Ch. 7 & 8 Flashcards
1
Q
Commonly used to transfer plasmids in plants?
A
Agrobacterium tumefaciens
2
Q
Where is the chromosome located in a bacteria cell?
A
Inside the Nucleoid
3
Q
What happens to the nuclear membrane when a cell divides?
A
It disappears as do the Chromosomes becoming chromatin
4
Q
When do chromosomes condense and appear visible?
A
only during cell division.
5
Q
What is gene density?
A
how many protein coding regions are there in a stretch of DNA
6
Q
What has higher gene density, E. coli or Humans?
A
E. Coli has a lot greater gene density.
7
Q
What is an intron?
A
Interspersed non-protein-coding-regions,
they are removed from the RNA after the transcription process: RNA Splicing
8
Q
In humans, about what percentage of a particular protein-coding gene directly encodes the desired protein?
A
5% actually encodes the protein, the remaining 95% percent is made up of introns.
9
Q
What percentage of the human genome is composed of intergenic sequences?
A
60%
10
Q
What are the two types of intergenic DNA?
A
Unique and Repeated
11
Q
What pseudogenes?
A
coding regions that look like genes but are never transcribed
12
Q
What are microsatellites?
A
repetitive regions of Intergenic regions in the genome
<13 bp
Tandem
Genome wide - forms of transposable elements
13
Q
What are unique regions of human genome?
A
regulatory
14
Q
What is a Gene?
A
A gene is the portion of a chromosome that effects a single phenotype.
Codes for single enzyme, protein, polypeptide, or “some gene product” (a structural RNA).
Can also be regulatory sequences
-A mutation of a regulatory sequence does not affect the sequence of the gene product, but the expression of a particular gene.
(if you remove a binding site on RNA polymerase, it will not affect the sequence at all, but it will affect the expression the gene)
15
Q
What components are required for Chromosome duplication and segregation?
A
- Origins of replication
- Centromeres
- Telomeres
- SMC’s (structural maintenance of chromosome proteins)
16
Q
What happens to a cell in G1 phase?
A
Prepares for cell division
gathering proteins, lipids etc…
17
Q
What are the key events in S phase?
A
Initiation of DNA replication ( a double stranded DNA molecule begins to separate and replicate at origins of replication
More replication ad establishment of cohesion by cohesin
Completely separated into sister chromatids, however still connected to each other by cohesin.
18
Q
Once replicated by origin of replication, how do you separate the chromosomes into new cells?
A
Need a centromere:
a region of DNA that can assemble a set of proteins called kinetochore, that can attach the spindle fibers
19
Q
What are the required components of Maintenance of Chromosomes in Cells?
A
Origin of replication, Centromere, Telomere
components can be DNA sequences and RNA and protein structures
20
Q
What is the basic function of the Origin of Replication?
A
DNA Replication
21
Q
What is the basic function of a Centromere?
A
Chromosome Segregation at cell division
22
Q
What is the basic function of a Telomere?
A
Maintaining individual chromosome integrity during condensation and segregation?
23
Q
How do sister chromatids hold together before cell division?
A
By cohesin
wraps around both sister double strand chromatids
24
Q
How do chromosomes condense from the loose coils within the nucleus to the X-shaped bodies during mitosis?
A
By Condensin:
cohesin makes loops, the conDensin links those loops together which brings them in even closer together
25
If sister chromatid cohesion is by cohesin,
and condensation is by condensin,
what must be cleaved in order to pull the sister chromatids apart from metaphase to anaphase?
Cohesin cleavage, while the chromsomes are still "condensed" by condensin.
26
When cohesin is cleaved, what happens to cohesin?
It is cleaved at a specific cleavage site that opens the loop, but is still available for later use in S-phase DNA replication.
27
How does a chromosome de-condense after the cell separates?
The bridge on Condensin is cleaved
28
???contradicted???what type of supercoil wraps around a histone?
A Left Handed supercoil wraps around a supercoil
29
What is a nucleosome and what is it made of?
```
A nucleosome is the "primary" structure of chromatin.
It is made up of:
A. Microfoccal nuclease
B. Histones, the octamer
C. N-terminal tails
D. The 10nM fiber
```
30
What is a confocal image?
Focal plane that is flat, only a section at a time to look at whats under the microscope
31
What is the approximate length of DNA in one cell, and what is the diameter which it must be contained?
about 2 meters of total DNA that must fit into a 10 x 10^-6 m (10 micrometer) nucleus.
32
If the approximate length of total DNA in a cell is 2 meters, what is the average length of DNA in each chromosome?
Humans have 46 chomosomes, therefore, 2 m / 46 = 4.4 cm.
33
When will you ever find histones free and floating by themselves?
In 1M NaCL
34
H2A-H2B part of a histone is made up of what, and classified as what type of protein?
One H2A, One H2B, (two H2A, two H2B histones in a nucleosome)
H2A-H2B Dimer (two H2A-H2B dimers in a nucleosome)
35
H3-H4 part of a histone is made up of what, and classified as what type of protein?
2 H3, 2 H4 (two H3 and two H4 histones in a nucleosome)
H3-H4 tetramer (one H3-H4 tetramer in a nucleosome)
36
Considering the following histone proteins with Molecular weight and % Lysine & Arginine listed respectively, why does it make sense for H1 to contain greater % of Lysine and Arginine?
H2A, 14,000Mt, 20% x2 in histone octamer (nucleosome)
H2B, 13,900Mt, 22% x2 in histone octamer (nucleosome)
H3, 15,400Mt, 23% x2 in histone octamer (nucleosome)
H4, 11,400Mt, 24% x2 in histone octamer (nucleosome)
H1, 20,800Mt, 32% not considered part of nucleosome
Since Lysine and Arginine are two of the basic amino acids, they are positively charged in the body, which act to stabilize any negative charge in the nucleosome.
37
What is the topological shape the the DNA assumes when it winds around an octamer?
Is it plectonemic or toroidal?
Supercoil, and toroidal (solenoid)
38
What can cleave lysine and arginine?
trypsin
-can use in vitro to cleave n-terminal tails
39
???contradicted??? what signs are histone octomer (nucleosome) supercoils?
left handed super coils are positive supercoils.
40
What fiber do you get when you have a histone octomer with no H1 present?
a 10 nm fiber
| 100 A in diameter
41
What type of fiber do you get when you have a histone octamer with H1 present?
a 30 nm fiber
-note that it is thicker because it is more dense with nucleosomes
42
If you trypsin treat N-terminal tails of histone octamer, can you form the 30nm fiber?
No, the tails (positively charged) serve to interact with the exterior of the nucleosome which allows for tighter packing.
43
What are two requirements to form a 30nm fiber?
H1 histone protein and N-terminal tails of histone octamer.
44
What are the two possible structures for a 30 nm fibers?
| How many nucleosomes are in each layer?
Solenoid and zigzag
Solenoid requires 6, I think zigzag requires 4
45
What is a first order structure?
the 10nm beads on a string formation of chromatin
| nucleosome and linker DNA
46
What is a higher order structure?
Beads on a string wrapped, 30nm fiber.
47
What is H2A.X?
A variant of H2A histone throughout the chromatin. When a double-stranded break in DNA occurs, the H2A.X near the break is "flagged" with a phosphoryl group and recruits DNA repair enzymes
48
What is CENP-A?
A variant that replaces H3 histone protein. The CENP-A tail binds kinetochore components. Kinetochore proteins are assembled in regions with CENP-A.
**Centromere regions are defined by specific sequences of DNA, there has to be some sort of protein that reads the sequence in this region and plants the CENP-A structure in this region.
49
with 147 bp of DNA, how many negative charges?
2 negative charges per base pair, 147x2 = 294 negative charges total for one histone octamer.
50
????confused????What is the average molecular weight for an amino acid?
How many amino acids in a 14,000Mt H2A protein?
average is about 100-110 AMU
H2A = 140AA, 20% Lysine arganine = 28 positive charges
51
What type of bond between DNA and nucleosome?
Ionic bond, or "Electrostatic Interactions"
52
How do you separate DNA from Nucleosomes?
High concentrations of NaCl
53
What type of interactions are between DNA and Histones, dynamic or static?
Dynamic, ionic bonds are charge neutralizations, and can roll and move reforming bonds (no net increase or decrease)
54
What is histone remodeling?
Moving histone octomer along DNA
55
What are they types of ATP-Dependent Nucleosome-Remodeling complexes?
```
SWI/SNF (8-11) (bromodomain)
ISWI (2-4) (bromodomain, SANT domain, PHD finger)
CHD (8-10) (chromodomain, PHD finger)
SWR1 (12-14) (bromodomain, SANT domain)
IN080 (10-12) (NONE)
```
(#-#) are number of subunits, the each also bind to specific histone domains (domain), and they all slide
56
What is the mechanism of a nucleosome remodeling complex?
The specific nuclesome remodeling complex (torsion subdomain, ATPase domain, tracking subdomain) attaches to the coiled DNA around the histone octamer like a clamp.
Torsion subdomain pulls coiled DNA which causes the linker to enter the region into the nucleosome
DNA rachets through the tracking subdomain
Torsion subdomain resets, binds new DNA. Under-twisted DNA wave moves to distal linker.
57
What can the histone N-terminal tails be modified to?
Serines, threonines, lysines and argenines
58
Acetylated histone tails will bind to what domain protein?
Acetylated histones tails bind to Bromodomain proteins.
59
Methylated histone tails will bind to what domain protein?
Methylated histones tails will bind to Chromodomain proteins
60
The bromodomain of the nucleosome-remodeling complex binds what specific modified histone?
Bromodomains - bind specific acetylated histones
61
The chromodomains of the nucleosome-remodeling complex binds what specific modified histone?
Chromodomains - bind specific methylated histones
62
The TUDOR domains of the nucleosome-remodeling complex binds what specific modified histone?
TUDOR domains - bind specific methylated histones
63
The PHD fingers of the nucleosome-remodeling complex binds what specific modified histone?
PDH fingers - bind specific methylated histones
64
The SANT domains of the nucleosome-remodeling complex binds what specific modified histone?
SANT domains - bind specific unmodified histones
65
How are nucleosome-remodeling complexes activated at specific locations in the chromatin of a nucleus?
histone acetyl transferase binds region of nucleosome directed by dna-binding protein on 30 nm fiber
acetyl transferase then transfers acetyl groups which opens 30nm fiber into 10nm fiber
(acetyls neturalize charges on lysines which opens it up)
A second DNA-binding protein then attaches to 10nm fiber which recruits nucleosome remodeling complex that attaches to directed sequece (as histone acetyl-transferase leaves). It further opens and rolls nucleosome to activate a gene.
66
How are histone octamers distributed during DNA replication?
Each strand gets half old histone proteins and half new.
Histone acetyltransferases binds acetylated histone tails using bromodomain -----> modification of "new" histones
67
What are common properties of all DNA polymerases?
1. DNAn - 3'OH + dNTP -----> DNAn+1 -3'OH
2. Nucleophilic attack of 3' OH at chain end upon the 5' alpha-phosphorus of the dNTP being added.
3. Requires a template to polymerize DNA
4. Requires a primer, a short fragment of nucleic acid complimentary to the template strand. Polymerase can add only to an existing chain at the 3'OH
68
What is the source of -∆G that drives the DNA polymerization reaction?
The enzyme pyrophosphatase that hydrolyzes PPi
Counting the number of phosphodiesters before and after the rxn, one is broken while one is formed. So the delta G is zero. However, pyrophosphate (PPi), is hydrolyzed (by the enzyme phyrophosphatase) which has a ∆G of -33 kJ/mol, which is very favorable.
69
In short, what is the Mechanism of DNA polymerase?
- Single active site
- mechanisms for choosing correct nucleotide
- processive
- proof-reading
70
What are 5 strategies for accurate replication?
1. balanced levels of dNTP's
2. two stage reaction:
a. dNTP pairs w/ template base.
b. enzyme closes around the site and catalyzes the reaction only if fit is correct.
3. 3'-5' exonuclease of DNA polymerase detects and removes mismatches.
4. repair enzymes replace errors on the newly syn. strand.
5. use of RNA primers to begin strand synthesis.
71
What other molecule must also be part of the active site in DNA polymerase to determine specific addition of nucleotides?
The template is part of the active site! which is complimentary for each dNTP that needs to be added.
72
How does the correct base pair match up to template strand?
The correct base pairing holds the alpha-phosphorous of the dNTP in the correct position for 3'OH to attack it
73
Why can't ribonucleotides add to template strand by DNA polymerase?
There is a discriminator amino acid on the structure of DNA polymerase that does not allow for the extra hydroxyl group of ribonucleic triphospates (rNTP's)
74
How do two metal ions help ensure correct base paring?
metal ions hold divalent cations in position (aspartates and triphospates) which activates the 3'OH to attack alpha-phosphorous
75
DNA Polymerases are Processive Enzymes. Define Processivity?
is a characteristic of enzymes that operate on polymeric substrates, and with DNA polymerase, it is the degree of processivity as the average number of nucleotides added each time the enzyme binds a primer:template junction.
76
In the Okazaki experiment, why was 3H-thymine used to label all DNA?
Because he was interested in in DNA, not the RNA?-I think
77
What is required to begin a leading strand or Okakzaki fragment?
A primer must be made!
78
What enzyme makes the primers, and what type of nucleic acid, and how does new synthesis begin?
Primase makes short RNA primers that DNA plolymerase adds onto to begin DNA synthesis.
79
What is the fate of the Okazaki fragment?
They are later removed and the gaps are filled in by a special DNA polymerase. Thi increases the fidelity of DNA replication (why would filling in a gap be more accurate than beginning a short fragment?)
80
What is the function of DNA helicase?
It separates the two strands of the double helix at the replication fork. It uses ATP to move down strand into the fork direction.
81
How can supercoiling be relieved that was caused by the helicase?
Topoisomerases relieve the supercoiling caused by the helicase.
82
How is reassociation of DNA prevented when helicase leaves single stranded regions?
Single-stranded DNA-binding proteins (SSBs)
-cooperativly bind to the single-stranded regions left by the helicase to prevent reassociation. SSBs bind to ssDNA and also to each other.
83
What are all the players at the replication fork?
2 DNA polymerases, sliding clamp (beta clamp) and clamp loader, Primase, Helicase, SSBs, Topoisomerases
84
What is the purpose of the Holoenzyme and what is it made of?
The Holoenzyme allows simultaneous leading and lagging strand synthesis. It consists of, 2 DNA Pol cores, t protein (two identicle parts attached to each Pol core), flexible linker (2), one gamma complex (clamp loader), one sliding clamp
85
Go through the steps of lagging strand DNA replication.
DNA helicase is bound at tProtein and unwinds dna (ssrp's bind) while lagging -DNA Pol III is creating an okazaki fragment
- Primase binds to helicase and places RNA primer on ssDNA (written as 5-3), while lagging strand Pol is creating previous okazaki fragment
- Primase released, while DNA pol releases DNA w/ sliding clamp after completion of okazaki fragment.
- Newly primed lagging strand ends up at the clamp loader which loads a clamp
- DNA Pol binds sliding clamp at "primer:template junction" and begins synthesis of new okazaki fragment (written 5-3)---then, again, DNA & clamp leave DNA pol as a complete Okazaki fragment.
86
How is Helicase and Primase interacting as an E. coli replisome?
Tau subunit binds helicase and stimulates its activity.
| Pimase associates with the helicase, and this stimulates the synthesis of a new primer.
