Midterm 2 Flashcards
(98 cards)
1
Q
What is mRNA
A
Messenger RNA which codes for proteins
2
Q
Where does transcription for mRNA synthesis begin?
A
Downstream of the promoter, but upstream of the coding region.
3
Q
What enzyme is responsible for transcription?
A
RNA Polymerase
4
Q
Which direction does RNA Polymerase synthesize RNA
A
5’-3’ direction
5
Q
What are the 3 parts of transcription?
A
Initiation, Elongation, and Termination
6
Q
What is transcription initiation guided by?
A
Sigma Factors
7
Q
How are prokaryotic genes often organized?
A
Operons
8
Q
Operon
A
A functioning of genomic DNA containing a cluster of genes under the control of a single promoter
9
Q
What does the transcription of the trp operon do?
A
Produces enzymes necessary to synthesize tryptophan
10
Q
What is the default state of the trp operon and how can this transcription be repressed?
A
Default state is ON and it can be repressed by the trp repressor
11
Q
What happens when tryptophan repressor binds to trp operator?
A
Binds tightly to DNA
12
Q
What happens when tryptophan repressor doesn’t bind to trp operator?
A
Doesn’t bind tightly to DNA
13
Q
What happens to the trp repressor when it’s bound to DNA?
A
It prevents RNA polymerase from binding to the promoter
14
Q
Is there transcription of the trp operon when tryptophan is present?
A
No
15
Q
You mutate the trp repressor so it can’t bind to DNA. What do you think will happen to the concentration of tryptophan in the cell?
A
It will increase
16
Q
Why don’t tryptophan levels change when you mutate the trp repressor?
A
The synthesis of tryptophan is regulated in additional ways
17
Q
How does tryptophan regulate feedback inhibition?
A
- Regulating enzyme activity (fast) and 2. Regulating enzyme production (slow)
18
Q
What happens to transcription when lactose is absent?
A
The lac repressor binds DNA, so transcription is OFF (Doesn’t transcribe when lactose is absent/ when glucose is present)
19
Q
What happens to transcription when lactose is present?
A
The lac repressor doesn’t bind to DNA, so transcription can proceed. (Transcribe when lactose is present/ when glucose isn’t present)
20
Q
When glucose is scarce what does E. Coli generate?
A
3’-5’ cyclic AMP (cAMP)
21
Q
What does cAMP do?
A
Binds to a protein called CAP & when bound, CAP activates transcription of the lac operon.
22
Q
What happens when CAP binds to cAMP?
A
Changes shape to bind to DNA and stimulate recruitment of RNA Polymerase (foot on gas pedal)
23
Q
Presence of lactose
A
Taking foot off of brake pedal
24
Q
What happens if glucose is abundant and cAMP levels are low?
A
CAP doesn’t activate transcription
25
What happens when lactose is present and glucose is scarce (cAMP level high)?
Abundant lac mRNA synthesized
26
What happens when lactose is present and glucose is present (cAMP level low)?
Little mac mRNA synthesized
27
What would happen if you completely got rid of lac operon (brake, unless lactose present)?
Transcription when glucose if absent, regardless of lactose concentration
28
What would happen if you completely got rid of CAP (gas pedal)?
No strong transcription ever, regardless of glucose or lactose concentration.
29
How does mRNA code for protein?
DNA template strand is transcribed, creating mRNA, which is then translated to make a protein
30
What is the DNA template strand used for?
RNA synthesis starting at 5' end
31
In mRNA, what does each triplet encode?
An amino acid
32
What do three RNA bases make up?
A codon
33
What does each codon in the mRNA comprise?
Three nucleotides: a triplet 64 possible codons (43) for 20 amino acids
34
How many amino acids does each codon encode?
1 (or START or STOP)
35
The genetic code is:
Triplet-based, redundant or unambiguous, non-overlapping, and universal
36
What is the start codon?
AUG/ MET
37
Which codons are not amino acids?
STOP codons
38
What does the small ribosomal subunit do?
Binds to mRNA
39
What does the large ribosomal subunit do?
Completes the initiation complex
40
Open Reading Frame
a DNA sequence between a start and stop codon
41
What does a stop codon do?
Leads to release of protein from tRNA
42
What is the structure of tRNA
Single-stranded RNA molecules
43
What charged tRNAs with amino acids?
Aminoacyl tRNA Synthetases
44
What does aminoacyl tRNA synthetases do?
There are 20 and each one recognizes one amino acid and all the associated tRNAs.
45
Where is the amino acid on the tRNA?
Covalently linked to 3' end of tRNA
46
If we mutate the anticodon of tRNAPhe from GAA to UCG, what codon would it recognize?
CGA (anticodon recognizes reverse compliments)
47
What codons does tRNAPhe recognize?
UUC and UUU
48
What are the three distinct RNA Polymerases in Eukaryotes?
RNA Polymerase I, RNA Polymerase II, (mRNA), and RNA Polymerase III (We are only focusing on II)
49
What do eukaryotic chromosomes exist as?
Chromatin
50
Histone Acetyltransferases (HATs)
51
ATP- dependent chromatin remodeling enzymes
Slide nucleosomes around & opening chromatin structure, allowing access to specific sequences in DNA
52
How do we "switch on" a gene?
Adding acetyl groups to tail of histone
53
What do we have when a gene is "switched on?"
Active (open) chromatin, unmethylated cytosines, and acetylated histones
54
What do we have when a gene is "switched off?"
Silent (condensed) chromatin, methylated cytosines, and deacetylated histones; methylation controls gene expression (C G sequences)
55
What represses transcription?
Some histone modifications and DNA methylation, which is known as EPIGENETIC Info.
56
Is opening chromatin necessary for transcription?
It is necessary but not sufficient
57
How are genes in euchromatin actively transcribed?
RNA Polymerase actively recruited to promoter of gene
58
When are we unable to recruit RNA Polymerase?
When there is a closed chromatin structure
59
What is transcription stimulated by?
Activators
60
Transcriptional activators are...
Modular
61
Modular Interactions
Allow different transcription factors to recruit same proteins to different DNA sequences.
62
DNA Binding Domain
(Charger Block) Confers sequence-specific DNA binding
63
Activation Domain
(Cable/ Charger) Interacts with other proteins
64
Eukaryotes have a nucleus, which causes transcription and translation to be...
Physically separated
65
Where is the transcript processed before being translated?
In the nucleus
66
Eukaryotic genes contain non-coding regions which are called?
Introns
67
What do eukaryotic genes have on the 5' and 3' ends?
5' Cap and Poly-A tail
68
In Eukaryotes, what is removed from the transcript?
Introns
69
How are introns removed from the transcript?
RNA Splicing (Spliceosome) before it's exported to the cytoplasm for translation
70
What codes for proteins?
Mature mRNA (with introns removed)
71
Does mRNA have to contain every exon?
No, it can be included or excluded (Alternative Splicing)
72
What's the average length of a mature human mRNA?
2200 bp, so about 46Mb of genome directly codes for protein
73
Which of the following would have the most severe impact on the sequence, structure, and function of the protein?
Insertion of 2 nucleotides into the first exon
74
Why do we care which genes are expressed in a given cell type?
Every cell in your body has the same DNA
Not every cell in your body is identical
Differential gene expression underlies all differences between cells
75
How can we monitor gene expression?
For RNA, use complimentary DNA or RNA molecule
For Protein, fuse with GFP or use antibody
76
How can we amplify RNA?
Make a cDNA copy by reverse transcription (RT), then amplify PCR (RT-PCR)
77
How is gene expression monitored by RT-PCR?
Degrade DNA with DNase (bc we only want to amplify sequence of coding RNA)
Synthesize cDNA
Degrade RNA with RNase
PCR. First cycle generates double-stranded DNA. Second Cycle similar to first cycle of normal PCR reaction
78
What is the purpose of actin experiments?
To show that your RT-PCR works
To show that you started with the same amount of stuff in every sample
79
How else can we monitor gene expression?
Northern and Western Blot
80
What does the size indicate in gel electrophoresis?
If it's bigger, it will move slow. If it's smaller, it will move fast
81
How do we monitor gene expression by Northern Blot?
Separate RNA by gel electrophoresis
Transfer RNA to nitrocellulose membrane
Ad probe, wash off unbound probe, detect signal from bound probe
82
How do we monitor gene expression by Western Blot?
Using an antibody specific for protein of interest.
1. Separate proteins using gel electrophoresis
2. Protein blot on nitrocellulose (transfer proteins to membrane)
3. Label with specific antibody (recognizes protein we care about
4. Detect antibody and reveals protein of interest
83
Are DNA and RNA positively or negatively charged?
Negatively charged (Mass:Charge ratio is same)
84
Do proteins have a constant mass:charge ratio, like DNA and RNA?
No, because some proteins have a positive, neutral, or negative charge along with different structures
85
How can we make sure that proteins have a constant mass:charge ratio?
Boil then in the presence of SDS and BMercaptoethanol
SDS is amphipathic and charged; keeps protein unfolded and adds charge
B-ME reduces disulfide bonds
86
Why might we want to clone DNA?
Make even more DNA and 'store' it
Introduce specific mutations into gene and put it back into the organism it originally came from
Sequence DNA/ cDNA
Use cDNA to make recombinant protein
Note- molecular cloning= cutting & pasting DNA- NOT cloning organisms
87
What occurs when DNA is added to a plasmid?
Cut plasmids with restriction enzyme(s)
Cut DNA to be inserted to generate compatible ends
Use DNA ligase to seal up plasmic, which now contains the insert DNA
88
What happens to bacteria that does or does not have an antibiotic resistance gene on the plasmid?
Does- will survive
Does not- will die
89
We want to express and purify a human protein in bacteria... What should we use to make the DNA?
An RT-PCR product amplifying the cDNA corresponding to the mRNA
90
We want to express and purify a human protein in bacteria... What promoter should we use?
A prokaryotic promoter whose expression we can induce
91
What do we need to express a eukaryotic gene in a prokaryotic cell?
We need prokaryotic promoter and terminator sequences.
We must only insert the coding sequence of the gene.
We need an affinity tag to allow us to purify the protein.
92
Why do we need an affinity tag?
There are many other proteins in the cell, so we get rid of these by fractioning using the affinity tag.
93
How can we induce protein expression?
IPTG binds the lac repressor like allolactose but is not broken down by the enzymes in lac operon. It's a gratuitous inducer
94
How do engineering complex genomes allow reverse genetics?
Fuse gene X to the GFP gene: use GFP fluorescence to see how much of protein X is present, in what cells, and where in the cell.
Inactivate gene X by mutation and see what happens to organismal development.
Selectively activate or inactivate the expression of gene X in a fully grown organism.
95
How can we make a transgenic cell or animal?
Knock out or mutate YFG; replace 'your favorite gene' with antibiotic resistance cassette
96
What is a dangerous kind of break in DNA?
Double-strand break; cells must repair double-strand breaks to survive
97
What are the 2 major pathways of DNA repair?
Non-Homologous End Joining (NHEJ)- DNA ends are glued back together; sometimes sequences are gained or lost.
Homologous Recombination (HR)- DNA from the homologous chromosome is used as a template for repair
98
What does CRISPR/Cas9 do?
It's a bacterial immune system that can cut DNA at essentially any sequence, guided by ~20bp of sequence complementarity with an RNA molecule
