1
Q
Cost of making proteins per second
A
- 120 ATP per second
2
Q
Constitutive expression
A
- genes that are needed at all times, and this are expressed at all times.
3
Q
Where can regulation occur?
A
- at all stages of protein synthesis - transcription, translation, post-translation
4
Q
Where regulation mostly occurs
A
- transcription initiation
5
Q
Why does regulation most regularly occur at transcription initiation?
A
- because it saves the cell the most energy
6
Q
What is there a trade off between when regulation occurs?
A
- energy savings and speed at which the change is put into effect.
- so altering protein activity doesn’t get that ATP back, but change is immediately noticeable
7
Q
Operon
A
Multiple protein coding regions under the control of a single promoter - a polycistronic mRNA
8
Q
Things coded for by the lac operon and their regions
A
- B galactosidase - Lac Z
- Lac Permease - LacY
- Transacetylase - LacA
- repressor protein that binds to the promoter region by default - LacI
9
Q
Function of B-galactosidase
A
- breaks down lactose into galactose and glucose
10
Q
Function of Lac permease
A
- allows passing of lactose from outside the cell to inside the cell.
11
Q
Relationship of glucose levels and cAMP levels
A
- if Glucose levels decrease, cAMP levels increase
12
Q
Describe the process of how glucose impacts expression of the lac operon genes
A
- CAP binds to cAMP
- CAP-cAMP complex binds to CAP site
- this complex interacts with the carbon terminal domain of RNA polymerase
- this increases rate of transcription of lac genes
13
Q
Describe what would happen if the levels of glucose in a bacterial cell went up (more glucose available than lactose)
A
- levels of cAMP would decrease
- thus less cAMP would bind to the CAP
- less complex (CAP-cAMP) would bind to CAP site
- less interaction between C terminal domain of a subunit of RNA pol
- thus lowers rate of transcription of the lac operon genes, less of the enzymes involved in lactose metabolism would be made.
14
Q
Ratio of protein ducts of the lac operon from B-gal, permease, transacetylase
A
- 10:5:2
15
Q
How can ratio of proteins be adjusted from a single mRNA
A
- level of mRNA stability
- level of translation initiation
16
Q
How can level of mRNA stability be adjusted from a single mRNA?
A
- lac mRNA degraded from 3’ end
- because bacterial mRNAs have rapid turnover, it allows for a quick response to changes in environment
17
Q
How can a single mRNA be adjusted at the level of translation initiation
A
- ‘strength’ of shine dalgarno sequence (how close is it to the consensus sequence?) affects how much protein is made from that gene
- also affected by availability of shine dalgarno sequence - is it folded into secondary structure or blocked by proteins etc.
18
Q
How do we define the lack of operon specifically?
A
- is inducible - is off by default, but turned on when needed.
19
Q
How can we define the trp operon by default?
A
- repressible
- as it is on by default, can be turned off when tryptophan is abundant.
20
Q
Tryptophan operon structure
A
- Trp Repressor
- followed by promoter and operator region
- followed by Trp L (Leader) - attenuator present in TrpL
- Trp L is followed by 5 different genes - E,D,C,B,A
21
Q
Initially, why can’t the Trp repressor bind to its operator
A
- because it is an aporepressor - it can’t work without another molecule
22
Q
What does tryptophan act as
A
- a co-repressor
- when lots of tryptophan is made, it binds to the trp repressor, which allows it to then bind to the operator and prevent transcription by not allowing RNA pol to transcribe the gene
23
Q
What is another example for allosteric regulation or feedback inhibition of proteins
A
- regulation of PFK in glycolysis
24
Q
Role of the mediator
A
- binds in between gene specific TFs and the core promoter complex (TFs and Pol II)
25
What are the facts of gene/tissue specific factors? (Activators/repressors)
Affect ability of GTFs to bind core promoter sequence
• Stimulate RNA polymerase II to proceed to elongation
• Recruit chromatin-remodeling complexes
26
How do Upstream TFs and GTFs work
1. An activator binds to an enhancer
2. The activator enhances the ability of TFIID to bind to the TATA box
3. TFIID promotes the assembly of the pre-initiation complex
4. Mediator binds to Pre-initiation complex, but initiation doesn’t happen
5. Activator bind to a distant enhancer and a coactivator complex to interact with mediator - this interaction causes RNA polymerase to proceed to the elongation stage of transcription.
27
What else can activators recruit
ATP-dependent chromatin remodeling complexes
28
Activators recruit ATP-dependent chromatin remodeling
complexes which in turn:
change locations/spacing of nucleosomes
• evict histones from nucleosomes
• replace standard histones with histone variants
29
What even more things can activators do
Can recruit histone modifying enzymes
(Covalent, but reversible fashion, involving methylation, acetylation, and phosphorylation)
30
Methylation leads to
Silencing, making genes inaccessible
31
Acetylation leads to what
- enhancing/increasing accessibility for transcription
32
Regulation of ferretin production
- when iron levels are low, IRP (iron response protein) binds to IRE (iron response element) inhibiting translation
- when iron levels are high, IRP binds to iron, causing conformational change that releases it from the IRE, allowing translation to proceed.
33
Virus characteristics (4)
- intracellular parasites
- can persist on their own under appropriate conditions (NOT SURVIVE.)
- can only reproduce within a host cell using tools from host - eg tRNA, ATP etc has to use from host cell.
- simple structures, nuclei acid, protein coat and enzymes
34
Viruses genomes (6)
- few dozen to a few hundred genes
- either DNA/RNA
- can be single stranded or double stranded.
- can be linear or circular
- single copy or multiple copies
- segmented or non segmented
35
Most common type of virus
- linear double-stranded DNA virus
36
3 tasks of viral replication cycles
- get in, replicate genetic material, get out.
37
7 steps of viral replication cycles
- attachment to host cell surface receptors
- entry - transfer of nucleic acid into host cell surface
- early gene expression - production of enzymes needed to replicate viral nucleic acid (sometimes shut down transcription/replication of host cell DNA)
- replication of viral nucleic acid
- late gene expression: production of capsid proteins needed for assembly of viral particles + enzymes needed for exit.
- assembly of new virus particles from replicated nucleic acid and newly synthesized capsid proteins.
- release of new virus particles from host cell.
38
T4
- virulent phage
- lytic cycle,
39
Lambda
- temperate phage
- can do either a lytic pathway, or establish dormancy (lysogenic path)
40
T4 Virulent replication cycle (incomplete)
- viral DNA injected into cytoplasm
- transcription by HOST RNA polymerase
- this makes viral mRNA (early gene expression phase)
- then transcription and translation of ‘late viral proteins’ - eg capsid proteins
- viruses are assembled, and host cell is lysed.
41
Temperate phage cycle in extreme conditions (on the verge of death cell, or VERY metabolically favorable)
- the phage in this case chooses a lytic cycle
42
Temperate phage cycle in meh conditions (normal)
- chooses a lysogenic cycle.
43
Phage in lysogeny called what
- prophage
44
What is viral replicase?
What has to be done to use it?
- RNA dependent, RNA synthesizing enzyme
- has to be brought along, or have to make it using host cell resources.
45
3 flavors of RNA viruses
(+)sense strand RNA viruses
(-)sense strand RNA viruses
Retroviruses
46
(+)sense strand RNA viruses
- RNA can be translated directly like it was mRNA
47
(-) sense strand RNA viruses
- RNA is complementary to mRNA
48
Replicates can’t tell the difference between (-) sense and +sense RNA strands - true or false
True
49
Positive sense RNA replicative cycle
- host ribosome will translate +s RNAto make replicases
- replicases will then make copies of -sense RNA
- but since replicase doesn’t know the difference between - and + sense RNA, it will produce several copies of + sense RNA from the few copies of - sense RNA, which the viral particles can now take out of the cell to infect new cells.
50
Negative sense RNA virus replication cycle
- these viruses bring in the replicase with them when entering the host cell.
- so now, the replicase makes copies of the - sense RNA to + sense RNA
- this + sense RNA will then be translated by host ribosomes, making more replicase, and then the replicase will produce even more copies of - sense RNA, which the phage can now take with it outside the cell to infect other cells.
51
Retrovirus (HIV) replicative cycle
- binds to CD4 receptors, releases its viral RNA genome and proteins (integrate, protease, reverse transcriptase)
- viral RNA is converted to double stranded viral DNA by reverse transcriptase
- reverse transcriptase has RNAase activity, thus it breaks down the RNA in DNA-RNA hybrid during copying, and then uses the DNA strand left behind as a template to synthesize the remaining second DNA strand.
52
What does integrase have that helps it integrate into the host cell DNA
An NLS
53
Plasmids (3)
- extra chromosomal molecules of circular double stranded DNA
- carry from a few to a few dozen genes
- found in some yeast, plants, protozoans etc
- nonessential
54
How are plasmids useful to the host cell
- can carry genes that enable bacteria to live in inhospitable conditions
- can carry resistance factors that destroy/modify antibiotics
- can carry genes for transferring plasmid via horizontal transfer (conjugation)
55
Plasmids don’t have their own ori - true or false
False. They do
56
Can plasmids be present in multiple copies in cell
- yes - copy number varies
57
Restriction enzymes (3)
- made by bacteria to degrade Foreign DNA
- cut double-stranded DNA in very specific sites - Recognition sites.
- absent in eukaryotes, present in most bacteria.
58
If restriction enzymes recognize certain sequences in double stranded DNA and bacteria have double stranded genomes, why don’t restriction enzymes degrade host DNA?
- recognition sites are methylated in host, preventing the restriction enzyme from cutting it.
59
Type II restriction enzymes
- they recognize palindromic sequences
60
2 types of ends formed by restriction enzymes
- sticky ends (staggered)
- Blunt ends
61
Requirements of plasmid vector molecules
- has to have ori
- has to have restriction sites for enzyme used (helpful if it has sites for lots of restriction enzymes)
- has to have some sort of selective marker - need a way to tell which cell has vector, and which one doesn’t have vector.
- has to have some way to distinguish the vector aloe vs the vector + insert.
62
Reporter gene
- a gene that is a way to screen for the insert
- most of the time you want to knock out the reporter gene, so that you can tell the difference between which vector has the insert, and doesn’t.
- eg LacZ
63
Polylinker
Artificially synthesized Collection of unique restriction sites
Lies in uncovered region of promoter site for reporter gene.
Insert of DNA destroys promoter (insertion mutation) and the reporter
64
Transformation
- when bacteria are made transiently permeable to DNA in its surroundings
65
Transduction
- phage being used as a vector to introduce new DNA into a host cell
66
Why do we clone in the first place?
- amplification
67
What can be done with cloned DNA
- produce large amounts of protein
- produce large amounts of DNA just to study
- create a genomic library
68
Preparation of a genomic library
- cleave genomic DNA with restriction endonucleases
- insert into vector cut with same restriction enzyme as genomic DNA
- introduce recombinant DNA (the vectors) and transform into prokaryotic cell
69
Preparation of cDNA library
- harvest appropriate tissue, isolate processed mature mRNA
- use reverse transcriptase to make RNA-DNA hybrid
- degrade RNA, leaving single stranded DNA
- use DNA polymerase to make complimentary strand, now you’ve got cDNA
- insert that cDNA into vector and transform into host cells
- gene can be transcribed and translated, doesn’t need to be able to remove interns anymore
- promoters and SD sequences come from vector, not from the cDNA.
70
Why wouldn’t a genomic library work for producing protein from a eukaryotic gene?
INTRONS - bacteria doesn’t know how to remove introns
71
PCR process
- heat to 90 degrees to break H bonds between bases
- cool back to 60 degrees to allow primers to anneal
- heat back up to around 72-74 degrees to allow DNA polymerase to add bases.
72
Advantages of PCR
- fast, easy to use
- all in vitro, so fewer variables
73
Limitations of PCR
- flanking sequences around target must be known
- contaminating DNA CANNNOOOTTT be present - likely will get copies of that as well.
74
To check sequence of cloned fragments use…
Dideoxy sequencing
75
To check size of cloned fragments use…
- gel electrophoresis
76
Dideoxy sequencing
- primer added to target DNA (sequence to be analyzed)
- many copies of the recombinant vector, primer, dNTPs, ddNTPs, and DNA polymerase added, incubated to allow DNA synthesis
- separate newly made strands using electropheresis
- output comes out as the 5’ - 3’ NEWLY SEQUENCED DNA.
77
SgRNA
Guide RNA tat directs cas9 to specific sequences in the human genome.
Basically a tracrRNA linked artificially repeat-spacer that’s complimentary to the target gene
78
To fix the broken DNA, what will the cell do in terms of gene editing with crisper-cas9
- One system - accidentally introduced error when fixing the break and knocks out the gene
- the second uses the other copy of that DNA as a template to fix the broken copy (Homology directed repair)
79
If the original disease-causing mutation in the DNA leads to an overactive protein, ———- is a good strategy
- cutting stuff
