Week 1 Flashcards
(162 cards)
1
Q
What type of bond is found in Acetyl CoA?
A
C-S
2
Q
What type of bond is found in ATP?
A
Phosphoanyhydride
3
Q
What type of bond is found in Phosphocreatine?
A
P-N
4
Q
What type of bond is found in Phosphoenol pyruvate?
A
C-O-P
5
Q
What is a nucleoside?
A
A nucleotide without a phosphate
6
Q
Which is more soluble- pyrimidine or purine?
A
Pyrimidine
7
Q
Which is more soluble- Nucleotide or nucleoside?
A
Nucleotide
8
Q
Which is more soluble- Nucleoside or base?
A
Nucleoside
9
Q
What is the implication with Chargaff’s rule?
A
A-T, C-G
10
Q
How does actinomycinD- doxorubicin act?
A
Blocks DNA replication through intercalation
11
Q
What is the purpose of a topoisomerase?
A
Relax DNA so that it doesn’t supercoil
12
Q
Why is RNA less stable than DNA?
A
RNA is susceptible to nucleophilic attack on the 2’OH
13
Q
How does puromycin act?
A
It’s a nucleotide analogue that mimics the tRNA acceptor region and terminates translation
14
Q
What are the three classes of RNA?
A
Structural, regulatory, informational
15
Q
Transcription is __________ and ___________
A
Unidirectional and processive
16
Q
What is the product of RNA Pol I?
A
rRNA
17
Q
What is the product of RNA Pol II?
A
mRNA, snRNA, miRNA, lncRNA
18
Q
What is the product of RNA Pol III?
A
tRNA, 5SRNA. . .
19
Q
What are the three major steps in transcription?
A
Initiation, Elongation and Termination
20
Q
What are the three major steps in initiation?
A
- Polymerase binds to promoter “closed complex”
- Pol. melts DNA and forms bubble “open complex”
- Pol catalyzes phosphodiester linkage of 2 rNTPs
21
Q
During elongation, RNA polymerase travels which direction?
A
3’ -> 5’
22
Q
What occurs during termination?
A
The Polymerase releases RNA and dissociates from DNA
23
Q
Alpha amanitin is which type of inhibitor to RNA pol II?
A
Non-competitive
24
Q
Alpha amanitin acts through which mechanism?
A
It blocks the RNA chain elongation by preventing translocation
25
How does Rifampicin act?
It binds RNA Polymerase and blocks the RNA exit channel
26
What is TFIIH important for?
Transcription and DNA repair
27
Mutations in TFIIH can cause
XP, Cockaynes syndrome and Trichothiodystr. . .
28
What are the three steps in adding the 5' cap?
1. Triphosphatase
2. Guanylyltransferase
3. Guanine 7 methyl transferase
29
Functions of the mRNA cap?
1. Regulation of nuclear export
2. Prevention of degradation by exonucleases
3. Promotion of translation
4. Promotion of 5' proximal intron excision
30
What does over-expression of eIF4E cause?
Malignant transformation because it's a cap binding protein which leads to increased translation
31
The 5' splice site of the intron is recognized by base pairing to?
U1snRNA
32
The 3' splice site of the intron is recognized by base pairing to?
U2snRNA
33
What are the two steps during splicing via a lariat intermediate?
1. Attack by the branch point 2'OH @ the exon's 3' phosphate
| 2. Attack by the 3'OH of exon 1 on the 5' phosphate of exon 2
34
What is the significance of Abnormal splicing of CD44?
It's a signal of tumor metastasis that is useful for diagnosis and prognosis
35
What is the causal mechanism for Marfan's syndrome?
Disruptive gene splicing in the fibrillin gene transcripts
36
What are the two steps in creating a PolyA Tail?
1. Cleavage
| 2. Polyadenylation
37
Alpha Thalassemia- how does it happen?
Mutation of AAUAAA consensus sequence
38
How can cancer be created by the poly A site?
Shortening of 3'UTR and elongating of polyA tail leads to deletion of oncogenes and continued life of the cell
39
3' end formation is coupled with?
Termination of transcription by RNA Pol II
40
What are the three types of excision repair?
1. Nucleotide
2. Base
3. Mismatch repair
41
Nucleotide excision repair is
1. good for
2. What happens when it goes wrong?
1. Thymine dimers
| 2. XP, CS and TTD
42
Base excision repair requires
glycosylases to recognize altered bases
43
Mismatch repair proteins in bacteria vs. mammalian counterparts
MutS and MutL vs. MSH and MLH
44
How does the MMR machinery recognize the new strand?
It is not yet methylated- in eukaryotes the MMR requires DNA nicks
45
What is the overarching goal of MMR?
Removal of the wrongly inserted base, not its mismatching partner.
46
What happens when MMR malfunctions
HNPCC
47
Lesion bypass usually fixes
excessive thymine dimers- not super effective as it's really mutagenic
48
DNA replication is __________ and _____________
bidirectional and semiconservative
49
DNA replication in Prok vs. Euk
Prok- one site of origin on each chromosome
| Euk- multiple site of origin on each chromosome
50
When helicases unwind the double helix ________ binds to each strand of DNA to hold it in place
single-strand binding proteins
51
DNA synthesis proceeds in which direction?
5' to 3'
52
DNA synthesis requires what?
RNA primers
53
Prokaryotic DNA replication is carried out by two DNA polymerases
What are their names? Which is more important? Why?
DNA Pol I and DNA Pol III. DNA Pol III is more important because it has a sliding clamp that keeps it attached to the DNA template over a long distance- higher processivity
54
DNA Pol I's purpose?
mediate replacement of RNA primers with DNA through it's 5' to 3' exonuclease activity and 5' to 3' DNA polymerase activity
55
Eukaryotic DNA replication requires what?
three polymerases
| one for primare and polymerase, one for lagging strands and one for leading strands
56
What is the function of primase?
Catalyzing the reaction needed to form the RNA primer during replication- it's a DNA dependent RNA polymerase
57
What are the three activities of DNA Pol I in E. coli?
1. 5' to 3' DNA polymerase activity requiring a 3' OH primer and a DNA template strand
2. a 5' to 3' exonuclease activity for RNA primer removal
3. a 3' to 5' exonuclease activity for proofreading
58
What is the function of DNA ligase?
an enzyme that catalyzes the formation of phosphodiester bonds between a 3' hydroxyl group and a 5' phosphate group of two polynucleotide chains
59
What are three types of DNA control elements?
1. TATA Box/ Initiator sequence
2. Promoter Proximal Elements
3. Enhancers
60
What is the difference between an enhancer and a promoter?
Promoters are about 20 bps and are about 200 bps upstream of transcription start sites whereas enhancers are 8-20 bps but can consolidate and be 100-200 bps long. Their location can vary upstream, downstream in the last exon or within an intron.
61
How does Beta Thalassemia occur?
Deficient production of Beta globin protein by ethyroid cells due to mutations in the B globin promoter
62
How does alpha Thalassemia occur?
Deletion of locus control region of B globin gene cluster
63
What is Hemophilia B Leyden?
X-linked clotting disorder due to inherited mutation in the DNA control element in the promoter of the Factor IX gene- alleviated during puberty due to active androgen receptor
64
What is Fragile X Syndrome?
CGG repeat in the 5' region of the FMR 1 gene which facilitates methylation of the cytosine residues in CpG islands and transcriptional inactivation of the FMR1 gene
65
What are the two classes of Activators and repressors?
1. Sequence specific DNA binding proteins
| 2. Co-factors
66
Where do SSBP's act?
They bind to promoter or enhancer elements to regulate transcription. They insert their alpha helices into the major groove of DNA
67
Where do co-factors act?
They do not bind directly to DNA, but bind sequence-specific DNA binding proteins and affect transcription through the contact
68
Sequence specific DNA binding proteins are modular, containing two major domains:
1. DNA binding domain
| 2. Activation or repression domain
69
What are the four families of DNA binding proteins?
1. Homeodomain proteins
2. Zinc-finger proteins
3. Basic leucine zipper proteins
4. Basic helix-loop-helix motif
70
Homeodomain Protein examples
Hox family, Pit1, Msx
71
Zinc-finger protein examples
estrogen receptor, androgen receptor, retinoic acid
72
Basic leucine zipper protein examples
c-fos and c-jun
73
bHLH examples
MyoD, myogenin, Myf5
74
What causes craniosynostosis?
mutation in the homeodomain protein MSX2 which is required for proper craniofacial development
75
What causes androgen insensitivity syndrome?
Mutations in the DNA binding domain or ligand binding domain of the androgen receptor (zinc finger DNA binding protein)
76
What causes Waardenburg Syndrome type II?
Mutations in microphthalmia-associated transcription factor gene which encodes a bHLH DNA binding protein- it's a transcription factor that plays a major role in the development of melanocytes
77
How do transcriptional activators or repressors stimulate transcription?
1. Regulate assembly of initiation complexes and rate of initiation of transcription
2. Regulate changes in chromatin structure influencing the ability of general transcription factors to bind promoters
78
What are the two classes or chromatin remodelling factors?
1. DNA-dependent ATPases (SWI/SNF) which disrupt histone octamers and DNA
2. Factors that reversibly modify histone through aceylation (HATs and HDACs)
79
The N-termini of histones are rich in _______ residues
lysine
80
How do HATS work?
They are co-activators which originally were thought to neutralize the positively charged ends and eliminate electrostatic interactions with DNA phosphates, but now we believe that it allows for the binding of specific transcription factors
81
How do HDACs work?
The are co-repressors which were originally believed to help histones retain positive charge at N-terminal ends.
82
How does Rubinstein-Taybi Syndrome occur?
results from mutations in one copy of the CREB binding protein gene (CBP). CBP is a transcriptional coactivator and is a HAT. Results in widespread transcriptional changes
83
How does Leukemia occur?
Result of chromosomal translocations leading to gain of function fusion proteins- some of which involved fusions of transcriptional regulators with HATs or HDACs
84
What are the two ways that transcriptional activators/repressors work?
1. Interact with TFs/Pol II associated proteins to influence initiation of elongation of the primary transcript
2. Interact with chromatin to regulate accessibility of DNA to Pol II transcriptional apparatus
85
What are five ways that sequence-specific DNA binding proteins are regulated?
1. The conformation of the DNA-binding protein can be altered by ligand binding
2. Entry into the nucleus can be regulated
3. The amount of transcription factor in the cell can be regulated
4. DNA binding can be regulated
5. Phosphorylation of the DNA-binding protein can alter various properties including protein degradation, recruitment of co-activators, and DNA binding.
86
What are examples of ligand binding effects?
The nuclear receptor family of Zinc finger transcription factors work by binding to steroid hormones which can affect dimerization of receptor, recruitment of coactivators/repressor and translocation into the nucleus i.e. esotrogen receptor activation and glucocorticoid receptor activation
87
How does estrogen induce transcriptional activity?
by causing ER dimerization
88
How does Tamoxifen work?
it binds to ER and prevents the recruitment of HAT co-factors
89
What is an example of regulation of nuclear entry?
NF-kB is usually held in the cytoplasm by IkB. if IkB gets phosphorylated, it gets degraded. Then NK-kB is released and moves into the nucleus where it turns on genes involved in inflammation.
90
How does aspirin work?
Aspirin inhibits phosphorylation and degradation of IkB which prevents the translocation of NFkB to the nucleus which inhibits transcription of genes involved in the inflammatory response.
91
What is an example of regulation of nuclear entry by NF-AT?
High intracellular calcium activates calcineurin's phosphatase activity which dephos's cytoplasmic NF-AT. This exposes the nuclear localization sequence, allowing NF-AT to enter the nucleus where it affects transcription of genes involve in immune response and in heart function.
92
Who to the immunosuppressants cyclosporin and FK506 work?
They inhibit calcineurin thereby inhibiting NF-AT action
93
What are examples of regulating amount of TF in cell?
Beta Catenin which in the absence of Wnt signaling is targeted for degradation through the ubiquitin-proteasome pathway via phosphorylation.
94
What happens in the presence of Wnt signaling?
The Axin-APC-GSK3 complex is destabililzed, preventing phsophorylation of B-catenin and leading to an increase in the cytoplasmic pool of the protein. This allows some of the B-catenin to move into the nucleus where it interacts with the TCF family of transcription factors and promotes the expression of Wnt responsive genes.
95
What's an example of regulating TF in the cell using p53?
p53 is downregulated by binding to the MDM2 protein which masks its activation domain and targets is for destruction by the ubiquitin- proteasome pathway
96
Example of regulating DNA binding
Id proteins- the Id family members negatively regulate DNA binding by heterodimerizing with other HLH proteins through their HLH domains, but preventing DNA binding due to their lack of a basic domain
97
Example of how phosphorylation affects activity of trans-acting factors
CREB- a series of events initiated by the binding of a ligand to a guanine nucleotide binding protein coupled receptor induces the phosphorylation of the CREB protein which, while present on the DNA, is inactive to promote transcription unless phosphorylated. Once phosphorylated, the CREB protein recruits the histone acetyl transferas CREB which has intrinsic HAT activity and recruits RNA Pol II- leading to transcriptional activation of the gene.
98
What is the mRNA start codon?
AUG
99
How do the mRNAs and tRNAs interact?
the tRNAs base-pair directly to the codons in the mRNA through the tRNA's anticodon loop
100
What is the function of the Aminoacyl tRNA synthetases?
they are protein enzymes that put the right amino acid on the right tRNA. They are important because each identifies the right tRNA and puts on the correct amino acid. Each AA/tRNA has its own synthetase associated with it.
101
What are the sizes of the ribosome subunits in bacteria and eukaryotic cells?
30S and 50S vs. 40S and 60S
102
What are initiation factors?
proteins that bring the ribosome to the message and assist in getting the machinery assembled. 3 in bacteria and over a dozen in eukaryotes.
103
What are elongation factors?
Proteins that deliver tRNAs and move the ribosome down the message.
104
What needs to come together for successful translation?
mRNA, tRNA, aminoacyl tRNA synthetases, ribosomes, initiation factors, elongation factors and partners and termination/recycling factors
105
What are termination/recycling factors?
proteins that end the process at a stop codon and dissociate the subunits so that they can be used again.
106
What are the four steps in translation?
1. initiation
2. Elongation
3. Termination
4. Ribosome recycling
107
What is the difference between translational initiation in bacteria and eukaryotes?
In bacteria the ribosome binds essentially right at the start codon due to the Shine-Dalgarno sequence and three initiation factors work to assemble the full ribosome. In eukaryotes, initiation factor eIF4E is required to bind to the 7-methyl guanosine cap on the 5' end of the mRNA which leads to the binding of many other eIF's. Then the ribosome scans down the message to find the AUG start codon. The large subunit joins the small the factors are released and the goal of initiation has been achieved.
108
What is the goal of initiation of translation?
The goal is to assemble a ribosome with the start codon AUG and initiator methionine tRNA in the P-site ready to receive the next aa-tRNA in the A-site
109
In addition to cap-dependent processes, there is a cap-independent process. What is it called and how does it work?
There are IRESs (internal ribosome entry sites) which are used by viruses to initiate translation after they shut down host cell cap-dependent synthesis.
110
At which step in the translation process does the most regulation occur?
Initiation
111
How does the peptide bond form?
The peptide bond forms when the protein chain moves from the P-site tRNA onto the A-site tRNA. Now the action of an elongation factor catalyzes the translocation.
112
What molecule deliver the aa-tRNA to the A-site?
EF1A in eukaryotes and EF-Tu in bacteria
113
What signals the start of termination?
The stop codon is detected by a recycling factor in the A-site
114
How do recycling factors work?
They fit into the same space as a tRNA, but when they do, they trigger the termination of the peptide chain
115
What is a missense mutation?
The codon is changed so it encodes a different amino acid
116
What is a silent mutation?
The codon is changed, but the same amino acid is encoded
117
What is a frame-shift mutation?
An addition or deletion of a nucleotide shifts the reading frame
118
What is a nonsense mutation?
A mutation resulting in a premature stop codon
119
What is a sense mutation?
The opposite of a nonsense mutation- now the stop codon is removed and the ribosome keeps going
120
What are examples of DNA frameshift mutations that manifest as disease?
Hemoglobin Wayne and Hemoglobin Constant spring from a frame-shift and sense mutation respectively. Both lead to proteins with longer C-terminal tails and are associated with chronic anemia
121
How can translation be regulated or altered?
1. Regulation by varying the mRNA structure and sequence
2. Regulation by altering the function of initiation factors
3. Regulation by protein binding to the mRNA
122
if pH < pKA, the ___________ form dominates
protonated
123
If pH > pKA, the ____________ form dominates
deprotonated
124
amino acids with non-polar aliphatic R groups
Glycine, Alanine, Valine, Leucine, Methionine, Isoleucine
125
amino acids with aromatic R groups
Tyrosine, Tryptophan, Phenylalanine
126
amino acids with polar, uncharged R groups
Threonine, Serine, Cysteine, Proline, Asparagine, Glutamine
127
amino acids with polar, positively charge R groups
Histidine, Lysine, Arginine
128
amino acids with Polar and negatively charged R groups
Aspartate, Glutamate
129
If you have hydroxyproline, you have what disease?
scurvy
130
If you add an extra carbonyl to glutamate, you have
carboxyglutamate which results in vitamin K deficiency and trouble clotting
131
N linked glycosylation?
sugars added to asparagine
132
O linked glycosylation?
Sugars added to ser or thr
133
How does Gleevec work?
competitively inhibits by binding to bcr-abl's active site which means that the substrate can't bind and the tumor cell can't proliferate
134
What is Bortezomib?
It's a proteasome inhibitor used to treat multiple myeloma
135
Which bond is the phi bond?
The amide- alpha carbon bond
136
Which bond is the psi bond?
The carbonyl carbon- alpha carbon bond
137
What are the functions of a peptide?
Hormones and pheromones (i.e. insulin and oxytocin)
Neuropeptides (i.e. substance P)
Antibiotics (polymyxin B for Gram plus and bacitracin for Gram minus)
Protection (i.e. amanitin in mushrooms, conotoxin in snails and chlorotoxin in scorpions)
138
What are two examples of medically important peptides?
Vasopressin and Angiotensin II
139
Features of the alpha helix?
Right handed screw- H bonds between CO of the n residue and the NH of the n+4 residue- residues 1 and 8 are on top of each other and all of the side chains are on the outside
140
Who wants to help form an alpha helix?
small hydrophobic residues like leucine and alanine
141
Who wants to break an alpha helix?
Proline and glycine
142
What is an example of an alpha helix that's important for life?
hemoglobin- mutations here cause thalassemia
143
What is an example of a left-handed alpha helix?
Collagen- it's important for collagen function- three left-handed helices intertwine into a right handed helix
144
What are the two types of Beta sheets?
Parallel and anti-parallel
145
Examples of alpha helices?
Structurally fibrous- keratin, myosin, toppomyosin, fibrinogen
Globular- hemoglobin and myoglobin
146
How are beta sheets formed?
hydrogen bonds between the backbone of neighboring strands
147
Examples of proteins composed mainly of beta pleated sheets?
Fibrous- spider silk and silk
| Globular- Immunoglobulin, Firbroblast growth, pepsin, HIV Protease
148
What is Circular Dichroism analysis?
CD measures the absorption difference of left and right circularly polarized light
149
What are the features of the B turn?
180 degree turn accomplished over four amino acids and is stabilized by a hydrogen bond to the carbonyl oxygen of the first amino acid to the amide proton of the 4th amino acid proline (position 2) and glycine (position 3) are common
150
What are the two major classes of proteins?
Fibrous and Globular
151
What are the characteristics of a fibrous protein?
Typically insoluble and made from a single secondary structure
152
What are the characteristics of a globular protein?
Water or lipid soluble
153
What is X Ray Crystallography?
It's a way to determine protein structure using electron density and diffraction data. The pro is that there are no size limits, the con is that crystallization is the rate limiting step
154
What is Biomolecular NMR?
A method for determining protein structure using NMR. Pros are that you don't need to crystallize the protein for the con is that we need very concentrated proteins and that it's difficult for large proteins
155
What are three examples of antibodies being used as drugs to inhibit protein-protein interactions and which diseases do they treat?
Bevacizumab (Avastin) Anti-VEGF for treatment of tumors
Infliximab (Remicade) Anti TNFalpha treatment of rheumatoid arthritis
Rituximab Anti-CD20 for B cell lymphoma
156
What are Hsp70 and Hsp40 (DNAK and DNAJ in prokaryotes)?
They are chaperones which bind to the hydrophobic region to prevent aggregation- induced at high temps- they help transport proteins in their unfolded states
157
What are GroES and GroEL?
chaperonin which uses ATP to fold proteins
158
Why is peptide prolyl cis-trans isomerase important?
It activates calcineurin. Cyclosporin inhibits cyclophilin which is a PPI and serves as an immunosuppressant
159
How is cystic fibrosis related to protein mis-folding?
The most common mutation is the deletion of F508 which leads to protein mis-folding
160
What are the three major protein purification approaches?
Gel-filtration based on size
Ion-exchange based on charge differences
Affinity based on ability to bind to specific ligands
161
What can you determine using Edman degradation?
The N-terminal sequence
162
What's the difference between a cofactor and a coenzyme?
Coenzymes have chemical groups that are used up- cofactors are not
