Topic 3 (PP 9-10) - The Genetic Code and Protein Translation Flashcards
1
Q
How many amino acid side R chains are there?
A
20
2
Q
At what pH are both the amino acid and carboxyl groups ionized?
A
7
3
Q
What is the bond between two amino acids?
A
peptide bonds
4
Q
What molecule is lost in the reaction of a peptide bond?
A
H20
5
Q
What are amino acid side chains called?
A
R-groups
6
Q
Denature protein
A
a protein in its unfolded configuration
7
Q
How can proteins be denatured with chemical exposure?
A
proteins exposed to high concentration of urea can denature the protein. Urea is a denaturing chemical that disrupts hydrogen bonds
8
Q
How can denatured proteins recover their natural shapes?
A
remove urea
9
Q
How can proteins be denatured with heat?
A
boiling proteins in solution
10
Q
Protein structure is determined by which weak interactions?
A
non polar side chains, polar side chains, hydrogen bonds, van der Waals attraction, electrostatic interactions
11
Q
What interactions affect protein structure in an aqueous environment?
A
nonpolar side chains packed into hydrophobic cores, polar side chains forming hydrogen bonds with water
12
Q
What are three types of non-covalent bonds that help proteins fold?
A
electrostatic attractions, hydrogen bond, van der Waals attraction
13
Q
primary structure of a protein
A
sequence of amino acids
14
Q
secondary structure of a protein
A
Presence of alpha-helices and beta-sheets
15
Q
Tertiary structure of a protein
A
folding of individual proteins into a 3-D structure
16
Q
Quaternary structure of a protein
A
assembled subunits
17
Q
Prp
A
prion protein
18
Q
PrPC
A
normal form of the prion protein, does not cause diseas
19
Q
PrPSC
A
disease form of prion protein
20
Q
What is the most common prion disease in the USA? Average cases per year? Average age onset?
A
Creuzfeldt-Jakob Disease (CJD), 350 cases per year, average onset of 60
21
Q
What are the types of CJD onset in humans?
A
spontaneous, genetic, transmissible (kuru, new variant nvCJD)
22
Q
How do prion diseases work?
A
- a single molecule of the normal form spontaneously converts to the diseased form (rare event)
- diseased form now acts like a catalyst to convert all normal form in the cell to diseased form
- diseased form aggregates, causing disease
23
Q
What is the name of the prion disease in humans (shared by other mamals) that affects neurons?
A
spongiform encephalopathy
24
Q
What is the Creuzfeldt-Jakob Disease?
A
a degenerative brain disorder that leads to dementia and death
25
Another protein folding disease is
cystic fibrosis
26
CFTR (cystic fibrosis)
cystic fibrosis transmembrane conductance regulator, regulates CI- ion transport
27
What are the consequences of CFTR
Loss of function mutations in CFTR lead to defects in epithelial fluid transport, causing compromised function in the lungs, pancreas, and other organs
28
What is trikafta?
a combination of novel drug mechanism, correctors, potentiators for treatment of cystic fibrosis
29
What is novel drug mechanism?
improve the function of a defective protein
30
What are correctors (elexacaftor, tezacaftor)?
improve folding and biogenesis ("pharmacological chaperones")
31
What are potentiators (ivacaftor)?
improve channel function of proteins that make it to the membrane
32
What does regulation of gene expression imply?
a particular step can vary in efficiency depending on specific signals, environmental conditions, or cell type
33
What does it mean if a promoter is closer to the consensus sequence?
it becomes more efficiently transcribed
34
Is is common for genes in bacteria to have perfect matches to consensus sequences?
very few have it, some genes are hard wired in their promoter to have specific transcription rates
35
What are the ways proteins structure can be depicted?
backbone, ribbon, wire, space-filling
36
What is a feature of independently folded domains?
retain shape and structure when separated from the protein
37
Proteins can be created from a single or many polypeptide domains?True or False
True
38
What is X-ray crystallography?
combined with amino acid sequence, a technique used to determine a proteins 3-D structure, and assess how this conformation changes as the protein functions by seeing the relative positions of proteins individual atoms. The proteins are coated to form crystals and x rays (diameter of H atom) shined through scattered the rays. The waves reinforce and cancel each other which is analyzed by electronic detectors and transformed into a spatial map. The position and intensity of each spot in the x-ray diffraction pattern contain information about the position of the atoms in the protein crystal.
39
What is NMR spectroscopy (nuclear magnetic resonance spectroscopy) ?
combined with amino acid sequence it is a protein structure method for very small proteins. Utilizes the magnetic nucleus of hydrogen atoms. Protein exposed to a magnet will cause nuclei to align with the field. Once hit with radio waves the nuclei wobble on axes and give off signals about their position.
40
What is cyro-electron microscopy?
a protein determination technique for large molecules that are hard to crystallize. A droplet of protein in water is placed on an electromagnetic grid in liquid ethane, freezing the proteins with a film of ice. The rapid freezing prevents water molecules from forming crystals and damaging the protein shape. The sample is examined through transmission electron microscopy with projected images through beams of electrons. The algorithm sorts the particles into sets oriented in the same direction and is transformed into a 3D structure.
41
transcription repressor
decreases or prevents transcription
42
feedback inhibition
product of a process or reaction inhibits an earlier step in its synthesis
43
What is tryptophan (trp)?
an essential amino acid that helps the body make proteins. In humans the body cannot synthesize it and it must be obtained through diet
44
How does the control of tryptophan work in bacteria? Gene regulation when tryptophan is low and high?
When trp is low, the inactive trp repressor doesn't bind, RNA polymerase is recruited to the promoter before the operon, produces mRNA, and the operon is turned ON
When trp is high trp binds to the active trp receptor bound to the operator, RNA polymerase does bind to the promoter before the operon, and the operon is turned OFF
45
What is the conformational difference between trp gene on vs off?
Trp repressor alters its conformation after binding tryptophan, allowing it to bind DNA, the gene is turned off
When the repressor is not activated it does not bind to DNA and the gene is on
46
What is positive control of gene expression by transcription activators?
activators directly or indirectly increase recruitment of RNA polymerase
47
What is positive control?
gene is off by default, requires activation to turn on
48
What is negative control?
gene is on by default, requires a repressor to turn it off
49
How does positive control of gene expression recruit activator proteins?
50
What is the importance of the gene regulation of the lac operon?
genes required for metabolism of the sugar lactose
51
In the lac operon what controls the positive control?
CAP activator
52
In the lac operon what controls the negative control?
lac repressor
53
When should the lac operon be turned on?
When only lactose is present
RNA polymerase binds to the promoter and synthesizes mRNA
54
When should the lac operon be turned off?
1. When there is glucose and lactose nothing binds
2. When there is just glucose, the lac repressor binds to the operator
3. When there is no glucose and no lactose, the lac repressor binds to the operator and the CAP activator protein and cylic AMP bind to the CAP binding site
55
What is the logic of the lac operon on/off regulation?
1. The genes should only be turned on if lactose is present
2. Glucose is a much more efficiently used energy source, if glucose is present the genes should not be turned on even if lactose is ctpresent
Positive control by CAP activator
Negative control by lac repressor
56
How does the glucose environmental factors in the lac operon relate to transcription?
57
What is coordinate control?
in prokaryotes related genes are expressed together in operons
58
In what kinds of cells can a single mRNA can be translated into multiple distinct proteins, usually with related function?
prokaryotes
ribosomes can bind internal sequences in prokaryotic mRNAs
59
What is the ribosome binding site in prokraryotes?
Shine-Delgarno sequence
60
What is the Shine-Delgarno sequence?
a consensus sequence that is recognized by the prokaryotic ribosome; a sequence of one of the ribosomal RNAs can base pair with the S-D sequence.
61
How do eukaryotic ribosomes translate proteins?
eukaryotes ribosomes recognize the 5’ cap and then scan for the first AUG
62
How is control of eukaryotic gene expression more complex?
requires enhancer, GTFs, Mediator, RNA Polymerase
63
What is an enhancer?
binding site for the activator protein
64
What is the chromatin structure formation in eukaryotes?
65
What is linker DNA in chromatin structure?
The DNA strands between nucleosomes
nuclease digests this linker DNA to free nucleosomes
66
chromatin
DNA plus all associated proteins
67
chromosomes
individual DNA molecules within a cell
68
What is the percent breakdown of DNA and protein in chromosomes?
Chromosomes are ~50% DNA and 50% protein
69
How many meters of DNA/cell are in mammals?
2 meters of DNA/cell in mammals
70
What do eukaryotic transcriptional activators recruit to help initiate gene transcription?
Histone-aceytl-transferase
Chromatin remodeling protein complex
71
How do steroid inducible genes work in eukaryotes?
72
How does steroid inducible gene expression work in eukaryotes? What does it show?
Gene expression is low when the cortisol receptor does not bind to the operator
Gene expression is high when the cortisol receptor binds to the operator and cortisol binds to the receptor
A single transcription regulator can coordinate the expression of many different genes
73
How does positive control of the lac operon my cAMP/CRP activator work?
* The lac operon is also under control by a positive regulator, the CRP protein (also known as CAP)
* Low glucose in the cell leads to high cAMP levels
* cAMP associates with CRP protein, allows it to bind the lac operator sequence
* CRP promotes binding of RNA polymerase; RNA polymerase binding is weak in the absence of CRP
* Full expression of the lac operon requires both the relaxation of lacI repression due to the presence of lactose, and the activation by CRP in the presence of low glucose
74
How does negative control of the lac operon by the lac repressor work?
75
How does the lac operon work?
76
What is the effect of histones/chromatin on gene expression?
Result is reduced transcription with the nucleosomal template (chromatin template)
77
How can we test the effect of histones/chromatin on gene expression in vitro?
- Establish an in vitro transcription system (RNA pol complex, associated transcription factors, DNA template)
- Compare DNA-only template with DNA + nucleosomes template
78
How can we test the effect of histones/chromatin on gene expression in vivo?
Approach: eliminate gene expression of one of the histones, reduce nucleosome number, observe the effects on gene expression (using yeast cells)
79
What is the effect of nucleosomes on gene expression?
overall repressive effect on gene transcription
because it coils parts of the DNA to make then inaccessible for gene translation and expression
80
How do chromatin remodelers alter the position of nucleosomes on chromatin?
The ATP dependent complex catalyzes the reaction taking ATP in and releases ADP
After many rounds of nucleosome sliding, the condensed chromatin becomes decondensed
81
How does the Swi/Snf chromatin remodeler work?
82
What do histone modifications do?
Histone modifications can alter the properties and structure of chromatin with methylation, aceylation, and phosphorylation
83
What is the histone code?
“histone code”: the specific post-translational modifications dictate gene expression states
84
What are the consequences of histone modification?
K is lysine (pos charged)
positive charge helps attract DNA
methylation doesnt alter change - unpredictable - but attracts proteins (in this case for a silencing affect)
acetylation neutralizes the pos lysine - affects DNA affinity - makes chromatin looser - transcription increases
85
what ways can we measure gene expression?
A. reporter genes
B. RT-qPCR
86
What is GFP?
a reporter protein that can be used to identify specific cells in a living animal
## Footnote
GFPs that fluoresce at different wavelengths help reveal the connections that individual neurons make within the brain.
87
How does lacZ work as a reporter gene?
lacZ codes for a protein (b-galactosidase) that cleaves lactose. X-gal is an artificial substrate of b-galactosidase that turns blue when cleaved
88
What are the two ways we can measure gene expression and what are examples of the first?
A. Reporter genes (GFP, LacZ)
B. RT-qPCR (reverse transcriptase quantitative PCR)
89
What is a reporter gene?
gene that researchers attach to a regulatory sequence of another gene of interest
90
What kind of effects does LacZ have on samples?
Bands of blue stripes where the regulatory region occurs in the gene sequence
91
What is the first step of RT-qPCR?
Step 1. cDNA synthesis
a. isolate RNA from cells by lysing
b. Use a reverse transcriptase (an RNA-dependent DNA polymerase) to make a DNA copy of every mRNA. Match the 3 prime poly adenylated tail with a poly T primer and derive new mRNA from cDNA template. Partially degrade the RNA with RNAse and synthesize the residual RNA into a complementary DNA strand by using DNA polymerase.
92
What is the second step of RT-qPCR?
What is PCR used for?
What does it require?
How many cycles are preformed?
2. PCR (polymerase chain reaction)
a. PCR is used to manipulate or detect DNA sequences
PCR requires a template, two DNA primers, a thermostable DNA polymerase, and dNTPs (nucleotide triposphates).
PCR runs for 25-35 cycles
Heat is used to separate DNA strands and cools to help primers anneal (specific to the gene of interest) to anneal to the DNA. The primers are on opposite ends of the strands. The complementary DNA is then synthesized. After the first round of amplification two double stranded DNA molecules are produced, then 4, 8 -\> becomes exponential.
93
What is an alternative to RT-qPCR after cDNA synthesis?
after cDNA synthesis, sequence all the DNA fragments using ”RNA-seq”
94
What is the genetic code?
The set of instructions that a nucleotide sequence of a gene through an mRNA is translated into the amino acid sequence of a protein
95
An mRNA sequence can be translated by one of how many reading frames
3
96
How does coordinate control work in prokaryotes?
prokaryotes related genes are expressed together in operons. In prokaryotes, a single mRNA can be translated into multiple distinct proteins, usually with related function.
Unlike in eukaryotes, ribosomes can bind internal sequences in prokaryotic mRNAs (remember: in eukaryotes ribosomes recognize the 5’ cap and then scan for the first AUG)
The ribosome binding site in prokaryotes is known as the “Shine-Delgarno sequence”, a consensus sequence that is recognized by the prokaryotic ribosome; a sequence of one of the ribosomal RNAs can base pair with the S-D sequence.
97
Why do prokaryotes and eukaryotes differ in their gene expression?
Unlike in eukaryotes, ribosomes can bind internal sequences in prokaryotic mRNAs (remember: in eukaryotes ribosomes recognize the 5’ cap and then scan for the first AUG)
98
What is the name of the ribosome binding site in prokaryotes and what is it?
“Shine-Delgarno sequence”
is a consensus sequence recognized by prokaryotic ribosome
a sequence of one of the ribosomal RNAs can base pair with the S-D sequence.
99
what is an enhancer?
binding site for activator protein in eukaryotes for gene expression
100
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102
103
104
105
106
107
108
what does crossing over result in? What kinds of combination of gene alleles?
In F1 there is a heterozygous dominant and a homozygous recessive
The heterozygous dominant can produce non crossover gametes, crossover gametes. From that in F2 it can produce parental phenotypes each 24% and recombinant phenotypes each 26%.
The homozygous recessive from F1 can only produce homozygous recombinant phenotypes
109
How is genetic diversity generated?
Sources of genetic diversity
1. Independent assortment of chromosomes in meiosis
2. Crossing over in meiosis (recombination of chromosomes)
3. Mutation - a randomly produced, heritable change in the DNA sequence of a chromosome
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