFIC ENZYMES
Who discovered DNA and how?
1869
Friedrich Miescher
-found NUCLEIN in the nuclei of white blood cells isolated from pus on old bandages
What was nuclein found to be made up of?
H O N P
(no S)
-had a unique ratio of P:N
Who discovered chromosomes and how?
1879
Walther Flemming
-used aniline dyes to stain cells of salamander embryos and saw threadlike material as the cells divided. This is where he started to describe chromosomal behavior during mitosis.
The study of chromosomes is called_____.
cytogenetics
What was the chromosomal theory of heredity?
Early 1900s
Thomas Hunt Morgan
-used drosophila to demonstrate that chromosomes carry the genetic material.
-but he didn’t know if it was DNA or protein?
-at the time, proteins were widely believed to be the genetic material because
1. DNA was though to lack the chemical diversity needed
2. DNA was thought to only provided a structural framework to the chromosome.
Briefly name the 3 classic experiments that demonstrate that DNA is the genetic material and proteins are not.
- Frederick Griffith
1920s - Avery, MacLeod, McCarty
1940s - Hershey and Chase
1950s
Describe the Frederick Griffith Experiment.
- english microbiologist
- worked with pneumonia-causing bacteria
- observed that nonpathogenic bacterial strains could be transformed to pathogenic strains when mixed with heat-killed pathogenic bacterial cells
Experiment:
- Living S-cells injected to mouse: S-colonies found in dead mouse tissue
- Living R-cells injected to mouse: R-colonies found
- Heat killed S-cells injected to mouse: mouse is healthy and no colonies found
- Living R-cells & Heat killed S-cells: both R and S colonies found
Concluded that something in the dead S cells “transformed” the live R cells to pathogenic S cells
What is pathogenicity of bacteria determined by?
The capsule gene
- R colonies: rough (this is the gene in its nonfunctional form
- S colonies: smooth**pathogenic cells have a carbohydrate layer around the capsule
Describe the Frederick Griffith Experiment.
- english microbiologist
- worked with pneumonia-causing bacteria
- observed that nonpathogenic bacterial strains could be transformed to pathogenic strains when mixed with heat-killed pathogenic bacterial cells
Experiment:
- Living S-cells injected to mouse: S-colonies found in dead mouse tissue
- Living R-cells injected to mouse: R-colonies found
- Heat killed S-cells injected to mouse: mouse is healthy and no colonies found
- Living R-cells & Heat killed S-cells: both R and S colonies found
Concluded that something in the dead S cells “transformed” the live R cells to pathogenic S cells.
Griffith did not know the chemical identity of this transforming principle in 1928.
What was nuclein of Friedrich Mieschers pus samples, found to be made up of?
H O N P
(no S)
-had a unique ratio of P:N
What is pathogenicity of bacteria determined by?
The capsule gene
- R colonies: rough (this is the gene in its nonfunctional form
- S colonies: smooth**pathogenic cells have a carbohydrate layer around the capsule
Describe the Avery, Macleod, and McCarty Experiment.
Extended Griffith’s experiment to determine whether the chemical identity of the “transforming principle” was protein, DNA or RNA.
-Made highly purified cell fractions from heat-killed S cells and found that the active fraction that had transforming capability had a C H N P composition consistent with that of DNA.
Describe the Avery, Macleod, and McCarty Experiment.
Extended Griffith’s experiment to determine whether the chemical identity of the “transforming principle” was protein, DNA or RNA.
-Made highly purified cell fractions from heat-killed S cells and found that the active fraction that had transforming capability had a C H N P composition consistent with that of DNA.
Exp: Took S-cell extract, treated with with enzymes and cultured it with R-cells.
- Treated with protease (found R and S colonies)
- Treated with RNase (found R and S colonies)
- Treated with DNase (found only R colonies)
Concluded that DNA was the transforming principle. Because only when the destroyed DNA, the transformation did not occur.
Describe the Avery, Macleod, and McCarty Experiment.
Extended Griffith’s experiment to determine whether the chemical identity of the “transforming principle” was protein, DNA or RNA.
-Made highly purified cell fractions from heat-killed S cells and found that the active fraction that had transforming capability had a C H N P composition consistent with that of DNA.
Exp: Took S-cell extract, treated with with enzymes and cultured it with R-cells.
- Treated with protease (found R and S colonies)
- Treated with RNase (found R and S colonies)
- Treated with DNase (found only R colonies)
Concluded that DNA was the transforming principle. Because only when the destroyed DNA, the transformation did not occur.
Describe the Hershey and Chase experiment.
- used T2 virus that infects E. coli bacteria by injecting it with its genetic material. This was composed of DNA and protein in approximately equal amounts. **The DNA contains P but not S
- *The Proteins contain S but not P
-Labeled using radioactive isotopes:
DNA with 32P
Proteins with 35S
What are isotopes?
same proton
different neutrons
different atomic mass
What are radioisotopes?
unstable
Their atomic nuclei spontaneously decompose to form nuclei with higher stability. The energy and particles released during the decomposition process are called radiation.
Describe the Hershey and Chase experiment.
- used T2 virus that infects E. coli bacteria by injecting it with its genetic material. This was composed of DNA and protein in approximately equal amounts. **The DNA contains P but not S
- *The Proteins contain S but not P
Exp: Labeled using radioactive isotopes
- Labeled phage DNA with 32P and let it infect E.coli. Saw that phage progeny contained 32P labeled DNA.
- Labeled phage proteins with 35S and let it infect E.coli. Saw that phage progeny did not contain any 35S labeled protein.
Concluded: Mainly DNA, not protein is inherited from parental phage.
What are isotopes?
same proton
different neutrons
different atomic mass
What are radioisotopes?
unstable
Their atomic nuclei spontaneously decompose to form nuclei with higher stability. The energy and particles released during the decomposition process are called radiation.
What are radioisotopes?
unstable
Their atomic nuclei spontaneously decompose to form nuclei with higher stability. The energy and particles released during the decomposition process are called radiation.
Who discovered DNA structure and composition?
1953
Watson and Crick
-proposed a 3D structure: double helix with paired subunits called nucleotides
monomer: nucleotides
polymer: DNA or RNA
What is a nucleotide composed of?
pentose sugar
phosphate group
nitrogenous base
Describe the structure of a pentose sugar.
1’ connects a base
2’ in DNA connected to H and in RNA connected to OH
3’ phosphodiester bonds
4’ H
5’ phosphates connects; also used to form phosphodiester bonds
How does the pentose sugar in a nucleotide differ in DNA and RNA?
DNA 2’ H
RNA 2’OH (less stable)
Purines
2 ringed structure
aromatic, hydrophobic
A and G
Pyrimidines
1 ring structure
C and T
Describe the structure of a pentose sugar.
1’ OH connects a base
2’ in DNA connected to H and in RNA connected to OH
3’ OH; phosphodiester bonds
4’ H
5’ phosphates connects; also used to form phosphodiester bonds
Pyrimidines
1 ring structure
C and T
What is the difference between a nucleoside and a nucleotide?
nucleoside: ribose sugar and base
nucleotide: ribose sugar, base, and phosphate
* When a ribose sugar connects its 1’C to a base and its 5’C to a phosphate group, it loses H20 through a dehydration reaction
What kind of bonds like nucleotides into a chain?
phosphodiester bonds
- covalent
- between 3’OH of one nucleotide and the 5’ Phosphate of another nucleotide
1. creates an alternating sugar-phosphate backbone
2. creates polarity of the chains (3’ end and a 5’ end)
What are the 2 Chargaff’s Rules?
- total purines= total pyrimidines
This means that the total number of A and G = the total number of T and C - Base composition ratios of A:T and G:C vary between species
Who helped solve the structure of DNA, in which Watson and Crick took credit for?
Rosalind Franklin
X-ray diffraction pattern
Explain the Model for DNA structure proposed by Watson and Crick.
- Double stranded
- sugar-phosphate backbone on the outside and bases in the middle
- 2 strands of the helix are held together by H-bonds
- A always found with T and G always found with C
- strands are antiparallel
- most DNA is R-handed
Explain the Model for DNA structure proposed by Watson and Crick.
- Double stranded
- sugar-phosphate backbone on the outside and bases in the middle
- 2 strands of the helix are held together by H-bonds
- A always found with T and G always found with C
- strands are antiparallel
- most DNA is R-handed
A-T
How many H-bonds?
2 H-bonds
G-C
How many H-bonds?
3 H-bonds
How is it possible that each base can exist in two alternative tautomeric states?
spontaneous flipping occurs at a very low rate between H-bond donors and acceptors on the base. If this happens at the wrong time, then it causes replication errors. If this happens when being copied, then a wrong base can come across it
What are 2 ways in which the double helix is stabilized?
- Base pairing: H-bonding
2. Base stacking: van der Waals/ hydrophobic effect
Do bases lie perpendicular/parallel to the helix?
bases lie perpendicular to the helical axis
Complementary base pairing between opposite strands gives DNA______.
self-encoding character where if you know the sequence of one strand, you can figure out the sequence of the other strand
Which elements are found in the middle of the helix and which are found on the outside?
outside: H, O, P, C (in phosphate ester chain)
inside: C and N in bases
Why do major and minor grooves even exist?
The angle at which the sugars point away from the base pairs is not 180 degrees. There is a narrow angle 120 and a wide angle 240.
Why do major and minor grooves even exist?
The angle at which the sugars point away from the base pairs is not 180 degrees. There is a narrow angle 120 and a wide angle 240.
Which groove is rich in chemical information?
the major groove
What kind of information can you get just from the major groove, minor groove, both?
BOTH: edges of each bp are exposed such as H-bond donors and acceptors and hydrophobic groups
MAJOR:
- can distinguish between AT/ TA/ GC/ CG
- proteins can recognize specific DNA sequences without altering the sequence
What are the unique patterns seen in the major and minor grooves?
minor:
- H-bond acceptors
- nonpolar hydrogens
major:
- H-bond donors
- H-bond acceptors
- methyl group (from T)
- nonpolar H (from C)
Gel Electrophoresis
- separates DNA
- size and shape
- pour
Gel Electrophoresis
- separates DNA
- size and shape
- porous gel and electric field
- load from - and migrates to + side
- DNA is - charged due to its P group and migrates towards the + pole
- **the distance each DNA fragment travels is inversely related to size: larger molecules have a harder time going through
- *smaller molecules migrate down further
Gel Electrophoresis
- separates DNA
- size and shape
- porous gel and electric field
- load from - and migrates to + side
- DNA is - charged due to its P group and migrates towards the + pole
- **the distance each DNA fragment travels is inversely related to size: larger molecules have a harder time going through
- *smaller molecules migrate down further
What experiment did they do to determine that the length of 1 helical turn was 10.5 bp?
1970s
The Mica Experiment
-Mica is a mineral that allows DNA to anchor to it through the 5’ terminal phosphate and it is stretched out in its natural form
*****DNase I is added to cleave phosphodiester bonds only on the DNA surface furthest from the mica. This allows it to cleave at the ‘cycle’ points of every turn. So cuts are made in multiples of 10 and 11 bp since 10.5 does not exist. These bands can be seen by doing gel electrophoresis.
What kind of charge does DNA have?
negative charge due to its - phosphate group
How do you see the color DNA bands in electrophoresis?
fluorescent dye is used -Ethidium: 1. planar 2. multi-ringed 3. cation (+) This dye slips itself into the stacks of bps of DNA. It fluoresces when exposed to UV light and increases its brightness after it slips itself in-between the stacks of bps.
What are the two types of gels you can use for gel electrophoresis and when do you know which to use?
- polyacrylamide (smaller strands)
- high resolution within 1 bp
- can only separate small DNA base pairs
What are the 3 forms of DNA that exist?
B DNA
A DNA
Z DNA
What are the 3 forms of DNA that exist?
B DNA
A DNA
Z DNA
B DNA
- major form
- R-handed
- 10 bp per turn
A DNA
- shorter and wider
- bp are tilted in respect to the helical axis
- RNA double helix
- R-handed
- 11 bp per turn
Z DNA
- sugar phosphate backbone is zig-zag
- no difference in major and minor grooves
- caused by purine/pyrimidine repeats
- not a stable helix
- exists only temporarily
- L-handed
- 12 bp per turn
Base flipping
- occurs during certain biological processes like DNA methylation or the removal of damaged bases
- enables the base to sit in the active site of the enzyme so it can be altered/ fixed
Base flipping
- occurs during certain biological processes like DNA methylation or the removal of damaged bases
- enables the base to sit in the active site of the enzyme so it can be altered/ fixed
Under what conditions can DNA denature?
When H-bonds are disrupted:
- high temp
- low salt concentration
- high pH (basic conditions)
Under what conditions can DNA renature or reanneal?
Conditions that favor H-bonds:
- low temp
- high salt concentrations
- low pH (acidic conditions)
DNA hybridization
The ability of complementary single strand DNA molecules to anneal allows formation of hybrid molecules if complimentary DNAs from different sources are mixed (this included DNA:RNA hybrids)
How is this done?
- DNA is denatured with heat
- A single stranded probe with a known sequence you want to find is labeled with a tag and thrown in
- as the molecules cool, a hybrid is formed
At what wavelength does DNA absorb max UV light? How?
260nm
due to its bases
Which absorbs more UV light at 260nm, single stranded or double stranded DNA?
single stranded
40% more
this is because of its exposed bases
Which absorbs more UV light at 260nm, single stranded or double stranded DNA?
single stranded
40% more
this is because of its exposed bases
Denaturation/melting occurs at a narrow or wide temperature range?
narrow range
a cooperative process
The midpoint of the transition of ds DNA to ss DNA is referred to as _____.
The melting point (Tm).
At this point, 50% of the DNA is denatured.
The midpoint of the transition of ds DNA to ss DNA is referred to as _____.
The melting point (Tm).
At this point, 50% of the DNA is denatured.
What does Tm for every DNA molecule depend on? Why?
- G: C content because there are 3 H-bonds here which means more heat and KE is needed to break the bonds.
- salt concentration of the solution. This matters because you have - charges on the phosphate groups. If they are not shielded, then they naturally repel each other. So in high salt concentrations, there are more cations to shield these negative charges on the backbone.
* *high salt concentration–> charges shielded—> more heat needed to melt
* *low salt concentration–> charges exposed–> less heat needed to melt
What does Tm for every DNA molecule depend on? Why?
- G: C content because there are 3 H-bonds here which means more heat and KE is needed to break the bonds.
- salt concentration of the solution. This matters because you have - charges on the phosphate groups. If they are not shielded, then they naturally repel each other. So in high salt concentrations, there are more cations to shield these negative charges on the backbone.
* *high salt concentration–> charges shielded—> more heat needed to melt
* *low salt concentration–> charges exposed–> less heat needed to melt
DNA topology
- B form
- Linear form
- cccDNA
- B form: relaxed with 10.5 bp per turn
- linear DNA: has free ends and can freely rotate to adjust the number of times the two chains twist around each other (in reality, it is topologically constrained because of its extreme length and order in chromosomes)
- cccDNA: covalently closed, circular DNA; the number of times the DNA strands twist around each other cannot change unless there is a break in the DNA backbone
DNA supercoiling
DNA strands must be separated during DNA replication or transcription. DNA supercoiling is the over or under winding of a DNA strand- and expression of the strain on that strand. This occurs when both ends are constrained/fixed.
-supercoiled DNA is no longer 10.5 bp per turn
DNA supercoiling
DNA strands must be separated during DNA replication or transcription. DNA supercoiling is the over or under winding of a DNA strand- and expression of the strain on that strand. This occurs when both ends are constrained/fixed.
-supercoiled DNA is no longer 10.5 bp per turn
Linking number (LK)
the number of times one strand would have to be passed through the other strand in order for the two strands to be entirely separated
-an invariant property for topologically constrained DNA
Twist (Tw)
the number of helical turns in the DNA
R-handed turn is +
L-handed turn is -
Writhe (Wr)
the number of times the double helix crosses over on itself (supercoils- positive or negative)
Does the linking number change for cccDNA?
no
formula for LK
LK= Tw + Wr
formula for LK for relaxed DNA
LK= Tw
What kind of supercoil does DNA prefer to be in, in its natural state?
-under-wound (negatively supercoiled) because positively supercoiled causes too much tension and strain and decreases the overall volume of DNA
Why is cellular DNA negatively supercoiled?
- has the tendency to unwind, making strand separation easier during DNA replication and RNA transcription
Why is cellular DNA negatively supercoiled?
- has the tendency to unwind, making strand separation easier during DNA replication and RNA transcription
- important for DNA packaging within cells. The length of DNA can be 1,000s of times longer than that of a cell so supercoiling makes it more compact and allows for much more DNA to be packaged.
Topoisomerases
- enzymes that introduce transient breaks into the DNA sugar-phosphate backbone
- changes the LK of topologically constrained DNA
- regulates the over and underwinding of DNA
What are the two types of Topoisomerases?
Type I:
- single strand breaks
- LK changes by 1
- does not require ATP
- can only decatenate if there is already and niche present
Type II:
- double strand breaks
- LK changes by 2
- requires energy from ATP hydrolysis for reaction
- **usually this is used to decatenate DNA (ring inside of a ring)
Gel Electrophoresis
- separates DNA
- size and shape (topology)
- porous gel and electric field
- load from - and migrates to + side
- DNA is - charged due to its P group and migrates towards the + pole
- **the distance each DNA fragment travels is inversely related to size: larger molecules have a harder time going through
- *smaller molecules migrate down further
-supercoiled cccDNA migrates the furthest (more write=more compact= faster migration), then relaxed cccDNA, then linear DNA, then circular on top migrate the least
What are the two types of Topoisomerases?
Type I:
- single strand breaks
- LK changes by 1
- does not require ATP
- can only decatenate if there is already and niche present
Type II:
- double strand breaks
- LK changes by 2
- requires energy from ATP hydrolysis for reaction
- **usually this is used to decatenate DNA (ring inside of a ring)
Where is chromosomal DNA located?
nucleus
Where does protein synthesis occur?
cytoplasm
Who discovered ribosomes and why are they important?
1955
George E. Palade
-cellular site for protein synthesis
RNA
information containing molecule that can obtain information from DNA in the nucleus and transfer that information to the cytoplasm to function as a template for protein synthesis
RNA
information containing molecule that can obtain information from DNA in the nucleus and transfer that information to the cytoplasm to function as a template for protein synthesis
How does RNA differ from DNA?
- sugar is ribose instead of deoxyribose
- uracil instead of thymine
- typically single stranded
- monomer: ribonucleotides (instead of nucleotides in DNA***)
How does uracil differ from thymine?
uracil:
- 5’ nucleotide: H
- 2’ ribose: OH
thymine:
- 5’ nucleotide: methyl group
- 2’ deoxyribose: H
How does uracil differ from thymine?
uracil:
- 5’ nucleotide: H
- 2’ ribose: OH
thymine:
- 5’ nucleotide: methyl group
- 2’ deoxyribose: H
How many ribonucleotides does RNA usually consist of?
4?
What was thought to be the direct role of RNA and then what was the adaptor hypothesis?
direct role: RNA template initially thought to fold up to create cavities on outer surface specific for the 20 different amino acids
adaptor hypothesis: Crick proposed that aa are first attached to adaptor molecules, which then interact with or “read” the RNA template
What was thought to be the direct role of RNA and then what was the adaptor hypothesis?
direct role: RNA template initially thought to fold up to create cavities on outer surface specific for the 20 different amino acids
adaptor hypothesis: Crick proposed that aa are first attached to adaptor molecules, which then interact with or “read” the RNA template
How was tRNA found?
- cell-free extracts used
- used radioactive labeled amino acids with 14C to follow protein synthesis
found:
1. particular RNA species became labeled with 14C amino acids and this labeled RNA was then able to transfer the amino acids to a growing polypeptide chain
2. Concluded that this tRNA acts as an intermediate carrier of amino acids in protein synthesis
How was tRNA found?
- cell-free extracts used
- used radioactive labeled amino acids with 14C to follow protein synthesis
found:
1. particular RNA species became labeled with 14C amino acids and this labeled RNA was then able to transfer the amino acids to a growing polypeptide chain
2. Concluded that this tRNA acts as an intermediate carrier of amino acids in protein synthesis
ribosomal RNA (rRNA)
- site of protein synthesis made up of proteins and rRNA
- 85% of cellular RNA is found in ribosomes
- thought to be the RNA template that orders aa during protein synthesis
- studies in E.coli demonstrated that rRNA populations in different ribosomes are not diverse enough in length or nucleotide composition to be the template for protein synthesis
Describe the Pulse-Chase Experiment.
**showed evidence that RNA is synthesized in the nucleus and moves to the cytoplasm
Exp:
- exposed cells to radioactively labeled ribonucleotides (the Pulse)
- added large excess of unlabeled ribonucleotides
* during the pulse, any mana that is synthesized is radioactively labeled and easy to detect - examined the location of the radioactive-labeled RNA at different times following the pulse (the Chase)
Found:
- after 15 min, new RNA in nucleus
- after 88 min, new RNA moved to the cytoplasm
