Exam 3 Flashcards
1
Q
Deoxyribonucleic acid (DNA)
A
Each nucleotide is made up of ribose, phosphate, and a nitrogen base
2
Q
Purine nucleotides
A
Adenine and guanine, 2 rings, pair with pyrimidines
3
Q
Pyrimidines
A
Cytosine and thymine, 1 ring, pair with purines
4
Q
What holds the sugar phosphate backbone together?
A
Phosphodiester bonds
5
Q
Chargaff’s rules
A
A+G=T+C
6
Q
double helix structure
A
creates a major and minor groove, important for the proteins that bind to its ability to access the DNA
7
Q
Nucleosomes
A
DNA is compacted 7x by winding it around these
They are the most basic units of compaction, made up of histone proteins.
8
Q
5 main types of histones
A
H1, H2a, H2b, H3, H4
9
Q
What histones make up nucleosomes?
A
2x2H2A, 2XH2B, 2XH3, 2XH4. 146 bp of DNA is wrapped around this core of proteins
10
Q
What is the purpose of the H1 histone?
A
Serves as a locket that seals the DNA/histone complex
11
Q
Chromatin
A
DNA + histones
12
Q
Structure of DNA
A
Antiparallel double helix
13
Q
Semiconservative replication
A
DNA is replicated by unwinding the 2 strands of the double helix and building up a new complementary strand on each of the separated strands of the original double helix
14
Q
Conservative replication
A
copy the whole chromosome - daughter DNA completely new
15
Q
Dispersive replication
A
randomly copy a bit here a bit there
16
Q
Meselson and Stahl experiment
A
grew E. Coli with a heavy isotope of nitrogen - integrated into the genome. then switch to a lighter nitrogen isotope, look at different weights of DNA
17
Q
Steps of DNA replication
A
- Initiation
- Elongation
- Termination
Bidirectionally
18
Q
Initiation
A
Unwind the double helix at the right spot and keep it unwound
19
Q
Helicase
A
Enzyme that breaks H-bonds and unwinds the DNA
20
Q
Single-stranded binding proteins
A
Keep DNA strands from binding each other or itself and protect them from being chewed up
21
Q
DNA gyrase (bacteria) / topoisomerase (eukaryotes)
A
Keeps DNA from tangling up as the replication fork moves along
22
Q
Origin of replication
A
The location where DNA replication is initiated
23
Q
Rolling circle replication
A
In circular bacterial chromosomes, replication occurs in both directions until they meet
24
Q
Specific DNA sequences at the ori
A
AT-rich - AT is easier to break because they only have 2 hydrogen bonds
25
DnaA
A protein that binds to 9-mer region, forcing unwinding of the 13-mer region to form an open complex
26
DnaB and DnaC
DnaC delivers DnaB protein to the open complex to initiate helicase activity
27
Which regions are replicated 1st?
Regions of genes actively expressed (euchromatin) are replicated earlier than those with repressed genes (heterochromatin)
28
Elongation
DNA polymerase, the enzyme that add nucleotides can only add them to the 3’ end and since DNA is antiparallel, replication occurs in opposite directions at the fork. DNA is added 5 —> 3 (of the new strand)
29
Primer
DNA pol III cannot start a chain of nucleotides, it can only add to a chain, so it needs to add on to primers. The primer is a short sequence of nucleotides that binds to the template strand to form a short duplex
30
Primase
The enzyme that adds the primer to the DNA chain; it is a type of RNA polymerase
31
DNA polymerase I
Enzyme that removes the primer - 5' to 3' exonuclease activity
5' to 3' polymerase - adds DNA nucleotides
32
Ligase
Stitches DNA back together
33
DNA topoisomerase
Relaxes supercoiling - recognizes supercoil, cuts DNA, rotates it to linearize it
34
Proofreading activity
DNA polymerase III finds incorrect bases and excises them because of its 3’>5’ exonuclease activity
35
Telomeres
Because DNA is synthesized only in 5’>3’ after the primer is removed, the end of the lagging strand is missing a bit of DNA at the telomere. Functions as a protective cap by sheltering the 3’ overhang of the DNA, which would be “seen” as a DNA break
36
Telomerase
Extends the template strand, primer is removed —> new 3’ overhang, makes new telomeres every cell division, made of an RNA and a protein (ribonucleoprotein), cancer cells reactivate telomerase expression
37
Hayflick’s limit
Somatic cells have a definitive number of replication cycles, dictated by the length of telomeres
38
The central dogma
DNA, RNA, protein
39
Ribozymes
RNA can catalyze biological reaction like proteins
- join amino acids to make proteins
- splicing pieces of DNA together
- tRNA processing
40
Messenger RNA (mRNA)
Used to encode the sequence of amino acids in a polypeptide. May be polycistronic (encoding two or more polypeptides) in bacteria and archaea. Encodes single polypeptides in nearly all eukaryotes
41
How many bonds are there between A and T?
2 hydrogen bonds
42
how many bonds are between G and C?
3 hydrogen bonds
43
histone charge
positively charged, DNA is negative --> interaction is favorable
44
histone structure
globular domain around which the DNA is attached, have tails that are accessible for things to bind to them, allows for modification of the interaction between nucleosomes and DNA
45
DNA polymerase III
adds new nucleotides in the 5' --> 3' direction
falls off of DNA - need the DNA polymerase III holoenzyme (catalytic core + accessory proteins), turns DNA pol III into a processing enzyme
46
Beta clamp
protein made of many dimers, latches DNA polymerase onto DNA by pushing it along
47
End replication problem
solution - add a DNA sequence that doesn't encode for genetic information
48
telomeres
made up of repetitive "non-coding" DNA of unique sequences for every species, 500-3000 repeats, G-rich overhang, function as a protective cap by sheltering the 3' overhang of the DAN into a T-loop
49
Sheltering complex
hides the 3' overhang
50
stem cells
essentially immortal unlike somatic cells
51
RNA vs DNA
RNA has an extra OH at the 2' carbon
52
What was the genetic info of the first organisms made of?
RNA: Acetyl-coA, Vit B12, dNTPs, primers
53
tRNA
decoders of DNA information into amino acids for proteins
54
rRNA
molecular machines that catalyze the assembly of proteins by using mRNAs and tRNAs
55
snRNA
process mRNAs
56
transcription direction
template: 3 -->5
RNA: 5 --> 3
57
+1 nucleotide
1st nucleotide of the coding region, not the first nucleotide that encodes information, upstream of the part that encodes info
58
transcription initiation prokaryotes
specific sequence mark for the start of the gene: -35, -10
- RNA pol holoenzyme scans DNA for the right start, opens the helix and binds it to start transcription
- core enzyme: synthesizes RNA
- sigma subunit: recognizes -35 and -10
59
bacteria RNA pol subunits
- sigma: bind to -35/-10 regions and unwinds the -10 to allow the polymerase to bind to the promoter
- beta' - binds DNA
- alpha - binds regulatory proteins
- beta - catalytic
- w - enzyme assembly and gene expression
60
sigma subunits
different sigma subunits recognize different sequences for "start" and "almost start", those will only turn on genes when there is a correct environmental signal (eg heat shock)
61
bacterial transcription elongation
the polymerase walks along the gene unwinding the DNA to copy it. The DNA that's already being copied rewinds, so only the part that's actively being copied, the transcription bubble, is unwound. The energy used for this process comes from the hydrolysis of nucleotides, energy rich triphosphate group
62
Intrinsic termination
bacteria
doesn't need help
40 bps, GC-rich stretch, followed by 8As
GD hybridize to make a hairpin
RNA polymerase pauses if the short DNA-RNA hybrid in the transcription bubble is weak and will backtrack to stabilize the hybrid, but finds the hairpin and detaches from the DNA
63
RHo-dependent termination
bacteria
binding of the protein Rho Factor to rut sites, the pausing of polymerase, and rho-mediate dissociation of the RNA polymerase
distinct termination sequences from the genes utilizing intrinsic termination
rut site - upstream segment in a sequence that is 40-60 nucleotides that is rich in c, poor in C
64
RNA Polymerase I
transcribes rRNA genes (excluding 5S rRNA)
65
RNA polymerase II
transcribes all protein encoding genes, for with the ultimate transcript is mRNA, and transcribes some snRNAs
66
RNA polymerase III
transcribes the small functional RNA genes (such as the genes for tRNA, some snRNAs, and 5S rRNA
67
RNA processing
modifications to eukaryotic RNA, including capping and splicing, that are necessary before the RNA can be transported into the cytoplasm for translation
68
common eukaryotic promoter consensus sequence elements
TATA box - -25
GC box - -90
CAAT box - -80
69
transcription - initiation - eukaryotes
RNA pol II cannot bind on its own and is recruited by general transcription factors (GTFs)
70
TATA box
a DNA sequence found in many eukaryotic genes that is located about 30 bp upstream of the transcription start site
71
TATA-binding protein (TBP)
A general transcription factor that binds to the TATA box and attracts other transcription factors and RNA polymerase II to promoters
72
transcription - elongation - eukaryotes
1. addition of a cap at the 5' end
2. splicing to eliminate introns
3. the addition of a 3' tail od adenine nucleotides (polyadenylation)
73
5' processing
a special structure, consisting of a 7-methylgaunosine residue linked to the transcript by three phosphate groups, that is added in the nucleus to the 5'end of eukaryotic mRNA
The cap protects an mRNA from degradation and is required for translation of the mRNA in the cytoplasm
74
3' processing
AAUAAA or AUUAAA sequence at the 3' end of the transcript, called the polyadenylation signal, recognized by enzyme which stops transcription 20 bases farther down, stretch of 150 to 200 adenine nucleotides called the poly(A)tail is added
Important for nuclear export, translation, and stability of mRNA
75
transcription - RNA - termination
RNase attacks and digests the residual RNA transcript that has remained attached to RNA pol II after 3' transcript cleavage. Following polyadenylation and 3' cleavage, the residual segment still attached to RNA pol II has not cap protecting it's 5' end
76
splicing
introns need to be cut out, exons need to be put together
77
5' splice site
GU
78
3' splice site
AG
79
branch point A
between 15-45 nucleotides upstream of the 3' splice site
80
spliceosome
binds and splices out introns in a few steps, protein recognizes 5' splice site and branch point, then other proteins come in to bing 5' and branch point together, nucleophilic attack by the hydroxy group, release 5' exon, form loop structure, hydroxy group attacks 3' splice site, degraded
81
Alternative splicing
different mRNAs and, subsequently, different proteins are produced from the same primary transcript by splicing together different combinations of exons
82
degenerate code
a genetic code in which some amino acids may be encodes by more than one codon each. 1961 - Marshall Nirenberg and Heinrich Matthaei mixed poly(U) with the protein-synthesizing machinery of E.coli in vitro and observed the formation of a protein.
83
tRNA structure
the clover-shaped RNA molecule that functions as a nuclear adapter
- anti codon loop recognizes RNA by complementarity (3-->5)
-Amino acid attachment site binds to unique amino acid
84
aminoacyl-tRNA
charged by aminoacyl-tRNA synthetases with the corresponding aa
20 enzymes in the cell, one for each of the 20 amino acids
85
wobble
pairing doesn't have to be perfect, loos pairing in the last nucleotide
86
prokaryotes ribosome
50S subunit and 30S subunit
87
eukaryotic ribosome
60S subunit and 40S subunit
88
rRNA funtion
ribozyme, catalytic core of the ribosome
89
decoding center
in the 30S subunit, ensures that only tRNAs carrying anticodons that match the codon will be accepted into the A site
90
peptidyltransferase center
in the 50S subunit, the site where peptide-bond formation is catalyzed
91
A site
acceptor site, where the tRNA enters, binds an incoming aminoacyl-tRNA whose anticodon matches the codon in the A site of the 30S subunit
92
P site
protein site, where the amino acid is added to the polypeptide chain, the tRNA in the P sire binds the growing polypeptide chain, part of which fits into a tunnel-like structure in the 50S subunit
93
E site
where the tRNA exits, contains a deacylated tRNA that is ready to be released from the ribosome
94
polysomes
multiple ribosomes on a single mRNA molecule
95
translation - initiation -prokaryotes
the first aminoacyl-tRNA is placed in the p site of the ribosome to establish the correct reading frame of the mRNA, the first amino acid in any newly synthesized polypeptide if methionine, specified by the codon AUG. It is inserted by a special tRNA called an initiator, symbolized tRNA Meti. Proteins called initiation factors play a key role in starting translation at the right frame.
96
Shine-Dalgarno sequence
in prokaryotes initiation codons are preceded by special sequences that pair with the 3' end of an rRNA, the 16S rRNA, in the 30S ribosomal subunit. This pairing correctly positions the initiator codon in the P site where the initiator tRNA will bind. Few nucleotides upstream of AUG
(AGGAGG)
97
translation - initiation - eukaryotes
- initiation factors: elF4A associated with the cap structure and with the 40S subunit and initiator tRNA to from an initiation complex
- once in place, the complex moves along the mRNA in the 5-->3 direction
- at the same time, the exposed sequence is scanned for AUG codon where translation can begin (Kozak sequence)
- after the AUG codon is properly aligned with the initiator tRNA, the initiation complex is joined by the 60 S subunit to form the 80S ribosome
98
summary of translation initiation
- small subunit binds the methylated cap of mRNAs and moves to the initiation site (P site)
- tRNA binds the initiation codon (AUG) and bridges the binding of the large subunit
- second aa-tRNA binds to the A site
- Met from tRNA on the P site is then moved and bound to the aa-tRNA on the A site
- first uncharged tRNA moved to E site (exits), then II aa-tRNA (which now has 2 aa) goes to the P site, and the third aa-tRNA is in the A site now
99
translation elongation
elongation factors (proteins) play a critical role, different factors but similar concept in prokaryotes and eukaryotes. GTP cleavage provides the energy for each step of elongation
100
termination
UGA, UAA, UAG
no tRNAs recognize these codons, instead proteins called release factors (RF1, RF2, RF3 in bacteria) recognize stop codons. Stop codons are recognized by tripeptides in the RF proteins, not by an anticodon. RFs fit into the A site of the 30S subunit but do. to participate in peptide-bond formation. Instead, a water molecule gets into the peptidyltransferase center, and its presence leads to the release of the polypeptide from the tRNA in the P site
101
germline mutations
passed down through germ cells
102
somatic mutations
mutations in non somatic cells
103
synonymous
no amino acid sequence change
104
missense
changes one amino acid
105
nonsense
creates stop codon and terminates translation
106
frameshift
wrong sequence of amino acids
107
promoter mutation
changes the timing or amount of transcription
108
polyadenylation mutation
alters the sequence of mRNA
109
splice site mutation
improperly retains an intron or excludes an exon
110
DNA replication mutation
increases (or less often, decreases) number of short repeats of DNA
111
point mutation
1 single nucleotide is changed
112
frameshift mutation
indels - insertion or deletion of one or more base pairs in the coding sequence of a gene leads to addition or deletion of mRNA nucleotides. This can alter the reading frame of the codon sequence, beginning at the point of mutation
113
cryptic splice sites
certain base-pair substitution mutations produce new splice sites that replace or compete with authentic splice sites during pre-mRNA processing.
114
spontaneous mutations
naturally occurring mutations that arise occasionally through errors during DNA replication or, much more often, through spontaneous changes in the chemical structure of nucleotide bases
115
Insertion or deletion of nucleotides
- string of repetitive nucleotides, destabilize the strand, one slips out, DNA polymerase adds an extra nucleotide, daughter slide inherits the mutation - slippage in the newly replicated strand
- slippage in the template strand, less nucleotides copied
- one portion that has already been replicated can slip out and form a large hairpin loop - partial rereplication of the template strand
- the more repeats you have, the more likely the strand will slip out - liked to neurodegenerative diseases
116
mispaired nucleotides
noncomplementary base pairing occasionally occurs during DNA replication. These so-called non-Watson-and-Crick base pairs can include the mispairing of guanine with thymine or the mispairing of cytosine with adenine. Both sets of mispaired nucleotides form two hydrogen bonds
117
Spontaneous nucleotide base changes
DNA nucleotide bases are organic chemical structures that can sustain damage or can spontaneously undergo structural alteration. These alterations embody damage to the duplex, but DNA replication may need to occur before they are preserved as mutations in the DNA sequence.
- depurination - loss of nucleotide base, the daughter strand filled opposite the apurinic site with a nucleotide, most commonly adenine: G-C --> T-A
- deamination - loses amino group --> uracil, replaces with whatever was on the other side T--> A, G-->C
118
trinucleotide repeat diseases
severity of the disease correlates with the number of repeat copies, repeat amplification in both coding and non-coding regions can lead to disease
119
mutagens can:
1. replace a base
2. alter a base so it mispairs with another
3. damage a base so that it can no longer pair with any base
120
mutagens - replace a base
a nucleotide base analog is a chemical compound that has a structure similar to one of the DNA nucleotide bases and therefore can work its way into DNA, where it pairs with a nucleotide base in the DNA duplex. DNA polymerases are unable to distinguish nucleotide base analogs form normal nucleotide bases due to their similarity in molecular size and shape. Thus, base analogs are incorporated into DNA strands during replication
(E.G. 5-Bromouracil)
121
Mutagens - alter/modify a base so it mispairs with another
EMS - addition of an alkyl group --> guanine can only make 2 H-bonds - binds with T
122
Intercalatating agents
mimic base pairs, causes insertions or deletions --> frameshift mutations
123
mutagens damage
UV damage - bonds 2 thymines together, and the opposite base can not bind
aflatoxin - adds to guanine, bulky base, changes DNA shape
124
mechanisms of DNA repair
1. repair/revert the nucleotide/base pair
2. replace it or part of the strand using base-pairing rule of the "healthy strand"
3. Proofreading activity of DNA polymerase
125
Enzymes repair
enzyme recognizes dimer caused by UV damage, cuts it out, and brings it back to what it is supposed to be
126
base-excision repair
initiate by damage to a DNA base or the presence of an incorrect base (non bulky)
damaged bases are removed and repaired through sequential action of DNA glycosylase, AP endonuclease, deoxyribophosphodiesterase (dRpase), DNA polymerase, and ligase
127
nucleotide-excision repair
bulky bases stop the DNA polymerase, replication block results in cell death
and excision-repair pathway that breaks the phosphodiester bonds on either side of a damaged base, removing that base and several on either side followed by repair replication
occurs during transcription or any other time
transcription - DNA breaks
complex cuts DNA on both sides and the remakes the portion by copying the bottom strand
128
nonhomologous end joining
Not very good
join 2 DNA molecules together and joins them, doesn't know if its the right parts
lose nucleotides - trims ends
129
homologous recombination
only during or shortly after DNA replication, during the S and G2 phase of the cell cycle
like crossing over
130
DNA damage checkpoints:
signaling cascades that block cell cycle progression in G1>S and G2>M and intra-S when DNA is damaged
results in a pause in the cell cycle to allow the cell time to repair the damage before continuing to divide
131
recombinant DNA
joining parts of DNA form different species and insert it into the DNA of a host cell
132
restriction enzyme
an endonuclease that will recognize specific target nucleotide sequences in DNA and break the DNA chain at those points
133
DNA palindrome
both strands have the same nucleotide sequence but in antiparallel orientation (reading 5' to 3' produced the same sequence on either strand)
134
cloning
take a plasmid that can replicate in your host, break it and make it have the DNA you want, ligate, put them into bacteria
135
directional cloning
use 2 restriction enzymes, one that's the 5' upstream, one in the 3' upstream
136
Polymerase chain reaction (PCR)
allows you to isolate and amplify the region of DNA that you want
1. denature
2. anneal - add 2 primers - 5-3 in each strand
3. elongate - Taq polymerase, from an enzyme that lives in deep ocean vents, so it can work at high temperatures
137
reverse transcription
turn RNA into DNA, complementary DNA (cDNA), synthesis is done using a reverse transcriptase, completely mature (no introns)
138
qPCR
calculate fluorescence over time to see how much RNA is there
139
DNA fingerprinting
where the restriction enzyme cuts will depend on the person
140
techniques to quantify DNA, RNA, and proteins
southern - DNA
northern - RNA
western - protein
SNOW DROP
141
Sanger sequencing
use ddNTPs that poison the reaching so it stops the reaction and you run it for all nucleotides, put them together,
142
Automated Sanger Sequencing
use fluorescent nucleotides to identify the sequence, the computer can tell which nucleotide was included
143
Next generation DNA sequencing
introduce a fluorescent nucleotide, different way of sequencing than Sanger
144
MinION
portable DNA sequencing machines - not as accurate, allows scientists to sequence in the field
145
snRNA
found in eukaryotic nuclei, where multiple snRNAs join the numerous proteins to form spliceosomes that remove introns from precursor mRNA
146
miRNA
Eukaryotic regulatory RNAs that function by base pairing with certain mRNAs, altering their stability and efficiency of translation
147
siRNA
Eukaryotic regulatory RNA made from long double-stranded molecules that are cut into shorter pieces used to regulate mRNA stability and translation
148
Telomerase RNA
located in the telomerase ribonucleoprotein complex, where it acts as a template to maintain and elongate telomere length of eukaryotic chromosomes
