Transcription and Translation - Campbell

Question 1

What is a mutation responsible for β-thalassemia that lies outside any globin gene?

Answer 1

mutation near the orgin of replication of beta-globin, which distrubes nucleosome location, which decreases the regulation of beta-globin, decreasing the amount of beta-globin, resulting in beta-thalassemia.

Question 2

The part of a gene that transcription factors bind to is known as what? Keep straight that this is made of DNA and transcription factors are proteins.

Answer 2

bind to the DNA in sequence specific manner at a certain place called the promoter region

Question 3

Which of the following is NOT necessarily a conserved sequence (only one correct
response this time)?

A. a nucleotide sequence (motif) that is the same or nearly the same in a promoter region from multiple species
B. a nucleotide sequence (motif) that is the same or nearly the same in a gene from multiple species
C. an amino acid sequence (motif) that is the same or nearly the same in a protein in multiple species
D. the TATA box
E. a nucleotide sequence (motif) in a promoter region that is actively transcribed in such a manner so as to allow an organism to conserve energy.

Answer 3

E. a nucleotide sequence (motif) in a promoter region that is actively transcribed in such a manner so as to allow an organism to conserve energy.

Question 4

A consensus sequence (only one correct response this time)

A. is the sequence that best agrees with a collection of conserved sequences.
B. is the sequence that allows an organism to conserve energy.
C. is the sequence that is calculated by bioinformatics software.
D. is not derived from conserved sequences.
E. cannot be an amino acid sequence.

Answer 4

A. is the sequence that best agrees with a collection of conserved sequences.

Question 5

Can a human disease (e.g. a β-thalassemia) result from a promoter region mutation in DNA sequence (hint- the promoter region is not part of the region of DNA that encodes a protein)? If so, what is the reason?

Answer 5

Yes. The promoter region is very important, so any mutation in the promoter by beta-globin can result in beta-thalassemia.

Question 6

Name three ways DNA synthesis (replication) and RNA synthesis (transcription) are alike.

Answer 6

Alike:

• both occur due to a large complex
• both have similar chemistry (5’->3’)
• both use base complementary rules

Differences:
DNA has much more proofreading than RNA
DNA has AGCT bases; RNA has RGCU

Question 7

Describe the transcriptional regulation of the LDL receptor gene by cholesterol.

Include in your description what occurs at the membrane that initiates the regulation, where in the cell this occurs, the name of the transcription factor that results, where the transcription factor goes to activate the gene, and the site to which the transcription factor binds.

Answer 7

When cholesterol is low, there will be low levels of cholesterol in the endoplasmic reticulum, SCAP and SREBP (sterol regulatory element binding protein) move from the ER to the Cis Golgi. Proteases will cleave SREBP, and a part of the SREBP (now a transcription factor) moves to the nucleus and binds to the DNA at SRE (sterol regulator element).

Question 8

What DNA abnormality causes Fragile X syndrome, and what does that cause to happen too much or too little? What is thought to be a large part of the reason reproductive cloning is so fraught with difficulty?

Answer 8

a Triple Nucleotide Repeat causes Fragile X syndrome. There is too many repeats (of CGG) and therefore there is more methylation, which throws off the regultaion and decreases the amount of FMR1 produced causing metal retardation.

Methylation is thought to be a large part of why reproductive cloning is so difficult in mammals.

Question 9

What does rifampicin do? What does α-amanitin do? Which is thought of as an antibiotic, and which a poison, and why?

Answer 9

rifampicin is an antibiotic translational inhibitor (works against bacteria).

alpha-amanitin is a poison that inhibits transcription that affects humans.

Question 10

What is the first step of RNA processing known as?

Answer 10

capping (adds methyl group to 5’ end to prepare strand for translation)

Question 11

How many diseases are the result of the long poly-dT stretches of DNA that encode the polyA tail of mRNAs (hint- this is a trick question)?

Answer 11

None. The tail is just used for mRNA stability that is a part of processing, so it does not come from DNA

Question 12

What is the function of polyadenylation?

Answer 12

It’s an enzyme that adds polyA tail to the 3’ end for mRNA stability

Question 13

How is it possible to make more than one different proteins having different properties from one gene?

Answer 13

alternative splicing allows for multiples proteins to be made that have different functions from one gene. (Ex. troponins)

Question 14

Can a human disease (e.g. a β-thalassemia) result from a mutation in DNA sequence in the intron (hint- the intron is a region of DNA that is spliced away before synthesis of a protein)? If so, what is the reason?

Answer 14

Yes. Sequences are important, even in intron information. In this case, splicing can be effected by the sequence of the intron, that can result in beta-thalassemia.

Question 15

How are tRNAs involved in “breaking” the genetic code? What is at one end of a tRNA and what is at the other end?

Answer 15

tRNA segments the genetic code into groups of three (codons) that code for different amino acids. Each tRNA is specific for a certain amino acid.

Question 16

What do we mean by “activation” of a tRNA? What is the enzyme that links the amino acid to the tRNA? What antibiotic has this step as a drug target?

Answer 16

Specific enzymes add amino acids (aminoacyl tRNA synthetases) to activate the tRNA

Mupirocin is an antibiotic that has this step as a drug target. (Isoleucyl aminoacyl tRNA synthetase)

Question 17

Does the entire mRNA encode a protein? What is the role of the 5’ UTR? What is the role of the 3’ UTR?

Answer 17

No, only the portion from the start codon to the stop codon is translated. 5’ UTR regulates transcription. 3’ UTR regulates stability (lifetime)

Question 18

Describe silent, missense, nonsense, and frameshift mutations. Which of these when mutated are likely to give rise to a genetic disease, and describe how the protein is changed? You may use globin proteins for your examples.

Answer 18

Silent mutation – point mutation resulting in the same A.A. being coded

Missense – point mutation resulting in a different A.A. being coded. (disease)

Nonsense – point mutation resulting in a stop codon. (disease)

Frameshift – insertion/deletion of one nucleotide that shifts the reading frame. (disease)

Question 19

The start codon of a gene related to MAP kinase kinase kinase is located in exon 2. If the DNA sequence in exon 3 of the MAP kinase kinase kinase-related gene is mutated from ATG (which normally codes for methionine in the amino acid sequence) to ACG, how does that alter the amino acid sequence (consult the genetic code in Table 37-1 on p396 of Murray et al. IF you need to)?

Would that be a silent, missense, or nonsense
mutation, or would there be the possibility of another kind of amino acid sequence
change? How about if the mutation inverted the AT to TA (ATG to TAG)?

Answer 19

ATC to ACG = missense

ATG to TAG = nonsense

Question 20

If a gene sequence in an INTRON is inverted from TGG to GGT, how does that alter the amino acid sequence (consult the genetic code in Table 37-1 on p396 of Murray et al. IF you need to)? Would that be a silent, missense, or nonsense mutation, or would there be the possibility of another kind of amino acid sequence change?

Answer 20

missense

Question 21

How many subunits are in a ribosome, and what are their names? There are ribosomes in eukaryotes, prokaryotes, and mitochondria. Which of these are more similar, and what is the medical relevance of this?

Answer 21

2 subunits (1 small subunit and 1 large subunit).

Prokaryotic ribosomes are similar to Mitochondria ribosomes. These two are pretty different than eukaryotic ribosomes. The medical relevance is that antibiotics rely on the difference between prokaryotic and eukaryotic ribosomes to work, but because of their similarity to mitochondria ribosomes, the will slightly affect eukaryotes.

Question 22

What are the three repeated steps of translation, repeated each time an amino acid is added to the growing polypeptide chain? What is the step that precedes these repeated steps, and what is the step that follows these repeated steps?

Answer 22

The repeated steps are:

1) binding of next tRNA
2) new tRNA is bound to the first tRNA and a peptide bond is formed.
3) mRNA and ribosome moves along, causing the first tRNA to leave and opening up the spot of binding at the new spot. (translocation)

Initiation phase begins translation and preceeds the repeated steps..

The step that follows these repeated step is called termination.

Accessory proteins IEF4E, EIF4G aid in termination
Accessory protein eEF2 aid in translocation.

Question 23

Which step of translation is affected by macrolides? Aminoglycosides? Tetracyclines? Chloramphenicol?

Answer 23

These are all antibiotics:

Aminoglycosides: blocks initiation of translation

Tetracyclines: affects binding of tRNA

Chloramphenical: affects bonding step

Macrolides: affects translocation

Question 24

What is the most serious adverse effect of chloramphenicol use?

Answer 24

fatal aplastic anemia

Question 25

What class of drugs shows ototoxicity and renotoxicity due to oxidative damage in which a primary role seems to be played by iron? In what cell types does this class of drugs preferentially concentrate to toxic levels?

Answer 25

Ototoxicity and renotoxicity due to oxidative damage in which a primary role seems to be played by iron is caused by Aminoglycosides. This class of drugs preferentially concentrate in kidney and ear cells.

Question 26

You will note that tetracyclines inhibit 30S and 40S subunits, i.e. both eukaryotic and prokaryotic small ribosomal subunits. How then can you explain its use as an antibiotic which does not harm animals?

Answer 26

Tetracyclines have efficient uptake in bacterial cells but has poor uptake in humans, which is why it does not harm animals.

Question 27

What has the indiscriminate use of antibiotics in past decades led to that is now a big concern for medicine?

Answer 27

antibiotic resistance

Question 28

Give three mechanisms by which antibiotic resistance in bacteria arise.

Answer 28

1) block binding site of drug molecule
2) degrades drug molecules
3) efflux pumps

Question 29

How can a mechanism of antibiotic resistance be readily passed between different strains and species of bacteria?

Answer 29

bacteria can share their DNA via plasmids

Question 30

What class of antibiotics does linezolid belong to? What is so special about linezolid that helps it avoid antibiotic resistance due to mutation of its binding site? How might this drug be susceptible to antibiotic resistance.

Answer 30

Linezolid is an antibiotic inhibitor.

Linezolid binds to a novel site, which has never been blocked before.

The bacteria can still block it, but less likely since it’s new.

Question 31

What class of antibiotics does telithromycin belong to? What is so special about telithromycin that helps it avoid antibiotic resistance due to mutation of its binding site? How might this drug be susceptible to antibiotic resistance.

Answer 31

Telithromycin is an antibiotic translation inhibitor.

Telithromycin has two binding sites. Antibiotic resistance can still block the molecule, but they would have to block two binding sites

Question 32

What class of antibiotics do the penicillins belong to? What is combined with one of the penicillins in tablet form that helps it avoid antibiotic resistance due to degradation of the antibiotic molecule?

Answer 32

Penicillin are cell wall synthesis inhibitors

Amoxicillin is combined with clavulanate. The bacteria will degrade clavulanate instead of degrading amoxicillin.

Question 33

Describe the regulation of mRNA stability and the translational regulation due to iron involving apoferritin and transferrin receptor.

Answer 33

Apoferritin intercepts iron coming into the body and holds the iron, keeping body Fe low. When iron is low, IREBP binds to IRE in 5’ UTR and apoferritin translation doesn’t occur. Iron bound by apoferritin is held until the cell is sloughed away.

Transferrin mRNA is stabilized by low Fe. When iron is low, IREBP binds to IRE in 3’ UTR and RNAse will be blocked and the transferrin RNA won’t be degraded.The transferrin receptor binds Iron and brings it into the body. It maintains cell Iron.

So when iron is low, apoferritin decreases and TFR increases.

If Iron is high, [apoferritin] increases and [TFR] decreases.

Question 34

You are in a lecture about cystic fibrosis in which the investigational drug Ataluren made by PTC Pharmaceuticals is described as a new wonder drug to eradicate the consequences of cystic fibrosis. You inquire further and find that Ataluren has indeed been successful in completing phase II clinical trials with cystic fibrosis, showing decreases in cough frequency and increases in pulmonary function, and is poised to continue further clinical trials preliminary to its becoming available as a prescription drug. Ataluren has a unique mechanism; in cystic fibrosis patients whose disease is due to a nonsense mutation it allows the ribosome to ignore the premature stop codon such that normal full-length CFTR protein is synthesized. Your take on this is that:

A. CFTR is not the protein responsible for cystic fibrosis so the drug would not be useful
for that indication.

B. because you know from biochemistry class that about 70% of cystic fibrosis patients suffer from a single amino acid deletion, and about another 20% have missense 
mutations, Ataluren would actually only be useful in around 10% of cystic fibrosis patients.

C. antibiotic resistance through mutation of the ribosome at Ataluren’s binding site will
almost certainly reduce the drug’s effectiveness over time.

D. Ataluren inhibits the binding step of translation at the ribosome, and thereby blocks
the docking of stop tRNAs on the ribosome.

E. because the mRNA still contains the defect, mis-regulation of transcription and/or mis-splicing will still cause some of the symptoms of cystic fibrosis, though these symptoms may then be more manageable with other drugs.

Answer 34

B. because you know from biochemistry class that about 70% of cystic fibrosis patients suffer from a single amino acid deletion, and about another 20% have missense 
mutations, Ataluren would actually only be useful in around 10% of cystic fibrosis patients.

Question 35

Which of the following antibiotics do not bind to ribosomes (more than one MAY be correct)?
A. macrolides B. aminoglycosides 
C. rifampicin D. telithromycin
E. mupirocin F. tetracyclines
G. linezolid H. chloramphenicol
I. amoxicillin/clavulanate

Answer 35

C. rifampicin
E. mupirocin
I. amoxicillin/clavulanate

Question 36

Among eleven children who underwent gene therapy several years ago in a trial that attempted to treat their X-linked severe combined immunodeficiency disorder, two of the children subsequently developed leukemia. These trials utilized retroviral vectors with the goal of integrating a functional copy of interleukin 2 receptor γ chain into the DNA of precursor blood cells. The trial was successful, with all children showing protection from bacterial and viral infections, presence of T lymphocytes, and so on. The leukemic children were successfully treated for leukemia and remain alive, last I heard, thank goodness. The leukemia can be explained by:

A. The virus causing even further collapse of the immune system of the children, making them prone to development of the leukemia.

B. integration of the virally-mediated DNA into a site within the promoter region of the proto-oncogene LMO2, causing up-regulation and therefore conversion to an oncogene.

C. retroviral killing of white blood cells, which the bone marrow responds to by uncontrolled growth and proliferation of white blood cells, and hence leukemia.

D. the destabilization of all host cellular mRNA species, and thus the loss of important functional tumor supressors.

E. apoptosis of the cells containing the retrovirus, causing them to become resistant to cancer chemotherapeutics.

Answer 36

B. integration of the virally-mediated DNA into a site within the promoter region of the proto-oncogene LMO2, causing up-regulation and therefore conversion to an oncogene.

Question 37

Characterize replication of DNA

Answer 37

• DNA polymerase

Question 38

Characterize transcription

Answer 38

• RNA polymerase

Question 39

Characterize translation

Answer 39

• ribosome
• tRNA, rRNA
• snRNA (small nuclear) that does RNA processing
• siRNA (small interfering)
• muRNA (micro) Regulation

Question 40

Name one elongation factor in translocation.

Answer 40

eEF2 (aka. translocase)

Question 41

What toxin binds ADP-ribosyl to eeF2 and stops translocation?

Answer 41

diphtherin