Molecular Biology Flashcards
1
Q
Describe the structure of chromatin.
A
- Negatively charged DNA loops twice around a positively charged histone octamer, forming a nucleosome.
- H1 (linker histone) binds to the nucleosome and to “linker DNA”, stabilizing the chromatin fiber.
- Chromatin fibers condense to form supercoiled structure.
2
Q
Histones are rich in which amino acids?
A
Lysine and Arginine (Note: positively charged)
3
Q
What is heterochromatin?
A
Chromatin that is condensed, transcriptionally inactive, and sterically inaccessible.
4
Q
What is euchromatin?
A
Chromatin that is less condensed, transcriptionally active, and sterically accessible.
5
Q
What are the 2 forms of chromatin?
A
Heterochromatin
Euchromatin
6
Q
What is the role of DNA methylation in prokaryotes?
A
Methylation of cytosine and adenine of the template strand during DNA replication allows mismatch repair enzymes to distinguish between old and new strands.
7
Q
What is the role of DNA methylation in humans?
A
Methylation of CpG islands in DNA represses transcription.
8
Q
What is the role of histone methylation?
A
Usually reversibly represses DNA transcription, BUT can activate it in some cases (depending on location).
9
Q
What is the role of histone acetylation?
A
Relaxes DNA coiling (by neutralizing positive histone charge), allowing for transcription.
10
Q
What is the most fundamental structural difference between purines and pyrimidines?
A
Purines have 2 rings and pyrimidines have 1.
11
Q
What are the purines?
A
A, adenosine
G, guanine
12
Q
What are the pyrimidines?
A
C, cytosine
T, thymine
U, uracil
13
Q
Which nucleotide has a methyl group?
A
Thymine
14
Q
How can uracil be produced from cytosine?
A
Deamination of cytosine.
15
Q
What nucleotides are found in DNA? RNA?
A
DNA: A, G, C, T
RNA: A, G, C, U
16
Q
What amino acid(s) are necessary for de novo purine synthesis?
A
Glycine
Aspartate
Glutamine
(and THF)
17
Q
What is the difference between a nucleoside and a nucleotide?
A
Nucleotide is a nucleoside with a phosphate group linked to (deoxy)ribose by a 3’-5’ phosphodiester bond.
18
Q
What DNA nucleotide bond is strongest? Why?
A
G-C is stronger than A-T given 3:2 hydrogen bonds.
19
Q
What amino acid(s) are necessary for de novo pyrimidine synthesis?
A
Aspartate
20
Q
Describe de novo purine synthesis in 2-3 steps.
A
1) Start with sugar + phosphate (PRPP).
2) Add base.
21
Q
Describe de novo pyrimidine synthesis in 2-3 steps.
A
1) Make temporary base (orotic acid).
2) Add sugar + phosphate (PRPP).
3) Modify base.
22
Q
Which metabolic pathway(s) involve carbamoyl phosphate?
A
De novo pyrimidine synthesis
Urea cycle
23
Q
How does leflunomide affect nucleotide synthesis?
A
It inhibit synthesis of orotic acid during de novo pyrimidine synthesis.
24
Q
How does mycophenolate affect nucleotide synthesis?
A
It inhibits IMP dehydrogenase during de novo purine synthesis.
25
How does ribavirin affect nucleotide synthesis?
It inhibits IMP dehydrogenase during de novo purine synthesis.
26
How does hydroxyurea affect nucleotide synthesis?
It inhibits ribonucleotide reductase (UDP -> dUDP) during de novo pyrimidine synthesis.
27
How does azathioprine (6-MP) affect nucleotide synthesis?
It inhibits de novo purine synthesis.
28
How does 5-FU affect nucleotide synthesis?
It inhibits thymidylate synthase (leading to decreased dTMP) during de novo purine synthesis.
29
How does methotrexate affect nucleotide synthesis?
It inhibits dihydrofolate reductase (leading to decreased dTMP) during de novo purine synthesis in HUMANS.
30
How does trimethoprim affect nucleotide synthesis?
It inhibits dihydrofolate reductase (leading to decreased dTMP) during de novo purine synthesis in BACTERIA.
31
How does pyrimethamine affect nucleotide synthesis?
It inhibits dihydrofolate reductase (leading to decreased dTMP) during de novo purine synthesis in PROTOZOA.
32
How does probenecid affect nucleotide metabolism?
It increases excretion of uric acid in urine.
33
How does allopurinol affect nucleotide metabolism?
It inhibits xanthine oxidase, thus inhibiting the degradation of purines to uric acid.
34
How does febuxostat affect nucleotide metabolism?
It inhibits xanthine oxidase, thus inhibiting the degradation of purines to uric acid.
35
What are the consequences of adenosine deaminase deficiency?
* Excess ATP and dATP leads to feedback inhibition of ribonucleotide reductase, creating an imbalance in the nucleotide pool.
* This prevents DNA synthesis and decreases lymphocyte count.
* One of the major causes of AR SCID.
36
What is the pathophysiology of Lesch-Nyhan syndrome?
* Defective purine salvage due to absence of HGPRT enzyme (which converts hypoxanthine -> IMP, and guanine -> GMP).
* Results in excess uric acid and increased de novo purine synthesis.
37
What are the signs/symptoms of Lesch-Nyhan syndrome?
```
HGPRT:
Hyperuricemia
Gout
Pissed off (aggression, self-mutilation)
Retardation
dysTonia
```
38
What is the treatment for Lesch-Nyhan syndrome?
Allopurinol (first line)
| Febuxostat
39
What are key features of the genetic code?
Unambiguous (each codon specifies one amino acid).
Degenerate (most amino acids are coded by multiple codons).
Non-overlapping (read from fixed starting point).
Universal (conserved throughout evolution).
40
What is an origin of replication?
Particular consensus sequence of base pairs in genome where DNA replication begins.
41
What is a replication fork?
Y-shaped region along DNA template where leading and lagging strands are synthesized.
42
What is a helicase?
An enzyme that unwinds DNA templates at the replication fork.
43
What is the role of single-stranded binding proteins?
They prevent DNA strands from reannealing.
44
What is the role of a DNA topoisomerase?
It creates a single- or double-stranded break in the helix to add or remove supercoils.
45
What is the mechanism of action of fluoroquinolones?
They inhibit prokaryotic topoisomerase II and IV.
46
What is the role of a primase?
It makes an RNA primer on which DNA polymerase III can initiate replication.
47
What is the role of DNA polymerase III?
Only prokaryotes.
* Elongates the leading strand by adding nucleotides 5' -> 3'.
* Elongates the lagging strand until it reaches primer of preceding fragment.
* 3' -> 5' exonuclease activity proofreads each added nucleotide.
48
What is the role of DNA polymerase I?
Only prokaryotes.
| * Degrades RNA primer and replaces with DNA.
49
What is the role of DNA ligase?
It catalyzes the formation of a phosphodiester bond (between Okazaki fragments) within a strand of double-stranded DNA
50
What is the role of a telomerase?
Only eukaryotes.
* RNA-dependent DNA polymerase that adds DNA to 3' ends of chromosomes to avoid loss of genetic material with every duplication.
51
In terms of nucleotide changes, what are the types of DNA point mutations?
Transition: pur -> pur or pyr -> pyr.
Transversion: pur -> pyr or reverse.
52
What are the 4 types of DNA mutations, in order of severity?
1) silent
2) missense
3) nonsense
4) frameshift
53
What is a silent mutation?
Nucleotide substitution that encodes the same amino acid. (Often in the 3rd, "wobble", position of codon.)
54
What is a missense mutation?
Nucleotide substitution resulting in changed amino acid.
55
What is a nonsense mutation?
Nucleotide substitution resulting in an early stop codon.
56
What is a frameshift mutation?
Deletion or insertion of a number of nucleotides not divisible by 3, resulting in misreading of all nucleotides downstream. (Often results in truncated, nonfunctional protein.)
57
What is the lac operon?
A classic example of a genetic response to an environmental change.
Activation of the lac operon in E. coli leads to switch from glucose to lactose metabolism in glucose-poor environments.
58
In a low-glucose environment, what mechanism leads to a change in the lac operon?
Low glucose results in the following sequence:
1) increased adenylyl cyclase activity.
2) increased generation of cAMP from ATP.
3) activation of CAP (transcription activator).
4) increased transcription along lac operon.
59
In a high-lactose environment, what mechanism leads to a change in the lac operon?
High lactose results in the following sequence:
1) unbinding of repressor protein from repressor/operator site.
2) increased transcription along lac operon.
60
Describe nucleotide excision repair.
* Specific endonucleases release the oligonucleotides containing damaged bases.
* DNA polymerase and ligase fill and reseal the gap, respectively.
61
When does nucleotide excision repair occur?
G1 phase of cell cycle.
62
What is the pathophysiology of xeroderma pigmentosa?
Defective nucleotide excision repair prevents repair of pyrimidine dimers caused by UV light exposure.
63
Describe base excision repair.
* Base-specific glycosylase removes altered base and creates AP site.
* One or more nucleotides are removed by AP-endonuclease, which cleaves the 5' end.
* Lyase cleaves the 3' end.
* DNA polymerase-β fills the gap and DNA ligase seals it.
64
When does base excision repair occur?
Throughout cell cycle.
65
What defects are typically repaired through nucleotide excision?
Bulky helix-distorting lesions.
66
What defects are typically repaired through base excision?
Spontaneous, toxic deamination.
67
Describe DNA mismatch repair?
* Newly synthesized stand is recognized and mismatched nucleotides are removed.
* The gap is filled and resealed.
68
When does mismatch DNA repair occur?
Predominantly in G2 phase of cell cycle.
69
What defect underlies HNPCC (hereditary nonpolyposis colorectal cancer)?
Defective DNA mismatch repair.
70
What is nonhomologous end joining?
Repair of double-stranded DNA breaks in which 2 ends of DNA fragments are brought together, without requirement for homology.
71
What defect underlies ataxia telangiectasia?
Defective nonhomologous end joining (of double-stranded DNA breaks).
72
What defect underlies Fanconi anemia?
Defective nonhomologous end joining (of double-stranded DNA breaks).
73
In which direction is DNA synthesized?
5' -> 3'
| 5' end of nucleotide bears the triphosphate
74
In which direction is RNA synthesized?
5' -> 3'
| 5' end of nucleotide bears the triphosphate
75
In which direction is protein synthesized?
N-terminus to C-terminus
76
In which direction is mRNA read?
5' -> 3'
| 5' end of nucleotide bears the triphosphate
77
What are the start codon(s)?
AUG (rarely GUG)
78
Which amino acid is encoded by the start codon?
Methionine (may be removed before completion of translation).
In prokaryotes, it's N-formylmethionine.
79
What role does N-formylmethionine play in the immune response?
N-formylmethionine is a prokaryotic amino acid which stimulates neutrophil chemotaxis.
80
What are the stop codon(s)?
UGA, UAA, UAG
81
What is a promotor (in gene expression)?
The DNA site where RNA polymerase II and multiple other transcription factors bind to DNA upstream from gene locus.
82
What nucleotide sequences are common in promotor regions?
AT-rich, with TATA and CAAT boxes.
83
What is an enhancer (in gene expression)?
* A stretch of DNA that increases gene expression by binding transcription factors.
* May be located close to, far from, or even within the gene whose expression it regulates.
84
What is a silencer (in gene expression)?
* A DNA site where negative regulators (repressors) bind.
| * May be located close to, far from, or even within the gene whose expression it regulates.
85
What are the RNA polymerase(s) in eukaryotes? What are their roles?
RNA polymerase I: makes rRNA
RNA polymerase II: makes mRNA
RNA polymerase III: makes tRNA
86
What are the RNA polymerase(s) in prokaryotes? What are their roles?
Single RNA polymerase: makes rRNA, mRNA, tRNA
87
What is the pathophysiology of death cap mushroom toxicity?
Contains α-amanitin which inhibits RNA polymerase II, causing severe hepatotoxicity.
88
What is the mechanism of action of rifampin?
Inhibits the prokaryotic RNA polymerase.
89
What is the mechanism of action of actinomycin D?
Inhibits RNA polymerases in both prokaryotes and eukaryotes.
90
How is an RNA transcript initially altered within the nucleus?
* Capping of 5' end (with 7-methylguanosine).
* Polyadenylation of 3' end.
* Splicing out of introns.
91
What is the primary location for mRNA "quality control"?
Cytoplasmic P-bodies (contains exonucleases, decapping enzymes, microRNAs).
92
What is the nucleotide signal for polyadenylation during RNA transcription?
AAUAAA
| Note: poly-A polymerase does not require a template.
93
Describe the process of pre-mRNA splicing.
* Primary transcript combines with snRNPs and other proteins to form spliceosome.
* Lariat-shaped intermediate is generated.
* Lariat is released to precisely remove intron and join 2 exons.
94
What is the target for anti-Smith antibodies?
Spliceosomal snRNPs.
95
What are exons?
DNA segments that contain the actual genetic coding for a protein.
96
What are introns?
Intervening noncoding segments of DNA.
97
Describe alternative splicing.
Varied splicing of exons from a common DNA segment, allowing the productions of a large number of unique proteins.
98
Describe the structure of tRNA.
* 75-90 nucleotides long.
* Has secondary structure, forming cloverleaf shape with 4 "arms/stems":
1) D-arm: recognition by correct aminoacyl-tRNA synthetase.
2) Anticodon arm: Opposite 3' aminoacyl end.
3) T-arm: tRNA-ribosome binding.
4) Acceptor stem: 5'-CCA-3' at 3' end accepts amino acid.
99
Explain wobble.
Accurate base-pairing is usually required only in the first 2 nucleotide positions of an mRNA codon, so codons differing at the 3rd "wobble" position may code for the same tRNA/amino acid.
100
What are the subunits of a eukaryotic ribosome?
40S and 60S
101
What are the subunits of a prokaryotic ribosome?
30S and 50S
102
Describe the initiation phase of protein synthesis.
* Initiation factors help assemble the 40S ribosomal subunit with the initiator tRNA.
* mRNA and 60S ribosomal subunit assemble with complex and initiation factors are released.
103
Describe the elongation phase of protein synthesis.
* Aminoacyl-tRNA binds to the A site.
* rRNA catalyzes peptide bond formation and transfers growing polypeptide to amino acid in A site.
* Ribosome advances 3 nucleotides toward 3' end of mRNA, moving peptidyl-tRNA to P site.
104
Describe the termination phase of protein synthesis.
* Stop codon is recognized by release factor.
| * Completed polypeptide is released from ribosome.
105
What are the 3 ribosomal sites relevant for protein synthesis?
A site: accepts incoming aminoacyl-tRNA.
P site: holds growing peptidyl-tRNA.
E site: holds empty tRNA as it exits.
106
What is post-translational trimming?
Removal of N- or C-terminal propeptides from zymogen to generate mature protein.
107
List common post-translational covalent alterations.
```
Phosphorylation
Glycosylation
Hydroxylation
Methylation
Acetylation
Ubiquitination
```
108
What is a chaperone protein?
An intracellular protein involved in facilitating or maintaining protein folding.
