Flashcards in Midterm 1 Deck (147):
The flow of genetic information goes from DNA to RNA to Proteins
Beadle and Tatum
They worked with bread mold and showed that each gene controlled a single protein (one gene: one enzyme hypothesis)
The lethal strain S has a smooth capsule that allows it to evade the host's immune system. The nonlethal strain was called the R strain. Griffith showed that the genetic material from the heat killed S strain turned the R strain into lethal cells.
The S strain was able to transform the R strain into a virulent strain.
Avery, Macleod, and McCarty
They took Griffith's experiment and did it in vivo. Then, they treated cells with DNase, RNase, and Proteinase to see which would affect the transforming ability. Since DNase prevented transformation, they concluded that DNA is responsible.
Hershey Chase Experiment
Used S and P radioisotopes to label the protein capsid and DNA of bacteriophage. They saw that most of the P was in the pellet where the cells are while the S was in the supernatant. This concluded that bacteriophages inject their DNA into the host cell and only this genetic info directs the creation of more progeny. DNA = genetic info!
Building blocks of DNA
Purines: Adenine and Guanine
Pyrimidines: Cytosine and Thymine
Polarity of DNA
Polarity comes from phosphodiester linkages, and has a net negative charge.
G and C are in same amount and A and T are in same amount
Rosalind Franklin and Maurice Wilkins
Performed X-ray diffraction studies on DNA and their data showed that DNA was in helical form with "ladder-like" rungs connecting parts of it
Watson and Crick
They proposed 3D structure of DNA to be double helix
Structural characteristics of DNA
Has sugar phosphate backbone, bases project inwards, one turn is about 10.5 bp, has major and minor grooves.
DNA has about 10.5 bp per turn in solution
Separates DNA molecules according to their weight. DNA travels to positive side because it is negatively charged
Intercalates between bases and can be seen under UV light
B form of DNA
Represents an ideal form of DNA with about 10 bp per turn. But DNA is not perfectly regular like this
Z form DNA
DNA is more elongated and slim, about 12 bp per turn. It is also left handed
Why is DNA so stable?
Large number of weak h-bonds, and also stacking interactions
Why is major groove rich in chemical information?
Proteins can tell by the order of hydrogen bonds/acceptors which base pairs are there. AADH = GC and ADAM = AT
Can be done with high heat or changing pH (the OH- concentration). Separates the double helix into single strands
Conditions for denaturation
high temp, lower salt concentration, high pH because they break the h-bonds
GC content and denaturation
The more GC present in DNA, the more stable it is and therefore the more heat is required for denaturation.
Wavelength that DNA absorbs
DNA sequence homology
similarity between the sequences of two DNA molecules
the pairing between complementary ssDNA or RNA. Only occurs when the strands have homology. (Used in southern blots)
Southern vs. Northern vs. Western blots
Southern = DNA size
Northern = RNA probed with DNA probe
Western = proteins probed with antibodies
relaxed circular DNA has about 10.5 bp per turn but supercoiled has more. It is caused by some sort of structural strain on the DNA, like underwinding
The number of times that each strand winds around the other. L = Twist + Writhe, when there are no supercoils, L=T
Enzymes that increase or decrease the linking number by underwinding the DNA. They break one strand, allowing the DNA to unwind and then religate
Type 1 and 2 topoisomerases
Type 1 relax supercoiled DNA without ATP
Type 2 need ATP to relax the DNA or to introduce supercoils
They are used in chemotherapy to stop fast cell division, and stop topoisomerases from doing their jobs
In the pulse, we expose the cells to labeled precursors which the cell will use when making macromolecules like DNA, RNA or proteins. In the chase, we wash out the label and let cell grow. Then, we use x-ray imaging to see where the labels end up
What labels are used in Pulse-chase experiments?
Protein = 35S-methionine
DNA = 3H-thymine
RNA = 3H-uracil
Conclusion of Pulse-chase experiments
RNA is synthesized in the nucleus and then migrates to the cytoplasm where it partakes in protein synthesis. Confirms central dogma
-Has ribose instead of deoxyribose
-Uracil instead of thymine
-Single stranded and has secondary structure
Why is RNA less stable in alkali?
Since the ribose sugar has a hydroxyl group, it can be deprotonated and this O- can attack the phosphodiester bond and degrade the RNA
Types of RNA
mRNA, tRNA, rRNA, miRNA, siRNA, and ribozymes
Secondary structures for RNA
Stem-loop, bulge, loop, RNA tends to fold on itself where there are complementary sequences
What form of helix does RNA take?
A form rather than B form like DNA
Does RNA follow Watson-Crick base pairing? Why or why not?
No, you can find GU and GA very commonly in RNA. Triple base-pairing is also possible for stabilization
Uses for Mg2+ and K+ in RNA
Since they are positively charged, they shield the negative charge of the backbone and provide stability. Also helps RNA pack more tightly
Its an endoribonuclease that cleaves off a leader segment from 5' end of precursor tRNA and changes it into functional mature tRNA
Another ribonuclease that is self-cleaving RNA
Primary structure of proteins
Chain of amino acids called a polypeptide
Either an identical amino acid, or an amino acid with similar properties. If an amino acid is similar enough, it shouldn't change the protein function or structure that much
Secondary structure of proteins
Beta sheets, alpha helices, random coils, and turns
Protein tertiary structure
Stable 3D structure
Protein quaternary structure
The number of polypeptide subunits together
Ex) hemoglobin has 4 subunits
What interactions are occurring in protein secondary structure?
Secondary structures are stabilized by H-bonds between the peptide bonds in the backbone
Both polypeptides are identical
Non-identical polypeptides (2) could be more if it wasn't a dimer
Composed of multiple polypeptide chains
Individual polypeptide chains
Structural domain of a protein
-A part of a single polypeptide chain that has folded onto itself.
-Domains can have independent functions, or all be used for one function
-Protein function is usually based on the combination of different domains
The region where two different polypeptides interact
Their quaternary structure creates an extremely specific antibody-antigen interaction
-contain disulfide linkages for added stability
A combination of secondary structures found in many proteins
EX) Beta barrel, coiled coil, and helix loop helix motif
4 Different types of protein folding
2) Chaperone assisted folding
How does major groove information?
Through its pattern of h-bond donor and acceptor groups. Bases can be read by this characteristic pattern
Do protein H-bonds interfere with base-pair bonding?
Protein to protein interactions
-may be affected by post-translational modifications like methylation or acetylation
Very specific catalyst that provides an environment for a reaction to occur rapidly
(Lower the activation energy of the reaction)
Protein levels modified by...
Removes certain peptide segments in order to produce usable insulin. It's activated in response to specific conditions
Def of Phosphorylation
Adds phosphate using ATP
Def of Adenylylation
Adds adenine using ATP
Def of Acetylation
Adds acetyl group from Acetyl-CoA
Def of Methylation
Adds methyl group from S methionine
Represents the dissociation constant and is the concentration of the ligand at which half of all the ligand binding sites on the protein are occupied
What does a high Kd mean?
It means the protein has a low affinity for the ligand
Ways to purify protein
Separation, detection using immunological techniques, and sequence or structural analysis
1) Lyse cells
4) Gel electrophoresis
What does detergent do to cells?
Makes holes in the plasma membrane
Ion exchange chromatography
Separates molecules according to their charge. Positively charged particles will come through column first and negatively charged particles are bound to the beads (can be eluted later). The opposite will happen if you use negatively charge beads
Gel filtration chromatography
Large proteins come out first because the smaller proteins go through the beads and can get stuck.
Use a competing molecule, salt or change in pH to remove the protein of interest from the column
Antibody affinity chromatography
Separated molecules according to their affinity for a specific ligand like an antibody. The beads have the antibody so the protein will stick to the beads. The antibody-binding proteins can be eluted by lowering the pH
Antibody is again coupled to the bead and this makes the protein heavier for centrifugation
A sequence of 7-10 amino acids recognized by an antibody
What wavelength do we measure for proteins?
How can we measure beta-galactosidase activity?
Instead of giving the cell lactose, we give it ONPG. Bgal will also recognize this fake sugar and will cleave it to create galactose and ONP which turns yellow
What does SDS do?
It linearizes the protein and coats it with a negative charge
Reduces disulfide bonds
What does SDS-PAGE separate by?
It separates proteins according to their molecular weights. Low MW will travel farther down and high MW will travel through the gel more slowly.
2D gel electrophoresis
The first gel separates the proteins by their IEP. While the second separates by size.
Stands for the isoelectric point. It is the pH at which the protein no longer has any charge.
Transfer the proteins from SDS PAGE gel to a nitrocellulose membrane. Then add an antibody that will bind tightly to the antigen. Next, add a secondary antibody that contains a marker that will bind to the primary antibody.
Wells are coated with an antigen. Next add plasma from the subject and if they have the antibody, it will bind to the antigen. Then add a secondary antibody to bind to that first one. Finally, if that secondary antibody binds, it should chnge the color of the solution
A type of protein sequencing that takes off one amino acid at a time and can identify which amino acid it was through column chromatogrpahy.
What is mass spectrometry used for?
It's a fast an accurate way to measure the molecular weight of a protein. It can also determine the sequence of proteins
The main goal is to identify the full set of proteins produced by a cell under a certain set of conditions
An x-ray beam is shot through a crystallized protein and from the diffraction pattern, we can determine the protein's structure.
Stands for the Electrophoretic mobility shift assay. The protein is mixed with radio-labeled DNA and if the protein sticks to a certain binding site, it will produce a different band in western blot than the rest of the DNA
Another way of finding out where the protein is binding to the DNA. Put a radio-labelon end of DNA and allow protein to bind. Then cute DNA at different sites and see where the DNA wasn't cut. This is usually the location of the protein.
1) Proteins are attached to DNA.
2) Antibody against the protein of interest is added
3) Precipitate the sample with the primary antibody attached
4) Take off protein and antibody and amplify the sequence of DNA using PCR
Bacterial enzymes that recognize specific, symmetric (palindromes) sequences in DNA and cleave at these sites
Genes with similar sequences that indicate a common ancestor
What year was the human genome finally sequenced?
DNA in bacteria
They have circular chromosomes and extrachromosomal elements like plasmids
What causes lower chromosomal gene density?
As an organism's complexity increases, it's gene density decreases because the DNA is filled with repeating sequences and introns rather than the actual gene
What happens to introns?
They are removed form the RNA after transcription during RNA splicing
Intergenic vs. Intragenic
Intergenic is within the gene itself while intragenic is between different genes
They are integrated into the genome after reverse transcription but can't be expressed because they lack the right regulatory sequences to direct their expression
Sequences that can move form one place in the genome to another. (Transposition)
Homologous genes within a species
Homologous genes between two species
Conserved linear order of the genes. It's a good illustration of common ancestry. We have quite a bit of synteny with the mouse genome
Proteins related in amino acid sequence and 3D structure
Single nucleotide polymorphism
A difference in one nucleotide
The four main factors that drive evolution
1) Mutation rate
2) Natural selection
3) Genetic drift
DNA mixed with protein
Number of nucleotides between nucleosomes
20 to 60 bp
How many base pairs per nucleosome?
200 base pairs per nucleosomes
Structure of nucleosome
Has 2 copies of each H2A, H2B, H3, and H4. Only one copy of H1. H2A and H2B form a dimer and H3 and H4 come together as a tetramer
What charge do nucleosomes carry?
They are positively charged
Has three alpha helices and two loops connecting them
Where do histone interact with the DNA?
They interact between the histone fold and the phosphide ester backbone of the minor groove
Function of histone tails
They stick out of the histone and are sites of post-translational modifications like methylation, acetylation, and phosphorylation. They aren't needed for nucleosome formation though. They also interact with adjacent nucleosomes to further pack the DNA tightly together
Function of H1
It binds to the linker DNA at the end of the nucleosome and also the middle of the associated 147 bp. It leads to more compact defined structure.
Order of DNA packing
DNA ➡ nucleosomes ➡ 30nm filament ➡ extended form of chromosome ➡ condensed section of chromosome ➡ the mitotic chromosome!
They are enzymes that loosen the interactions between the DNA and the nucleosomes in order to increase its accessibility
Slides the DNA off the nucleosome to expose the genes needed
Transfers nucleosomes to a different DNA so that that strand is now free
H2A and H2B exchange
The third form of unwrapping DNA exchanges the subunits in a nucleosome in order to loosen up the DNA
Signal of acetylation
It tells cells to start transcription of this gene. It is done by adding an acetyl group from acetylcoA to a lysine or other amino acid
Signal from Methylation
It's usually tells the cells that this gene is not needed and signals the nucleosome to bind more tightly to the DNA. It is done by adding a methyl group to arginine
Method of DNA replication