DNA and RNA

Question 1

Function of SSBP (single stranded binding protein)

Answer 1

Prevents DNA from reverting to duplex form (ie - re-annealing)

Question 2

Function of Helicase (prokaryotic)

Answer 2

Unwinds DNA at replication fork

Question 3

Stop codons for TXN (prokaryotic)

Answer 3

UGA UAA UAG

Question 4

Nucleotide vs Nucleoside

Answer 4

Nucleotide = nitrogenous base + 5-C sugar + 1 or more phosphate groups

Nucleoside = nitrogenous base + 5-C sugar WITHOUT ANY PHOSPHATE GROUPS

Question 5

Requirements to make Purines

Answer 5

Glycine
Aspartate
Glutamine
PRPP

Question 6

Requirements to make Pyrimidines

Answer 6

Orotic Acid
PRPP

(together make orotidine monophosphate)

Question 7

Major causes of DNA damage?

Answer 7

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

Question 8

Common causes of mutation or modification of DNA bases?

Answer 8

Deamination: Nitrous acid can greatly speed this process up

Depurination: the phosphate backbone is more sensitive to breakage

Question 9

Relative Solubility of different components of nucleotides?

Answer 9

Pyrimidines > Purines

Nucleotides > Nucleoside > bases

Question 10

Types of nucleotides not in DNA or RNA

Answer 10

Coenzyme A
cAMP
cGMP
NAD

Question 11

Types of Structural RNA

Answer 11

Ribosomal RNA (rRNA) - most abundant type
transfer RNA (tRNA) - smallest type
Small nuclear RNA (snRNA)
Small nucleolar RNA (snoRNA)

Question 12

Types of regulatory RNA

Answer 12

micro RNA (mRNA)
small interfering RNA (siRNA)

Question 13

Information-containing RNA

Answer 13

messenger RNA (mRNA) - largest type

Question 14

Types of high energy bonds

Answer 14

1.) Thioester bonds (C-S) …. acetyl CoA
2.) Hi-energy phosphate bonds:
P-O-P … ATP
P-N … phosphocreatine
C-O-P … phosphoenolpyruvate

Question 15

Definition of “high energy bond” in biochemistry?

Answer 15

The bond, when hydrolyzed in standard conditions, releases >6 kcal/mol

Question 16

2 types of mitochondrial-linked diseases

Answer 16

LHON - Levers Hereditary optic neuropathy

MERRF - Myoclonic epilepsy and ragged-red fiber disease

Question 17

Factors that affect DNA melting Temperature (Tm)

Answer 17

1. ) 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
2. ) Extremes of pH: it interrupts H-bonds on bases
3. ) DNA change length ∝ Tm
4. ) [G-C] ∝ Tm
   - G:C have 3 H-bonds; A:T have 2 H-bonds

Question 18

Diseases affected by the low solubility of Purines?

Answer 18

1. ) Gout
   - Defect in phosphoribosyl synthetase
2. ) Lesch-Nyhan disease
   - Defect in Hypoxanthine-guanine phosphoribosyl transferase

Question 19

Function of Gyrase (a topoisomerase type II enzyme) (prokaryotic)

Answer 19

Introduces negative supercoils, thereby relaxing positive supercoils that form during helicase unwinding

Question 20

Function of DNA Primase (prokaryotic)

Answer 20

Synthesizes a short RNA segment on the ssDNA template (no DNA polymerase can start synthesis w/o a DNA or RNA primer)

Question 21

Function of DNA Polymerase III (the main prokaryotic polymerase)

Answer 21

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

Question 22

Function of DNA polymerase I (prokaryotic)

Answer 22

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

Question 23

Function of DNA topoisomerases (prokaryotic)

Answer 23

Create a nick in the helix to relieve supercoils

Question 24

DNA Pol α acts in a complex with another enzyme, ______ . (eukaryotic)

Answer 24

RNA Primase

Question 25

DNA Pol α and RNA primase work together to do what? (eukaryotic)

Answer 25

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.

Question 26

Function of DNA Pol δ (eukaryotic)

Answer 26

Its the main eukaryotic DNA polymerase. It has 3’→5’ exonuclease proofreading and is analogous to prokaryotic DNA Pol III.

Question 27

Function of Telomerase (eukaryotic)

Answer 27

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

Question 28

Clinical conditions associated with defective excision repair of DNA?

Answer 28

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.

Question 29

Function of Nucleotide excision repair (NER)

Answer 29

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.

Question 30

Steps in Base excision repair for DNA

Answer 30

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.

Question 31

How is mismatch (G-T, C-A, etc) repair done?

Answer 31

Mismatch repair recognizes mispaired bases (G-T or A-C pairs). The daughter strand is identified by lack of methylation.

Question 32

Disease(s) associated with mismatch repair?

Answer 32

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

Question 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.

Answer 33

1. ) homologous recombination (HR)

2. ) non-homologous end joining (NHEJ)

Question 34

What is Homologous recombination

Answer 34

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

Question 35

What is Non-homologous end joining

Answer 35

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.

Question 36

The nucleosome consists of a ______ + _____.

Answer 36

histone octamer (8 histones) + 2 loops of dsDNA.

The nucleosome is the “bead” in the “beads on a string description” of DNA.

Question 37

Ineffective double strand repair leads to a ______ . Without a functional _____, cells, especially those in the _____, inherit mutations that cause cell death or cancer.

Answer 37

ataxia-telangiectasia (ATM gene)

ATM gene

brain

Question 38

What does the Histone octamer (core histones) consist of?

Answer 38

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.

Question 39

How do histones stay attached to DNA?

Answer 39

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

Question 40

Transcription is controlled via 4 cis-acting elements: ______, _____, _____, and _____.

Answer 40

promoters, enhancers, silencers/operators, and response elements

Question 41

Whatre promoters?

Answer 41

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)

Question 42

What’re enhancers?

Answer 42

Enhancers also bind transcription factors, and can significantly ↑ the rate of transcription (can be located upstream, downstream, or a distance from the gene)

Question 43

What’re silencers?

Answer 43

Silencers repress transcription when repressors, a subset of transcription factors, bind to them (known as operators in prokaryotes

Question 44

What’re response elements?

Answer 44

Response elements bind specific transcription factors (e.g. heat shock response element, estrogen response element) and modulate transcription

Question 45

What’re operators?

Answer 45

It’s where negative regulators (repressors) bind

Question 46

Different types of Eukaryotic RNA polymerases and their primary functions

Answer 46

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)

Question 47

RNA polymerase in prokaryotes?

Answer 47

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

Question 48

Transfer RNA (tRNA) is composed of _____ nucleotides.

Answer 48

75-90

Question 49

tRNA’s secondary structure is ______ while the tertiary structure is _____ shaped.

Answer 49

“cloverleaf,”

“L”

Question 50

The “bottom” of the tRNA cloverleaf houses the _____ , which pairs with _____ when brought together in a ribosome.

Answer 50

anti-codon

mRNA codons

Question 51

The 3’ end of tRNA has a _____ sequence that is recognized by ______, the enzyme responsible for charging the tRNA with an amino acid.

Answer 51

CCA

aminoacyl-tRNA synthetase

Question 52

Aminoacylation _____ an amino acid to the 3’ end of the tRNA

Answer 52

covalently bonds

Question 53

_____ proofread before and after charging; if the wrong amino acid is on the tRNA, the covalent bond is _____.

Answer 53

Aminoacyl-tRNA synthetases

hydrolyzed

Question 54

What is the wobble hypothesis

Answer 54

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.

Question 55

Transition vs transversion

Answer 55

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.

Question 56

Order of increasing severity of different types of mutations

Answer 56

silent < missense < nonsense < frameshift.

Question 57

The RNA produced by RNAP II is actually called _____ until processing has occurred

Answer 57

hnRNA (heterogeneous nuclear RNA)

Question 58

5’ capping adds a _____ to the 5’ end of the transcript

Answer 58

7-methylguanosine

Question 59

_____ adds the 3’ poly-A tail, as many as 200 adenines (does not require a template)

Answer 59

Poly (A) polymerase

Question 60

Splicing is catalyzed by the _____ and _____.

Answer 60

spliceosome

snRNPs (small nuclear ribonucleoproteins – nuclear b/c all of this occurs in the nucleus).

Question 61

UVA-induced thymine dimers in DNA can be repaired by _______ and _____

Answer 61

nucleotide excision repair and TF2H

Question 62

What’re Okazaki fragments?

Answer 62

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

Question 63

Origin binding proteins bind to the _____ and become part of the complex, also recruits _____

Answer 63

origin

Pol III.

Question 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).

Answer 64

Processivity clamp

exonuclease

Question 65

Steps in transcription

Answer 65

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.

Question 66

Describe how alpha-amanitin blocks transcription.

Answer 66

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.

Question 67

Describe how a rifampicin blocks transcription.

Answer 67

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.

Question 68

Name 4 components of the RNA polymerase II pre-initiation complex.

Answer 68

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.

Question 69

Describe the clinical syndromes caused by mutations in TFIIH subunits.

Answer 69

Problems with nucleotide excision repair:
Cockayne’s syndrome,
Trichothiodystrophy,
Xeroderma pigmentosum

Question 70

List the functions of the 5’ cap of the mRNA.

◦

Answer 70

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

Question 71

List the three reactions required to add a 5’ cap to pre-mRNA.

Answer 71

(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.

Question 72

Provide examples of genetic disorders caused by splicing defects.

Answer 72

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.

Question 73

Memorize the conserved sequences at the 5’ and 3’ ends of most introns and the consensus sequence at the polyA site.

Answer 73

◦ 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.

Question 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:

Answer 74

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

Question 75

Describe the two reactions that make the mature 3’ end of mRNA’s.

Answer 75

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)

Question 76

Provide an example of how alternative poly A sites can be used to make more than one protein from a single gene.

Answer 76

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).

Question 77

Cap independent initiation

Answer 77

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)