DNA and RNA Flashcards
(77 cards)
1
Q
Function of SSBP (single stranded binding protein)
A
Prevents DNA from reverting to duplex form (ie - re-annealing)
2
Q
Function of Helicase (prokaryotic)
A
Unwinds DNA at replication fork
3
Q
Stop codons for TXN (prokaryotic)
A
UGA UAA UAG
4
Q
Nucleotide vs Nucleoside
A
Nucleotide = nitrogenous base + 5-C sugar + 1 or more phosphate groups
Nucleoside = nitrogenous base + 5-C sugar WITHOUT ANY PHOSPHATE GROUPS
5
Q
Requirements to make Purines
A
Glycine
Aspartate
Glutamine
PRPP
6
Q
Requirements to make Pyrimidines
A
Orotic Acid
PRPP
(together make orotidine monophosphate)
7
Q
Major causes of DNA damage?
A
Oxidative damage: OH- from cellular respiration
Ultraviolet: covalently linkes adjacent thymines (creates thymidine dimers –> xeroderma pigmentosum)
Alkylating Agents: Carcinogens (Cisplatin or mustard gas) are attracted to nucleophilic groups
8
Q
Common causes of mutation or modification of DNA bases?
A
Deamination: Nitrous acid can greatly speed this process up
Depurination: the phosphate backbone is more sensitive to breakage
9
Q
Relative Solubility of different components of nucleotides?
A
Pyrimidines > Purines
Nucleotides > Nucleoside > bases
10
Q
Types of nucleotides not in DNA or RNA
A
Coenzyme A
cAMP
cGMP
NAD
11
Q
Types of Structural RNA
A
Ribosomal RNA (rRNA) - most abundant type
transfer RNA (tRNA) - smallest type
Small nuclear RNA (snRNA)
Small nucleolar RNA (snoRNA)
12
Q
Types of regulatory RNA
A
micro RNA (mRNA) small interfering RNA (siRNA)
13
Q
Information-containing RNA
A
messenger RNA (mRNA) - largest type
14
Q
Types of high energy bonds
A
1.) Thioester bonds (C-S) …. acetyl CoA
2.) Hi-energy phosphate bonds:
P-O-P … ATP
P-N … phosphocreatine
C-O-P … phosphoenolpyruvate
15
Q
Definition of “high energy bond” in biochemistry?
A
The bond, when hydrolyzed in standard conditions, releases >6 kcal/mol
16
Q
2 types of mitochondrial-linked diseases
A
LHON - Levers Hereditary optic neuropathy
MERRF - Myoclonic epilepsy and ragged-red fiber disease
17
Q
Factors that affect DNA melting Temperature (Tm)
A
- ) Salt Concentration: [salt] ∝ Tm
- phosphate groups need Na+ or Mg+2, without them they’re exposed to more (-) charges which tears them apart at a low Tm - ) Extremes of pH: it interrupts H-bonds on bases
- ) DNA change length ∝ Tm
- ) [G-C] ∝ Tm
- G:C have 3 H-bonds; A:T have 2 H-bonds
18
Q
Diseases affected by the low solubility of Purines?
A
- ) Gout
- Defect in phosphoribosyl synthetase - ) Lesch-Nyhan disease
- Defect in Hypoxanthine-guanine phosphoribosyl transferase
19
Q
Function of Gyrase (a topoisomerase type II enzyme) (prokaryotic)
A
Introduces negative supercoils, thereby relaxing positive supercoils that form during helicase unwinding
20
Q
Function of DNA Primase (prokaryotic)
A
Synthesizes a short RNA segment on the ssDNA template (no DNA polymerase can start synthesis w/o a DNA or RNA primer)
21
Q
Function of DNA Polymerase III (the main prokaryotic polymerase)
A
Adds DNA nucleotides to the hydroxyl group on the 3’ end of the new strand (5’→3’ synthesis)
DNA Pol III also has a 3’→5’ proofreading ability w/ exonuclease function to correct mistakes
22
Q
Function of DNA polymerase I (prokaryotic)
A
When DNA Pol III reaches the “prior” RNA primer on the lagging strand, DNA Pol I degrades the primer and fills in appropriate DNA nucleotides → DNA ligase then closes any remaining breaks in the new strand
23
Q
Function of DNA topoisomerases (prokaryotic)
A
Create a nick in the helix to relieve supercoils
24
Q
DNA Pol α acts in a complex with another enzyme, ______ . (eukaryotic)
A
RNA Primase
25
DNA Pol α and RNA primase work together to do what? (eukaryotic)
1. ) RNA primase lays down a short series of RNA molecules
2. ) DNA Pol α elongates the RNA primer with ~20 DNA nucleotides
3. ) Once this short strand of nucleotides is finished, the DNA Pol α/RNA primase dissociates and DNA Pol δ takes over.
26
Function of DNA Pol δ (eukaryotic)
Its the main eukaryotic DNA polymerase. It has 3’→5’ exonuclease proofreading and is analogous to prokaryotic DNA Pol III.
27
Function of Telomerase (eukaryotic)
A reverse transcriptase enzyme with an intrinsic RNA template that adds DNA to the 3' end of the lagging strand of a replicating chromosome to avoid chromosome shortening with each round of replication.
Stem cells and cancer cells upregulate telomerase activity → no shortening of chromosomes → increased replicative ability
video of telomerase action: https://www.youtube.com/watch?v=AJNoTmWsE0s
28
Clinical conditions associated with defective excision repair of DNA?
Xeroderma pigmentosum: Autosomal recessive
Defective ecvision repiar such as uvr ABC endonuclease results in inability to repair thymidine dimers (which form in DNA when exposed to UV light)
Associated with dry skin and with melanoma and other cancers
NER enzymes are important in removal of UV-induced damage (i.e. thymine dimers) and defects in these enzymes cause xeroderma pigmentosum. Patients with xeroderma pigmentosum have a greatly increased risk of developing skin cancer, often during childhood.
29
Function of Nucleotide excision repair (NER)
NER enzymes recognize bulky distortions in the shape of the DNA double helix. A small region of DNA on either side of the damaged base (about 20 base pairs total) is removed from the DNA helix. Sequential action of DNA polymerase and DNA ligase fills in the gap left by the NER enzymes.
30
Steps in Base excision repair for DNA
Base excision repair:
1.) Glycosylase enzymes recognize and remove incorrectly paired and chemically altered bases without interrupting the phosphodiester backbone.
2.) AP-endonuclease (AP can stand for both apyrimidinic and apurinic) enzymes detect that a base is missing and begin the process of excision by making an endonucleolytic cut on the 5′-side of the AP location.
Lyase cuts at the 3'-end to remove the baseless sugar-phosphate molecule.
3.) DNA Pol I (prokaryotic) or DNA Pol β (human) then replaces the damaged base and DNA ligase seals the new DNA strand.
31
How is mismatch (G-T, C-A, etc) repair done?
Mismatch repair recognizes mispaired bases (G-T or A-C pairs). The daughter strand is identified by lack of methylation.
32
Disease(s) associated with mismatch repair?
hereditary nonpolyposis colorectal cancer (HNPCC):
an autosomal dominant condition in which a defective mismatch repair gene causes a microsatellite repeat replication error to go unfixed.
Inheriting a defective copy of one of several mismatch repair genes puts the patient at high risk for cancer
33
___(1)___ and ___(2)____ are the two methods of correcting double strand DNA breaks; ___(1)___ results in accurate repair while ___(2)____ can cause significant errors.
1. ) homologous recombination (HR)
| 2. ) non-homologous end joining (NHEJ)
34
What is Homologous recombination
Damaged strands are matched to non-damaged strands and DNA polymerase fills in the damaged strand using the non-damaged strand as a template.
HR is also used in meiosis to generate genetic diversit
35
What is Non-homologous end joining
It's when broken ends of two DNA strands are brought together by a group of proteins and joined without checking for homology. Thus there is significant potential for error and mutations.
36
The nucleosome consists of a ______ + _____.
histone octamer (8 histones) + 2 loops of dsDNA.
The nucleosome is the "bead" in the "beads on a string description" of DNA.
37
Ineffective double strand repair leads to a ______ . Without a functional _____, cells, especially those in the _____, inherit mutations that cause cell death or cancer.
ataxia-telangiectasia (ATM gene)
ATM gene
brain
38
What does the Histone octamer (core histones) consist of?
2 each of H2A, H2B, H3, and H4
Adjacent nucleosome cores are linked by intervening DNA associated with histone H1. This is the "string" part of "beads on a string.
39
How do histones stay attached to DNA?
DNA is negatively charged while histones are primarily lysine and arginine (positively charged).
Acetylation of positively charged histones weakens the DNA-histone bonds and makes DNA accessible to transcription factors and RNA polymerases
40
Transcription is controlled via 4 cis-acting elements: ______, _____, _____, and _____.
promoters, enhancers, silencers/operators, and response elements
41
Whatre promoters?
Promoters are where RNA polymerase and transcription factors (TFs) bind to initiate transcription (often located 25 to 50 bases upstream of the gene, and often contains A-T rich sequences of TATA or CAAT boxes)
42
What're enhancers?
Enhancers also bind transcription factors, and can significantly ↑ the rate of transcription (can be located upstream, downstream, or a distance from the gene)
43
What're silencers?
Silencers repress transcription when repressors, a subset of transcription factors, bind to them (known as operators in prokaryotes
44
What're response elements?
Response elements bind specific transcription factors (e.g. heat shock response element, estrogen response element) and modulate transcription
45
What're operators?
It's where negative regulators (repressors) bind
46
Different types of Eukaryotic RNA polymerases and their primary functions
RNAP I transcribes rRNA (most abundant)
RNAP II transcribes mRNA; opens DNA at promoter site (AT-rich upstream sequence - TATA and CAAT)
α-amanitin (deadly toxin found in certain mushrooms, 'death cap mushrooms'): inhibits RNAP II → liver damage when ingested
RNAP III transcribes tRNA (shortest RNA)
47
RNA polymerase in prokaryotes?
RNA polymerase is made of 5 subunits and can synthesize all the 3 kinds of RNA and they do not require primers--they bind directly to promoter sequences
48
Transfer RNA (tRNA) is composed of _____ nucleotides.
75-90
49
tRNA's secondary structure is ______ while the tertiary structure is _____ shaped.
"cloverleaf,"
"L"
50
The "bottom" of the tRNA cloverleaf houses the _____ , which pairs with _____ when brought together in a ribosome.
anti-codon
mRNA codons
51
The 3’ end of tRNA has a _____ sequence that is recognized by ______, the enzyme responsible for charging the tRNA with an amino acid.
CCA
aminoacyl-tRNA synthetase
52
Aminoacylation _____ an amino acid to the 3’ end of the tRNA
covalently bonds
53
_____ proofread before and after charging; if the wrong amino acid is on the tRNA, the covalent bond is _____.
Aminoacyl-tRNA synthetases
hydrolyzed
54
What is the wobble hypothesis
the 3rd position of the mRNA codon isn’t as critical to pairing and is allowed some "wobble" with respect to nucleotide base pairing with the tRNA. tRNAs that code for the same amino acid often differ in this "wobble" position.
55
Transition vs transversion
In a transition, a purine is substituted for a purine or a pyrimidine is substituted for a pyrimidine.
In a transversion, purine → pyrimidine or pyrimidine → purine.
56
Order of increasing severity of different types of mutations
silent < missense < nonsense < frameshift.
57
The RNA produced by RNAP II is actually called _____ until processing has occurred
hnRNA (heterogeneous nuclear RNA)
58
5’ capping adds a _____ to the 5’ end of the transcript
7-methylguanosine
59
_____ adds the 3’ poly-A tail, as many as 200 adenines (does not require a template)
Poly (A) polymerase
60
Splicing is catalyzed by the _____ and _____.
spliceosome
snRNPs (small nuclear ribonucleoproteins – nuclear b/c all of this occurs in the nucleus).
61
UVA-induced thymine dimers in DNA can be repaired by _______ and _____
nucleotide excision repair and TF2H
62
What're Okazaki fragments?
Small stretches of DNA synthesized during replication in the 5' to 3' direction (on the lagging strand). Since the synthesized strand is running in the 3' to 5' direction, and since new dNTPs can only be added at the 3' hydroxyl group, the DNA synthesis process takes a kind of "leapfrogging" approach whereby small segments on that strand are copied 5' to 3' and then melded together later
63
Origin binding proteins bind to the _____ and become part of the complex, also recruits _____
origin
Pol III.
64
DNA polymerase III synthesizes a DNA strand from its complement on both leading and lagging strand. High processivity due to a ____ mechanism that holds the polymerase tightly to the DNA . No 5' to 3' _____ activity (thus can't be used to remove RNA primers).
Processivity clamp
exonuclease
65
Steps in transcription
1: RNA polymerase binds to promoter sequence on the helical DNA in a "closed complex."
2: Polymerase melts DNA strands apart near transcription start site, forming an "open complex” aka the "transcription bubble."
3: Polymerase catalyzes phosphodiester linkage of two initial rNTPs.
4: Polymerase advances 3' to 5' down template strand, melting DNA and linking rNTPs.
5: At transcription stop site, polymerase releases completed RNA and dissociates from DNA.
Note that steps 1-3 are called initiation, step 4 is elongation, and step 5 is called termination.
66
Describe how alpha-amanitin blocks transcription.
alpha-amanitin: extremely toxic substance found in death cap mushrooms. Acts by inhibiting the movement of RNA Pol II, binding its bridge substructure so that translocation of the polymerase down the DNA chain can't happen.
67
Describe how a rifampicin blocks transcription.
rifampicin: broad-spectrum antibiotic. Acts by binding the beta subunit of bacterial RNA polymerase, plugging up the exit chamber where assembled RNA exits the transcriptional complex. Thus elongation is prevented from going farther than a few base pairs due to having nowhere to go.
68
Name 4 components of the RNA polymerase II pre-initiation complex.
1.) TFII [transcription factor II] A, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
- Of these, TFIID and TFIIH are of particular interest:
▪ TFIID (TBP) binds the TATA box on the DNA sequence.
▪ TFIIH facilitates nucleotide excision repair, adds PO4 to C-terminal domain of Pol II, and act as helicases to open DNA strands.
69
Describe the clinical syndromes caused by mutations in TFIIH subunits.
Problems with nucleotide excision repair:
Cockayne's syndrome,
Trichothiodystrophy,
Xeroderma pigmentosum
70
List the functions of the 5' cap of the mRNA.
| ◦
1. ) It makes the 5' end resistant to exonucleases (which target the "lone ends" of single D/RNA strands).
2. ) It helps with splicing and processing through a cap-binding complex that recognizes the cap. (primes for splicing, 3’ tail, translation)
3. ) Translation factor eIF4E (eukaryotic initiation factor 4E) recognizes the cap for transport to the ribosomes.
4. ) When the cap is eventually removed, it signals for the mRNA to be degraded.
Decapping is a key event in RNA degradation
71
List the three reactions required to add a 5' cap to pre-mRNA.
(1) Cut off the last PO4 from the triphosphate group at the 5' end of the mRNA.
(2) Add guanosine triphosphate (GTP) backwards via guanylyl transferase. It loses two PO4 groups of its own (making GMP) and forms a 5'-to-5' triphosphate bond (2 PO4 from mRNA, 1 PO4 from GMP) to the end of the mRNA.
(3) Methylate the 7-position of the guanosine cap via S-adenosyl methionine (SAM; nearly universal methyl donor in cell; donates methyl to form homocysteine) to form 7-methyl-guanosine at cap.
72
Provide examples of genetic disorders caused by splicing defects.
Provide examples of genetic disorders caused by splicing defects.
Marfan's syndrome
Abnormal splicing of CD44 (cell-surface glycoprotein) is a predictor of tumor metastasis. Used as diagnostic and prognostic marker.
73
Memorize the conserved sequences at the 5' and 3' ends of most introns and the consensus sequence at the polyA site.
◦ Splice site at 5' end of intron: GU. Mark the beginning of every intron.
◦ Splice site at 3' end of intron: AG. Last two bases of every intron.
◦ Consensus sequence at poly-A site: AAUAAA.
74
Identify on a diagram of a gene the following:
a) transcription start site:
b) introns:
c) 5' splice sites:
d) 3' splice sites:
e) branch points:
f) exons:
g) 5' UTR:
h) 3' UTR:
i) initiation codon:
j) termination codon:
k) poly A site:
Identify on a diagram of a gene the following:
a) transcription start site: Look for either the +1 position or the little bent arrow coming out of the sequence at this point.
b) introns: Should be between the GU (5') and AG (3') splice sites.
c) 5' splice sites: Look for GU.
d) 3' splice sites: Look for AG.
e) branch points: should be in the introns, between the GU (5') and AG (3').
f) exons: should be between the introns. Mark the introns with GU at 5' and AG at 3', then look between them for the exons.
g) 5' UTR: look for a region between position +1 (start of transcript) and the start codon (see below) on the processed mRNA strand.
h) 3' UTR: look for a region after the stop codon (see below) until the end of the transcript, including the consensus sequence and the poly-A tail.
i) initiation codon: also called start codons. Encodes methionine. Look for 5' AUG.
j) termination codon: also called stop codons. Look for 3' UAG, UAA, or UGA.
k) poly A site: look for consensus sequence AAUAAA
75
Describe the two reactions that make the mature 3' end of mRNA's.
Describe the two reactions that make the mature 3' end of mRNA's.
1) recognition of consensus sequence at pre-mRNA's 3' end (AAUAAA) and cleavage of the mRNA soon after this sequence.
(2) polyadenylation of free hydroxyl at 3' end. (not coded for)
76
Provide an example of how alternative poly A sites can be used to make more than one protein from a single gene.
Similar to alternative splicing! Depends on where the poly A tail gets added.
Two different forms of immunoglobulin M (IgM), membrane-bound and secreted, are formed by alternative poly-A sites in their common gene. This can occur because transcription continues past poly-A site depending on which one is used it will make 2 variants (heavy and light chains).
77
Cap independent initiation
sometimes eukaryotes don’t need a cap. IRES can allow transcription to occur without a cap. A virus might want to do this in order to hijack the host cell’s machinery. It inhibits the ability of the cell to initiate translation. The virus can then use the IRES to make its own proteins. Cellular mRNA’s that don’t need a cap are often stress related. The cell can shut down cap dependent translation and begin cap independent translation (hypoxia or other stresses). Perhaps cancer related (tumors are hypoxic)
