Lecture 7- DNA/RNA/Protein Flashcards
(151 cards)
1
Q
What encompasses the “Central Dogma?”
A
The path from DNA being converted to a protein (Transcription and translation)
2
Q
How are the individual deoxyribonucleotides of DNA held together? (type of bond and where bonded)
A
Covalent phosphodiester bond from 3’–>5’
3
Q
What can cleave DNA?
A
deoxyribonuclease
4
Q
What can cleave RNA?
A
ribonuclease
5
Q
How are ribonucleotides linked in RNA? (type and where on structure)
A
covalent phosphodiester bonds from 3’–>5’
6
Q
What type of backbone does DNA have?
A
sugar-phosphate backbone
7
Q
What are the two grooves in DNA?
A
Major and minor groove
8
Q
What binds to the major groove and minor groove?
A
Major- Regulatory proteins
Minor- Drugs
9
Q
Number of bonds between A-T and G-C?
A
A-T 2 bonds
G-C 3 bonds
10
Q
What type of bonds hold together the two strands of DNA?
A
hydrogen bonds
11
Q
melting temperature Tm defines what?
A
The temperature at which half of the helical structure is lost
12
Q
What is the loss of helical structure called?
A
denaturation
13
Q
Which DNA structure has a higher Tm, one with many ATs or GCs?
A
The one with GCs.
14
Q
What are the 3 forms of DNA and which one do we care about?
A
A, B and Z-DNA
We are about B-DNA (right handed helix)
15
Q
Where is closed circular DNA found in eukaryotes?
A
mitochondria
16
Q
In eukaryotes, what types of proteins are associated with the long linear dsDNA?
A
histone and non-histone proteins
17
Q
In prokaryotes, (typically) is the DNA linear/circular, single/double stranded, and what type of proteins are associated with it?
A
circular
double
non-histone proteins
18
Q
in prokaryotes, in association with the circular chromosome, what other genetic factor resides?
A
plasmids- small circular extrachromosomal DNA
19
Q
Why are plasmids so important in bacteria?
A
They may carry genes that convey antibiotic resistance
20
Q
What is meant when it is said that DNA replication is “semiconservative?”
A
Each daughter DNA contains one strand from the parent DNA and one newly synthesized daughter strand
21
Q
What are the steps in Prokaryotic DNA synthesis?
A
- separation of 2 complimentary strands
- formation of the replication fork
- Direction of DNA replication
- RNA primer5. Chain elongation
- Excision of RNA primers and replacement by DNA
- DNA ligase
22
Q
Prokaryotes: Where does separation begin of the two complimentary strands, and what characteristics does it have?
A
Origin of replication
This “consensus sequence” has almost exclusively AT base pairs that facilitate melting (AT bonds = less bonds to break than GC)
23
Q
T/F: Eukaryotes have a single site of origin of replication.
A
False; they have many for rapid replication
24
Q
Prokaryotes: What does DNA-A protein do?
A
binds to the origin and causes the AT rich regions to melt
25
Prokaryotes: What protein binds to the origin and causes the AT rich regions to melt?
DNA-A protein
26
Prokaryotes: What unwinds the double helix?
DNA helicases
27
Prokaryotes: What does DNA helicase do?
unwinds the double helix of DNA
28
Prokaryotes: What keeps the strands of DNA apart and protects the DNA from nucleases that degrade ssDNA?
ssDNA-binding proteins
29
Prokaryotes: What do ssDNA-binding proteins do?
keeps the strands of DNA apart and protects the DNA from nucleases that degrade ssDNA
30
Prokaryotes: What cuts and rejoins one strand of double helix to remove supercoils?
Topoisomerase I
31
Prokaryotes: What does Topoisomerase I do?
cuts and rejoins one strand of double helix to remove supercoils
32
Prokaryotes: What cuts and rejoins both strands of DNA?
Topoisomerase II
33
What does DNA gyrase do?
cuts and rejoins both strands of DNA
34
What direction do DNA polymerases read the parental strand?
3'-->5' direction
35
Which direction does DNA synthesis occur?
5'-->3' direction
36
Leading/Lagging strand
Leading strand- in direction of replication fork (continuous)
Lagging- opposite direction of fork (discontinuous)
37
One segment of DNA on the lagging strand not yet connected is called...?
Okazaki fragment
38
What do DNA polymerases require, and what is needed in their chemical structure?
RNA primer.
| need a free OH on the 3' end
39
What enzyme makes RNA primers?
primase
40
T/F: RNA primers are needed for both DNA and RNA polymerases.
False; only needed for DNA polymerases
41
Prokaryotes: What enzyme catalyzes elongation, and how does it bind to the template?
DNA polymerase III
| bound by its b-subunit
42
Prokaryotes: describe DNA pol III and proofreading
DNA pol III has 3'->5' EXONUCLEASE activity to correct mismatch bases (can come back)
43
What happens when DNA pol III gets to the end of the segment? (blocked by an RNA primer)
RNA (from the RNA primer) is then excised and the gap is filled by DNA pol I
44
What does DNA ligase do, specifically?
makes the phosphodiester linkage between the 5'-phosphate on the DNA chain synthesized by DNA pol III and the 3'-OH on the chain made by DNA pol I
45
In eukaryotic DNA replication, what removes RNA primers?
RNase (rather than DNA polymerase)
46
What are the steps of the cell cycle?
```
M (Mitosis)
G0 (enters into G1)
G1 (can go to G0)
S (DNA synthesis)
G2
```
47
What happens in G1 phase?
Cell prepares to initiate DNA synthesis
| Biosynthesis occurs
48
What happens in S phase?
DNA is replicated (DNA content doubled)
49
What happens in G2 phase?
Biosynthesis for mitosis occurs
| preparation
50
What happens in G0 phase?
Cells stop dividing
- but can enter back into G1 phase later on
ex: Brian and heart cells
51
Polymerase a (alpha) function
Contains primase
| Initiates DNA synthesis
52
Polymerase b (beta) function
Repair
53
Polymerase y (gamma)
Replicates mitochondrial DNA
54
Polymerase δ (delta)
Elongates Okazaki fragments of the lagging strand
55
Polymerase ε (epsilon)
Elongates the leading strand
56
What are telomeres and what is their function?
Complexes of noncoding DnA plus proteins located at the ends of linear chromosomes
- Maintain structural integrity of the chromosome, preventing attack by nucleases
- Allow repair enzymes to distinguish between a true end from a break in dsDNA
57
Describe the physical structure of telomeres
Consist of several thousand tandem repeats of noncoding AGGGTT base paired with a complementary region of Cs and As
-The GT strand is longer than its complement, leaving a few hundred nucleotides in length at the 3' end
(If it is not there, it means the DNA is broken)
58
In somatic cells, what happens to telomeres upon each successive division?
They are shortened
59
What happens once telomeres are shortened beyond a critical length?
The cell is no longer able to divide and is said to be SENESCENT
60
What is unique about stem cells and cancer cells in respect to telomeres?
They have the enzyme TELOMERASE which maintains telomeric length (can divide forever)
61
What is reverse transcriptase?
The enzyme that synthesizes DNA from RNA
62
Where do we typically see reverse transcriptase (association)?
With RNA viruses
63
Describe the organization of eukaryotic DNA
Eukaryotic DNA is associated with tightly bound basic proteins called histones
The histones order the DNA structural units called nucleosomes
Nucleosomes are further arranged into chromosomes
64
What are histones primarily made from?
Basic amino acids
| Lysine and Arginine mostly
65
What are nucleosomes? (and structure)
A structural component of DNA composed of (H2A, H2B, H3, H4)2
66
What are the different types of histones?
H2A, H2B, H3, H4, H1
67
Which histone protein is the linker, and not associated with the nucleosome core?
H1
68
What are the 4 steps in eukaryotic DNA structural organization
1. Naked DNA
2. Nucleosome "bead" (histones + DNA)
3. Nucleofilament (30 nm fiber)
4. Nucleofilament is coiled and anchored to scaffold protein
69
What are the 5 types of DNA damage?
1. hydrolysis
2. oxidation
3. methylation
4. UV light
5. Ionizing radiation
70
What does Hydrolysis of DNA lead to (examples)?
Leads to deamination of:
- Cytosine to Uracil
- Adenine to Hypoxanthine
- Guanine to Xanthine
71
What happens when DNA is oxidized?
Leads to the formation of
- 8-hydroxyguanine from Guanine
- 5-hydroxymethyluracil from Thymine
72
What happens when DNA is methylated?
Leads to the formation of
- N7-methyl dG (guanine)
- N3-methyl dA (adenine)
73
What happens to DNA in the presence of UV light?
Pyrimidine dimers form
| thymidine in particular
74
What happens to DNA in the presence of ionizing radiation?
- damages DNA directly
| - strand breaks
75
What are the 3 types of DNA repair?
1. Base Excision Repair (BER)
2. Nucleotide Excision Repair (NER)
3. Mismatch Repair
76
Describe base excision repair
removes and replaces individual damaged bases
77
Describe nucleotide excision repair
- removes and replaces damaged BULKY lesions (2-30 nucleotides)
- recognize physical distortion rather than specific base sequences
78
Describe mismatch repair
removes nucleotides that do not form correct base pairs
79
DNA repair diseases, and describe them
1. Xeroderma Pigmentosum
- pyrimidine dimers formed in skin cells exposed to UV light
- defects in excision repair due to a mutant UV-specific endonuclease
2. Ataxia Telangiectasia
- defects in excision repair
- neurodegenerative disease
- poor coordination
80
What are the species of ribosomal RNA in eukaryotes?
```
5S
5.8S
18S
28S
(comprise about 80% of RNA in cell)
```
81
What are the units for ribosomal RNA?
Svedberg
82
What are the species of ribosomal RNA in prokaryotes?
5S
16S
23S
83
What is the smallest type of RNA?
Transfer RNA (4S)
84
How many different types of tRNA are there?
At least one for each of the 20 amino acids
85
What is the function of tRNA?
carries its amino acid on the 3' end to the site of protein synthesis
(comprises about 15% of RNA in the cell)
86
What is the function of mRNA?
To carry genetic information from the nucleus to the cytosol, where it is used as a template for DNA synthesis
87
Notes on the structure of mRNA
- has a poly A tail on the 3' end
| - has a 5' cap consisting of a 7-methylguanosine attached via triphosphate linkage
88
How many species of RNA polymerase does it take to synthesize RNA in prokaryotes?
One species
| -One synthesizes all of the RNA (except for the short RNA primers needed for DNA replication)
89
What is, and what does RNA polymerase do?
A multisubunit enzyme that recognizes a nucleotide sequence (promoter) at the beginning of the length of DNA to be transcribed
90
What is the sense strand?
The DNA strand with the same sequence as the mRNA
91
What is the antisense strand?
The DNA strand that is being transcribed into mRNA (the copy)
92
What direction does transcription occur?
5' --> 3'
93
What is the first step in transcription (prokaryotes)
begins with the binding of RNA polymerase to the promoter region (which is not transcribed)
(promoter contains characteristic consensus sequences)
94
Are promoter sequences upstream or downstream from the start of transcription? and what is an example? (prokaryotes)
Upstream
| -Pribnow box
95
Does RNA polymerase require a primer?
NO
96
Which DNA strand does RNA polymerase transcribe?
anti-sense strand
97
Where does transcription terminate in prokaryotes?
at the site of a hairpin loop, followed by a string of Us
98
T/F: Transcription in Eukaryotes requires a single polymerase.
False:
| separate polymerases are used in the synthesis of rRNA, tRNA and mRNA
99
Where do Transcription Factors (TFs) bind to?
Bind to either:
- within the core promoter region
- proximally (close to it)
- distally (some distance away)
100
T/F: Each eukaryotic RNA polymerase has its own promoters and TFs.
True
101
What is the active form of transcribed genes?
relaxed chromatin called EUCHROMATIN
102
What is the inactive form of gene segments?
condensed chromatin called HETEROCHROMATIN
103
What is the term used for interconversion between euchromatin and heterochromatin?
chromatin remodeling
104
What is a major mechanism of chromatin remodeling?
acetylation of lysine residues
105
What enzyme catalyzes the acetylation of lysine residues?
Histone acetyltransferases
106
What enzyme catalyzes the deacetylation of lysine residues?
Histone deacetylases
107
What types of groups does histone acetylation block? +/-
histone acetylation blocks positive (+) groups
108
What does RNA polymerase I do?
Synthesizes the precursor of the 5.8S, 18S and 28S rRNA in the nucleolus
109
What does RNA polymerase II do?
Synthesizes mRNA
110
What does RNA polymerase III do?
Synthesizes tRNA and 5S rRNA
111
What transcribes RNA from all mitochondrial genes?
Mitochondrial RNA polymerase
112
Describe RNA processing in prokaryotes vs eukaryotes
```
Prokaryotes
-RNA is used as unaltered primary transcript as soon as it is made (no processing)
Eukaryotes
-capping at 5' end
-addition of poly A tail at 3' end
-removal of introns
```
113
What is the 5' cap, and what is its function?
7-methylguanosine at the 5' end to the 5'ribose via a triphosphate linkage
-it protects the 5'end from exonucleases and promotes mRNA translation by ribosomes
114
What is the poly A tail comprised of?
40 to 200 adenine nucleotides attached to the 3' end
115
When is the poly A tail added?
after transcription
116
What enzyme catalyzes the reaction of the addition of the poly A tail?
Polyadenylate polymerase
117
What are the functions of the poly A tail?
-Helps stabilize the mRNA
-Facilitates mRNA exit from the nucleus and aid in translation
(gradually shortened after entering the cytosol; no longer needed)
118
In RNA processing, which segments are removed? How is this done?
introns
| -via RNA splicing (introns cut out of the primary transcript and exons are linked together to form functional product)
119
Where does transcription happen?
nucleus
120
Where does translation happen?
cytosol
121
Where is the anti-codon located?
on the tRNA
122
What is the start codon and what does it code for?
AUG
| Methionine
123
What are the stop codons?
UAG
UGA
UAA
124
What are the two forms of substitution? describe them
Transition- replace purine with purine or pyrimidine with pyrimidine
Transversion- replace purine with pyrimidine or vice versa
125
What do insertions or deletions lead to (regarding genetic code)?
frameshift mutations
126
What are the 3 different types of point mutations, and what do they do?
Silent mutation- base change still codes for same AA
Missense- now codes for different AA
Nonsense- now codes for a stop codon (terminal codon)
127
What is the organelle where protein synthesis occurs?
Ribosomes
128
What transfers amino acids?
tRNA
129
What catalyzes the attachment of AA to tRNA?
Aminoacyl-tRNA synthetases
130
What is the template for protein synthesis?
mRNA
131
If no ends are labeled when looking at a genetic code, what is assumed?
that it is being read left to right 5' --> 3'
132
Where is the attachment site on the tRNA and which part of the AA binds to which part of the tRNA?
The attachment site is at the tRNA 3' end
| The carboxylic group of AA binds to the OH of ribose at 3' end of tRNA
133
When a tRNA has a covalently attached AA; the tRNA is ___? and the AA is ___?
the tRNA is CHARGED
| the AA is ACTIVATED
134
What would you read on the tRNA when the start codon is present?
the tRNA would read 3'->5' UAC (anticodon), because the start codon AUG would be read on the mRNA from 5' -> 3'
135
What is the prokaryotic ribosome and its subunits?
70S
Large subunit- 50S
Small subunit- 30S
136
What is the eukaryotic ribosome and its subunits?
80S
Large subunit- 60S
Small subunit- 40S
137
What does the small ribosomal subunit do?
binds mRNA and is responsible for the accuracy of translation by ensuring correct base pairing between teh codon and anticodon
138
What does the large ribosomal subunit do?
catalyzes peptide bond formation
139
Wobble hypothesis
States that tRNAs can recognize more than one codon for a specific AA.
-First base of the anticodon (5' end) or (3rd of codon; auG) can code for the same AA with different bases
140
First initial steps of protein synthesis
Recognition of AUG start codon
-A special initiator tRNA recognizes AUG (eukaryotes)
-Initiator tRNA enters P site on small subunit of ribosome carrying methionine
-A site is empty
Large ribosomal subunit then joins the complex
141
What are the sites in the ribosomal complex?
E P A, mRNA is moving from right to left so "E" is the exit site
142
During elongation of protein synthesis, which end do new AAs bind to, and what enzyme catalyzes the rxn?
New AAs bind to the carboxyl end of the growing chain
| Catalyzed by Peptidyltransferase
143
Process of protein degradation?
Defective proteins are attached by a chain of small proteins, UBIQUITIN
Then degraded by the PROTEASOME in the cytosol
(degraded by proteasome after being ubiquitinated)
144
What does gel electrophoresis measure?
separates macromolecules based on size and charge
145
What does Western blotting measure?
Detects PROTEINS
146
What does Southern blotting measure?
detects DNA
147
What does Northern blotting measure?
detects RNA
148
What does PCR stand for and do?
Polymerase Chain Reaction
| -amplifies small samples of DNA
149
What does High Performance Liquid Chromatography (HPLC) do?
separates the components of a mixture via a column
150
What does X-ray Crystallography and Nuclear Magnetic Resonance do?
determines 3D structure
151
What are two methods used to determine an AA sequence?
Edman Degradation- method (label each AA and cleave off one at a time
Mass Spectrometry
