Unit 2 Deck Flashcards
(331 cards)
1
Q
What types of genomes can microbes have?
A
one or more circular or linear chromosomes and plasmids
2
Q
What is a structural gene?
A
produces a functional RNA that normally encodes for a protein
3
Q
What is a DNA control sequence?
A
regulates expression of a structural gene
- doesn’t encode for a RNA or protein
Ex: promoters and regulatory protein binding sites
4
Q
What are the 5 ways that genome compaction is achieved?
A
1) Polymer Dynamics
2) cellular confinement
3) molecular crowding
4) Supercoiling (topoisomerase)
5) Nucleoid-Associated Proteins (NAPs) / Structural Maintenance of Chromosomes (SMC) proteins
5
Q
What is the role of polymer dynamics in genome compaction in microbes?
A
chromosomes should spontaneously shape into a globule
- this leads to a 100-fold compaction
- this compaction is due to Brownian motion of chromosomes
6
Q
What is the role of molecular confinement in genome compaction?
A
The cell of microbes is highly compact because of all the macromolecular machinery confined in the cytoplasm.
- this high density of cytoplasm forces the genome to be pushed and compacted
7
Q
What is the role of cellular confinement in genome compaction?
A
The cell walls of microbes can be relatively strong and provide a border to hold the genome internally
8
Q
What is the role of NAPs in genome compaction?
A
Bacteria pack their DNA into nucleoids. Loops of nucleoids are anchored in the center by NAPs.`
9
Q
What are nucleoids?
A
bacterial DNA packed into a series of loops or domains
10
Q
What is the role of supercoiling in genome compaction?
A
allows for the twisting of DNA strands to fit it all in better
11
Q
What are topoisomerase?
A
enzymes that twist DNA into supercoils or relieve supercoils
- the enzyme cleaves both strands of DNA at one site
- the intact strands are pulled between the ends of the cut strands
free ends are reconnected by the enzymes
12
Q
What is negative supercoiling?
A
when the DNA strands are twisted in the opposite directions of their grooves
13
Q
What is positive supercoiling?
A
when the DNA strands are twisted the same direction as the grooves in the DNA strands
14
Q
Is DNA mostly negatively or positively supercoiled?
A
negatively
15
Q
What are type 2 topoisomerases?
A
Enzymes that form negative coils/ relieve positive coils
16
Q
What are type 1 topoisomerases?
A
Enzymes that form positive coils/ relieve negative supercoils
17
Q
What is GyrAB?
A
a type 2 topoisomerase
- GyrA breaks the double stranded DNA
-GyrB grabs the strands prior to breakage
18
Q
What are SMCs?
A
proteins that loop around the strands to cause looping of the DNA
19
Q
What are some actions of NAPs?
A
- bending
- Bridging
- Coating
- condensing into a globule
20
Q
What are some actions of SMCs?
A
- Zipping
- loop of extrusion
- cohesion
21
Q
What are plasmids?
A
- generally smaller than chromosomes
- found in all 3 domains of life
- usually circular, some can be linear
- require host machinery for replication
22
Q
what 2 kinds of extragenomic DNA molecules can interact with bacterial genomes?
A
1) plasmids
2) genomes of bacteriophages
23
Q
What are advantages to having plasmids in cells?
A
1) carry genes fro antibiotic and toxin resistance
2) carry genes for pathogenesis
3) symbiosis (gene providing other growth advantages)
24
Q
What is the genome like in the Borrelia Burgdoferi prokaryote?
A
22 linear and circular plasmids and chromosomes
25
What is the size range for bacterial and archaeal chromosomes?
130-14000 kilobase pairs
26
What is the genome size range in eukaryotic chromosomes?
2900kb to over 100million kb
27
What is noncoding DNA?
non-structural gene
28
What percent of eukaryotic genomes are noncoding?
>90%
29
What percentage of prokaryotic genes are noncoding?
<15%
30
What is it important for prokaryotes to have less noncoding genes?
for replication efficiency to compete and be able to pump out their most essential genes ASAP in different working conditions
31
What is an operon?
unit of other genes that operate and produce together
32
What does monocystronic mean?
RNA is produced from a single gene
33
What does Polycistronic mean?
RNA is produced from an operon
34
What does semiconservative mean in DNA replication?
that both strands are used as template strands for copying; end product has one parental strand and one new daughter strand
35
How many replisomes are used in DNA replication for prokaryotes?
2 - 1 for each of the replication forks in the replication bubble
36
How many DNA polymerases are used in DNA replication?
4: 2 for each of the lagging strands + 2 for each of the leading strands of the replication bubble
37
What is dnaA?
the initiator protein
- binds to DNA and melts apart OriC to form replication bubble
38
What is dnaB?
-helicase
- unwinds DNA upstream of polymerase
- gives polymerase access to the single parental strands
39
what is DnaC?
-loads DnaB helicase ring onto the ss Parental DNA at the melted OriC
- basically holds helices ring into place
40
What is DNA Primase?
synthesized RNA primers for the beginning of the leading strands and each of the Okazaki fragments
41
What is DNA Pol 3?
the is the major DNA replicator enzyme
42
What is the sliding clamp?
- Prevents DNA pol 3 from falling off of the template strand
43
What is the clamp loader?
Carries 2 DNA pol 3 enzymes to make up the replisomes and loads the sliding clamp
44
What is DNA Pol 1?
Replaces the RNA primer with DNA
45
What is DNA Gyrase?
Relieves positive supercoiling upstream of replisome
46
What are SSBs?
single-stranded binding proteins that protect ssDNA that has yet to be replicated from being degraded.
47
In E.coli, what triggers the initiation of replication?
- DnaA accumulates during cell growth, then gets to a high enough concentration to trigger DNA replication
48
What part of the DNA does DnaA-ATP complexes bind to?
9-bp repeats within the Origin
49
What does DnaA binding do to the shape of the parental DNA strands?
-basically melts it open/pulls it apart by looping/bending the DNA
50
Where does strand separation from the DnaA occur?
At the 13-mer repeats
51
What is Tus (Terminus utilization substance) ?
-binds to ter sites and acts as a counter-helicase
52
What do ter sites do?
multiple ter sites ensure that polymerases do not escape and continue replicating simultaneously
53
Because there are so many ter sites, how is it decided which ter site is used?
Depends on whether replication of one fork lags behind the replication of the other = so where the ends meet.
54
What is a Catenane?
A pair of linked rings of sister chromosomes after replication is complete
55
How are catenanes unlinked?
Topo |V recognizes Dif sites on the sister chromosomes and cuts the double-stranded DNA, then rejoins them once one chromosome has passed through the other.
56
What are the two ways plasmids can replicate?
1) bidirectional
2) rolling-circle replication
- some plasmids use others, but this is rare
57
What is bidirectional replication?
Starts at a single origin and occurs in two directions simultaneously
58
What is rolling-circle replication?
Starts at a single origin and moves in only one direction.
59
True or false: replicated genomes must segregate prior to cell division.
True
60
What does high-copy-number mean?
Plasmids or chromosomes can segregate randomly to daughter cells with high number of plasmids
61
Low-copy-number definition:
Plasmids or chromosome must be actively segregated equally to daughter cells (partioning)
62
What are plasmids and chromosomes segregated by ?
- ParA ATPase
- ParB protein
63
What is ParA ATPase?
- forms dynamic protein waves in response to its stimulator protein, parB
- binds to nucleoid
- segregate and position bacterial chromosomes
64
What does ParB do?
Binds to cargo on the centromere
65
A high-copy plasmid is inherited by which mechanism?
Passive diffusion
66
Do parA proteins create high or low-copy plasmids?
Low-copy
67
What are some common features shared between eukaryotic microbes (protists) and prokaryote chromosomes ?
Both have double stranded DNA that is replicated bidirectionally
68
What are some big differences between eukaryotic and prokaryotic chromosomes?
- eukaryotic are only linear with telomeres
- eukaryotic are contained within a nucleus
- eukaryotic replication involves mitosis
69
What is telomerase?
Eukaryotic chromosomes require a reverse transcriptase to replicate their linear ends
70
What strand is used to carry out transcription ?
DNA template strand
71
In bacteria, what is the RNA polymerase holoenzyme made of?
- core polymerase: a1, a2, B, B’ (required for the initiation and elongation phase
- sigma factor (required only for the initiation phase)
72
What is the B’ subunit and its role?
- houses the catalytic site for RNA synthesis and binds to
1) rNTP substrate
2) DNA substrate
3) RNA product
73
What do sigma factors do?
- first binds to the polymerase, then helps the polymerase bind and detect a promoter, signaling the beginning of an open reading frame
- separates DNA strands to make a transcription bubble in preparation for RNA synthesis
74
True or false: different sigma factors recognize a different promoter
True
75
True or false: every cell has a common, housekeeping sigma factor
True
76
What is the housekeeping sigma factor in e.coli?
Sigma-70
77
What does the sigma-70 factor recognize?
Consensus sequences -10 and -35 positions relative to the rna transcript (+1)
-10: TATAAT
-35: TTGACAT
78
What are the 3 phases of transcription?
1) initiation
2) elongation
3) termination
79
What happens generally during the initiation phase of transcription?
RNA pol holoenzyme binds to the promoter and melts the helix to create transcription bubble
- synthesis of first RNA nucleotide
80
What happens generally during the elongation phase of transcription?
RNA chain is extended using the 3’ OH
81
What happens generally during the termination phase of transcription?
RNA Pol detaches from the DNA after the transcript is made
82
What are the steps of the transcription initiation phase?
1) RNA pol forms a loosely bound, closed complex with DNA by binding to the promoter sequence
2) closed complex becomes an open complex through unwinding of one helical turn
3) RNA pol in open complex becomes tightly bound to DNA, beginning transcription
4) RNA pol retains sigma factor until ~9 bps have been joined/ created, then it dissociates
83
What are the characteristics of transcription elongation?
- the sequential addition of ribonucleotides from nucleoside triphosphates
- RNA pol continues to move along the template strand, synthesizing RNA at 45bases per second
-the unwinding of DNA ahead of the moving complex forms a 17-bp transcription bubble
-positive super coils ahead are removed by DNA topoisomerases
84
What are the 2 types of transcription termination signals?
1) rho-dependent
2) rho-independent
85
What is Rho-dependent termination?
Relies on the rho protein and a strong pause site at the 3’ end of the mRNA transcript
86
What is rho-independent termination?
Requires a GC-rich region of RNA and 4-8 consecutive uridine residues
87
What are the steps of rho-dependent termination?
1) rho binds to the c-rich region
2) RNA transcript is threaded through the rho hexamer, pulling the rho towards the RNA polymerase
3) contact between rho and the RNA pol causes termination
88
How does termination work in rho-independent cases?
NusA protein forms a hairpin of the mRNA transcript until the rna pol comes in contact, causing termination
89
What is rifamycin?
A transcription antibody that:
- selectively binds to the beta subunit of bacterial rna pol
- blocks rna exit to inhibit further transcription initiation
- polymerase can still bind to the promoter
90
What is the average half-life for mRNA
3-5 minutes
91
Why are tRNAs and rRNAs much more stable?
Have modified bases that are less susceptible to RNase digestion
92
How many possible codons are there?
64
93
How many codons are stop codons?
3
94
What are the 3 functional regions of tRNA molecules?
1) anticodon
2) 3’ acceptor end
3) sidle loops
95
What does the anticodon region of tRNA do?
Forms hydrogen bonds with the mRNA codon to specify an amino acid
96
What does the 3’ acceptor end do on tRNA?
Binds to the amino acid
97
What does the sidle loops on tRNA do?
Is recognized by enzymes that match tRNA to the proper amino acid
98
True or false: each tRNA must be charged with proper amino acid before it encounters a ribosome
True
99
What charges tRNAs with amino acids?
Aminoacyl-tRNA synthetases
100
True or false: there are 20 aminoacyl-tRNA synthetases in each cell. One per amino acid type
True
101
What are the 2 subunits of the ribosome in prokaryotes?
30S(small subunit) and 50S(large subunit)
-combined to make the 70S ribosome
102
What bonds are between amino acids?
Peptide bonds
103
What enzyme forms peptide bonds between amino acids?
Peptidyltransferase
104
What is peptidyltransferase?
A ribozyme: an RNA molecule that carries out catalytic activity
- part of 23S rRNA of the 50S large ribosomal subunit
105
What is a benefit of rRNA between all organisms?
Differences in rRNA sequences increase when there is more evolutionary distance between organisms
- used as a molecular clock
106
What are the 3 sites of the 70S ribosome?
1) A (acceptor) site
2) P (peptidyl-tRNA) site
3) E (exit) site
107
What does the acceptor site in the ribosome do?
Binds to incoming aminoacyl-tRNA
108
What does the P site in the ribosome do?
Harbors the tRNA with the growing polypeptide chain
109
What does the E site do in the ribosome?
Binds a tRNA recently stripped of its polypeptide or amino acid
110
how do ribosomes find the right reading frame?
- with 3 potential reading frames, the ribosome utilizes the Shine-dalgarno sequence (Ribosome Binding Site or RBS)
111
How does the shine-dalgarno sequence allow for the right reading frame by the ribosome?
It is a purine-rich consensus sequence (AGGAGGU) located 3-9bases upstream of the start codon
- complementary to the 3’ sequence of 16S rRNA of the 30S subunit
- the N-formyl-methionyl-tRNA pairs with the start codon in the P site
112
What is a polysome?
Multiple ribosomes can jump onto the RBS, one after another, allowing many ribosomes to move along the mRNA, all translating simultaneously
- ribosomes are closely packed
- protects mRNA from RNases
- speeds up the production of protein from a single mRNA molecule
113
What is the energy form used in translation/protein synthesis?
GTP
114
What are the 3 stages of protein synthesis?
1) initiation
2) elongation
3) termination
115
What happens during the initiation phase of protein synthesis?
Brings two ribosomal subunits together, puttin the first amino acid in position
- n-formylmethionyl-tRNA is the first to bind to the ribosome (by the 30S subunit)
- this is the only tRNA to bind directly to the P site
116
What happens during the elongation phase of protein synthesis?
1) an aminoacyl-tRNA binds to the A site
2) a peptide bond forms between the amino acids in the A and P sites
3) the bonding which uses GTP cause a message shift by 1 codon
4) repeat
117
What happens during the termination phase of protein synthesis?
Releases the completed protein and recycles ribosomal subunits
118
What does streptomycin do?
An antibiotic that binds to the A site and allows for improper codon-anticodon matchups that result in the wrong protein sequence
119
What does tetracycline do?
It’s a protein synthesis antibiotic that binds near the A site to prevent aminoacyl-tRNAs from binding to the A site
120
What does chloramphenicol do?
It’s a protein synthesis antibiotic that binds to the P site and inhibits peptide bond formation
121
What does erythromycin do?
A protein synthesis antibiotic that binds to the peptidyltransferase site and alters peptide bond formation
122
What can proteolytic cleavages do to a protein
May activate or inactivate a protein
123
What amino acid may be removed after protein synthesis
The starting amino acid, methionine.
124
What are Dnak chaperones?
Clamps down on polypeptide to assist folding
125
What are GroEL and GroES chaperones?
Protein to be folded fits inside a stacked ring with a hollow center
- uses ATP to release the protein
126
What are degrons?
Degradation signals on proteins
127
What is the N-terminal rule?
The n-terminal amino acid of a protein directly correlates with its stability
128
How do proteases recognize misfiled proteins?
Due to the hydrophobic regions being exposed on the protein
129
What are Lon and ClpP in bacteria?
ATP-dependent endoproteases
130
What are proteosomes?
Protein-degrading machines found in eukaryotes and archaea
131
True or false: Clp protease is not similar to the archaea and eukaryotic proteosomes
False, they are similar
132
What is the fold-or-destroy triage system?
The chaperone-based remolding pathway or degradation pathway used until the protein is repaired or destroyed
- critical for a microbe to survive environmental changes and stress.
133
What are proteins meant for the cell membrane tagged with?
A hydrophobic N-terminal signal sequence of 15-30 amino acids
- bound by the SRP
- undergo cotranslational insertion into the inner membrane/ complete export from the cell
134
What is the SecA-dependent general secretion pathway?
The process in which proteins are delivered to the periplasm
135
What are the steps to the SecA-dependent pathway?
1) peptide is completely translated in the cytoplasm
2) the complete protein is captured by the SecB protein
3) SecB unfolds and delivers the protein to SecA, which is associated with the SecYG translocon.
136
What are the 2 types of gene transfer?
1) vertical transmission
2) Horizontal transmission
137
What is vertical transmission?
Passing DNA from a parent to a child
138
What is horizontal gene transfer?
Transfer or small pieces of DNA from one cell to another in the same cell population.
139
What is the mosaic nature of microbial genomes?
The result of horizontal gene transfer, recombination, and a variety of mutagenic and DNA repair strategies
140
What percent of proteins in some archaeal species are also found in bacteria and eukaryotes?
40%
141
What is a benefit to gene transfer?
Microbes can acquire genes that might be useful against environmental changes
142
What is transformation?
The process of importing free DNA into bacterial cells
- a mechanism of horizontal gene transfer
143
What is required for cells to undergo transformation ?
They need to be competent
144
What do naturally competent bacteria have that lab made competent cells don’t?
Specific protein complexes called transformasomes
145
How do you make cells artificially competent?
Perturbing the membrane by chemical (CaCl2) or electrical (electroporation) methods
146
Why do some bacteria undergo natural transformation?
1) use DNA as a food source
2) use specific DNA sequences to repair damaged genomes
3) acquire new genes through horizontal gene transfer
147
How do transformasome complexes form?
Across the cell membrane in response to competence factors
148
What are transformasomes composed of?
1) a protein fishing line that captures extra cellular DNA
2) a transmembrane pore
149
What are the steps of transformation?
1) pill act like microscopic harpooners that cast their line through the pores in the cell membrane
2) spear a stray piece of DNA at the very tip
3) the pili then reel the DNA into the bacterial cell through their membrane
150
What is conjugation?
The transfer of DNA from one bacterium to another following cell to cell contact
- typically initiated by special pills protruding from the donor cell
- occurs in bacteria and archaea
- tip of pious attaches to the receptor on the recipient cell and pulls them closer together
151
What is required for conjugation to occur?
At least one of the cells need to have the F factor (fertility factor)
152
What is the F factor?
A plasmid
153
What are the steps of conjugation?
1) f+ plasmid donor extends sex pilus and makes contact with f- recipient
2) contraction of the pilus pulls the two cells together. Relaxase unwinds the DNA near the oriT
3) one strand of the F factor is nicked at the nic site on the oriT region. Another relaxase attaches to the 5’ end of the snipped strand and transfers it to the other cell
154
What is a Hfr strain?
F plasmid integration into the host chromosome makes a high frequency recombination strain
155
What is recombination in Hfr strains?
Donor dna fragment replaces recipient dna fragment or degrades
- allowing the mapping of relatives according to time of transfer
156
True or false: gene transfer only happens within microbes of the same domain.
False, some bacteria can transfer genes to different domains
157
What is agrobacterium tumefaciens?
A bacteria that causes gall disease
- contains a tumor inducing plasmid that can be transferred via conjugation to plant cells
- used as a tool for plant breeding and engineering
158
What is phage transduction?
The process in which bacteriophages carry host DNA from one cell to another
- occurs as an offshoot of a phage life cycle
159
What are the 2 types of phage transduction?
1) general transduction
2) specialized transduction
160
What is generalized transduction?
Can transfer any gene from a donor to a recipient cell
- phage have a difficult time distinguishing viral versus bacterial genomes
- phages can pack up either host or phage dna and spread it
161
What is specialized transduction?
Can transfer only a few closely linked genes between cells
162
What is the impacts of biofilms on HGT?
Make it easier
163
What is a characteristic of our gut microbes?
Serve as reservoirs of antibiotic resistance gene pools
164
What happens to a new piece of DNA in a cell?
- DNA is degraded by nucleases
- some plasmids can stabilize replicate and coexist with chromosomes
- the dna can get incorporated into host chromosomes via recombination
165
What are the two types of dna recombination?
1) generalized recombination
2) site-specific recombination
166
What is generalized recombination?
Requires two recombining molecules with a considerable stretch of homologous dna sequences
167
What is site-specific recombination?
Requires very little sequence homologous between the recombining dna molecules
- requires a short sequence recognized by recombination enzyme
168
What are the 3 functions for generalized recombination?
1) likely first evolved as an internal method of repair to fix mutations or restart stalled replication forks
2) cells with damaged chromosomes use dna donated by others of the same species to repair damaged genes
3) a self-improve,ent program that samples genes from other organisms for an ability to enhance competitive fitness
169
What is RecA?
Central protein in generalized recombination
- scans dna molecules for homology and aligns homologous regions to form a triplex dna molecule, or synapse
170
What is RecA role in deinococcus radiodurans?
To reconstruct it’s chromosome after extreme radiation damage
171
What is a transition mutation?
A purine base to another purine, and the same for 2 pyramidine bases
172
What is a transversiin mutation?
From purine base to a pyramidine base and vice versa
173
What is an inversion mutation?
Dna sequence is flipped in orientation
174
What is a reversion mutation?
The dna base or sequence mutates to the first original sequence
175
What is a silent mutation?
A mutation that doesn’t change the amino acid sequence
176
What is a missense mutation?
Changes the amino acid sequence
177
What is a nonsense mutation?
Changes the amino acid sequence early to a stop codon
178
What are types of lesions that can lead to mutation?
- base damage
- apurinic/apyrimidic sites
- dna strand breaks (sing,e and double), which happen from removal of base from sugar group
- cross links ( inter strand, intrastrand, and protein to dna)
- ribonucleotide misincorporations and mispaired bases (mismatches)
179
What are endogenous sources of dna damage?
Results from byproducts of cellular metabolism
- ROS and RNS can chemically alter dna
180
What are exogenous sources of dna damage?
Chemical or radiation damage like uv rays, gamma rays, and secondary metabolites
181
How often do replication errors (mismatches) occur?
Once every 16 rounds of replication = 1 error every 60 million bases replicated
- 1 error per 1000 rounds or replication gets missed with mismatch repair
182
What are the two types of DNA repair?
1) error-proof repair pathways
2) error-prone repair pathways
183
What is error-proof repair pathway?
Prevents mutations
- methyl mismatch repair, photoreactivation, nucleotide excision repair, base excision repair, and recombinational repair
184
What is the error-prone repair pathway?
Risk inducing mutations
- operates only when damage is so severe that the cell has no other options but to mutate or die
- mainly tries to keep dna intact while sacrificing accuracy
185
What is recombinational repair?
- takes place at the replication fork
- a single-stranded segment of undamaged daughter strand is used to replace a gal in damaged daughter strand
- carried out by RecA
- works on any damage that causes gaps during replication
186
What is the SOS repair?
Induced by extensive dna damage that destabilizes the genome
- many dna repair enzymes are expressed
- two main sloppy pol lack proofreading activity
- RecA coprotease activity stimulates auto digestion of the LexA repress or
- lexA repress or degrades, allowing for the expression if SOS genes
187
What are the genes in the SOS system?
- SulA: inhibitor of cell division
- UmuDC: error prone polymerase
- UvrA: part of the NER pathway
188
What are transposable elements?
Move from one dna molecule to another
- exists in all life forms
- can hop within and betweeen chromosomes
- cannot exist outside of a large dna. Olecule
189
What is the insertion sequence?
Transposable elements contain a transposable gene that is flanked by short inverted repeat sequences
- they are targets for the transposable enzyme
- a sequence that is excised and moved to the target site.
190
What are the two transpose mechanisms?
1) replicative
2) nonreplicative
191
What are complex transposons?
Transposable elements that carry other genes in addition to those required for transposition
192
What is a composite transposon?
Consists of two insertion sequences that flank a drug resistance gene or one or more catabolic genes
- carry out nonreplicative transposition (cut and paste)
193
What can transposon mutagenesis be used for?
To identify a genes function
- did a transposon particular gene decrease fitness?
194
What revealed the genes that are essential for carbon fixation?
Transposon mutagenesis
195
What are the ways gene expression is controlled?
1) Transcriptional level: increase/decrease gene expression
2) translational level: translate or degrade mRNA
3) post-translational: activate/degrade/sequester regulatory proteins
196
What are the 5 levels of gene expression control?
1) alteration of dna sequence: flipping a dna segment
2) control of transcription: repressors, activators, sigma factors, srnas
3) control of mRNA stability: activity, srnas
4) translational control: hiding rbs sites or other mRNA sequences
5) post-translational control: cleavage, phosphorylation, methylation, etc.
197
What is the least reversible and most drastic level of gene expression control?
At the dna sequence level
198
What gene expression control level is the most rapid and reversible?
The protein level
199
What are regulatory proteins?
- help a cell sense internal changes and alter its gene expression to match
- bind to specific ligands
- different regulatory proteins bind to different ligands
200
What are the 2 forms of regulatory proteins?
1) repressors
2) activators
201
What are repressors?
Bind to regulatory sequences in dna to prevent transcription of target genes
- some must first bind to a small ligand
202
What are activators?
Bind to regulatory sequences in dna and stimulate transcription of target genes
- most must bind to a small ligand first
203
What is the process of repressors without a corepressor ligand?
- repressors bind to dna target sequence and reduces expression of genes
- inducer ( ligand) binds to the dna bound repressor and causes it to release from the dna = expression of target genes
204
What is the process of repressors with corepressor ligands?
- repressor binds to ligand and binds to the dna, reducing expression of target genes
- when ligand leaves repressor, the repressor leaves the dna = expression of target gene
205
What is the process of activators?
Activators bind to ligand and binds/ activates the target gene
- when ligands pop off, the activators leave = no expression of target gene
206
What are the two components in transduction systems that sense the external environment?
1) sensor kinase
2) response regulator
207
What is a sensor kinase?
- found in the cell membrane
- binds to environmental signals
- activates itself via phosphorylation
208
What are response regulators?
- found in the cytoplasm
- takes the phosphate from the sensor
- binds to chromosome to alter gene expression via transcription rate.
209
Are enzymes that metabolize lactose inducible or constitutive?
Inducible
210
Are enzymes that metabolize glucose constitutive or inducible?
Constitutive
211
What is the LacZYA operon?
An operon that controls the catabolism of lactose
Without it, the energy from lactose is unavailable to the cell
212
True or false: the lacI and LacZYA operons share the same promoter.
False: lacI has a different promoter on the same operon from LacZYA
213
What happens when there is an absence of lactose?
- LacI binds as a tetramer to the operator region
- represses the lac operon by preventing the open complex formation by RNA polymerase
214
What happens when lactose is present?
LacZ at low levels cleaves and rearranges lactose to make the inducer allolactose
- allolactose then binds to LacI and reduces its affinity to the operator, allowing induction of the operon
215
When does cAMP accumulate in a cell
When the cell is starved for carbon
216
What governs the level of LacZYA transcription?
Cyclic AMP
217
What is required for the maximum expression of the lac operon?
1) cAMP
2) cAMP receptor protein (CRP)
218
What does the cAMP-CRP complex do?
- binds to the promoter and interacts with the RNA polymerase to increase the rate of transcription initiation
219
What is catabolism repression?
An operon enabling the catabolism of one nutrient is repressed by the presence of a more favorable.nutrient (normally glucose)
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What is diauxic growth?
The biphasic curve of a culture growing on two carbon sources
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How does glucose presence affect the lac operon?
- glucose transport by the phosphotransferase system causes catabolite repression by inhibiting LacY permeate activity, which allows for lactose transport into the cell
- this is inducer expression
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What are dual regulatory proteins?
Perform as both activators and repressors under different conditions
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What is attenuation?
Idling ribosomes that send chemical signals that affect the expression of many genes and operons
- coupling of transcription and translation can cause this
- a regulatory mechanism in which translation of a leader peptide affects transcription of a downstream structural gene
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What does the arabinose operon do?
Converts arabinose into xylulose-P: an intermediate Jo a bio synthetic pathway
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What is AraC?
The major regulator of the arabinose operon.
- a dimer that can repress or activate gene expression, depending on whether arabinose is available
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What happens when arabinose is absent?
The AraC dimer is elongated = it represses the expression of genes that break down arabinose
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What happens when arabinose is present?
The AraC dimer is compact = it stimulates the binding of RNA polymerase to transcribe the genes
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Is repressing anabolic pathways similar or different to repressing catabolic systems?
Different
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What are aporepressors?
Binds to a corepressor, which is the end product of the biosynthetic pathway
- complex binds to the operator sequence upstream of target gene or operon
- blocks RNApol, and so transcription is off
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What happens when trp levels exceed cellular needs?
Excess trp binds to the inactive TrpR aporepressor to block RNA polymerase
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What is a characteristic of the attenuator region of the trp operon?
It has two trp codons and is capable of forming stem-loop structures
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What is a quicker response to decreasing expression levels: attenuation or repression?
Attenuation because it halts transcription in progress
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What is the stringent response?
Produce fewer ribosomes by lesser transcription of rRNA genes and other genes because of idling ribosomes (ribosomes with no work) synthesizing the signal molecule ppGpp
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What are the 2 main types of untranslated RNA?
1) small RNA (srna)
2) cis-antisense RNA l as RNA)
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What is the function of small RNA and asRNA?
- binding to complementary sequences of target transcripts to stimulate or prevent translation
- interacting w/ proteins
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What is the origin of sRNA?
Encoded by regions of intergenic spaces
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What is the origin of asRNA?
Transcribed from the DNA strand opposite of the mRNA encoding strand template strand
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What are the mechanisms of RNA function?
1) inhibits translation by binding to the RBS
2) promotes mRNA degradation by binding to the mRNA
3) promotes processing by stabilizing mRNA transcripts
4) activates translation by binding to the mRNA and making the RBS site available
5) prevents mRNA degradation by binding to cleavage sites
6) sequesters regulatory proteins by binding to them
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What is the average length of small rnas?
100-200 nucleotides
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What are the benefits of sRNAs?
- energetically inexpensive
- no protein synthesis required like regulatory proteins
- sRNAs diffuse rapidly
- sRNAs typically act on preexisting messages
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What are ribozymes?
3D folded RNA molecules that catalyze enzymatic reactions
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What are riboswitches?
3D folded RNA molecules that can control gene expression
- found at a 5' end of an mRNA molecules upstream of a coding sequence
- has two alternative stem loop structures that switch back and forth in response to a target metabolite
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What is affected by a riboswitch?
translation of the message or the transcription of the gene
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What does a riboswitch's role in translational control look like?
- forms a big stem loop with a hole in the center for a ligand to bind, stabilizing this position.
- here, the RBS is not available to translation machinery
- in the other form, the RBS site is released and can be used to continue translation
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What is a riboswitch's role in transcriptional control?
- it forms a big stem loop with a ligand in the center. This form prevents the transcription termination stem loop from forming
- in the alternative form, the termination stem loop is allowed to form
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What is the heat shock sigma factor in e.coli?
- sigma H
- regulated at translational and degradation levels
- levels of sigma H is regulated by two temperature dependent mechanisms
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What are some characteristics of cells that are subjected to heat?
they express heat-shock proteins like chaperones that refold damaged proteins and those that affect DNA and membrane integrity.
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What are sigma H two temperature dependent mechanism?
1) RNA thermometers controls access to RBS of sigma H
2) chaperones direct sigma H degradation
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What are the general steps of E.Coli Heat-shock response?
1) rpoH is transcribed, but partially folded so the RBS is hidden = very little sigma H is made
2) chaperones shunt the little amount of sigma H to degradation
3) when heated, the mRNA melts, revealing the RBS = sigma H is made
4) the chaperones that would typically denature and degrade sigma H are unavailable as they are trying to protect other denatured proteins
5) sigma H can now drive expression of heat-shock genes
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What does the inductions of quorum-sensing genes require?
accumulation of a secreted autoinducer
- at certain extracellular concentrations the autoinducer will reenter cells
-
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What does the autoinducer do once it has reentered the cells?
- binds to a regulatory molecule (in vibrio Fischer it's LuxR)
- the LuxR-autoinducer complex then activates transcription of the luciferase target genes that confer bioluminescence
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How do pathogens use quorum-sensing?
- to control virulence genes
- virulences proteins like proteases and other degradative enzymes
- not produced until cell density is fairly high and can overwhelm the host
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Can quorum-sensing be used in interspecies communication?
- yes
- tells bacteria if they are the majority (put up a fight) or the minority (lay low)
- can use antibiotics to prevent them from talking and growing
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What is chemotaxis?
and integrated gene circuit that has many bacterial genes regulated and switches
-ability of an organism to sense chemical gradients and modify its motility in response
- swim toward higher concentrations favorable conditions and away from unfavorable conditions
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What are the two types of flagellar movement?
- counter-clockwise = smooth, directional swimming
- clockwise = tumbling, everywhere nonspecific
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What happens as bacteria continue to move towards an attractant?
it runs longer which = directions, CCW movement
- when there is a decrease in attractant, bacteria change to CW swimming = tumbling
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How does different concentration levels of attractant flip the rotor on flagella?
- random movement towards an attractant causes a drop in CheY-P levels = CCW rotation and swimming
- when CheY is phosphorylated by CheA kinase, the increase in CheY-P cause CW rotation and tumbling
-CheZ continues to dephosphorylate CheY
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What are Methyl-accepting chemotaxis proteins (MCPs)?
-clusters at cell poles and binds to chemoattractants
- the cytoplasmic domains of MCPs bind to the CheA kinase and controls its activity
- initiates a series of events lowering CheY-P levels
-methylation desensitizes and demethylation sensitizes MCPs (reversible)
259
What are the 3 main components of the coronavirus?
- protein
- fats
- nucleic acid
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What is the coronavirus genome?
single stranded RNA
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what is the nucleoprotein in coronavirus?
a proteins that protects the ssRNA viral genome from degradation
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What is the viral envelope in coronavirus?
a protective lipid bilayer stolen from the last cell it infected
- allows the virus to hide and become deposited into the next cell it infects
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What is the spike proteins in coronavirus?
binds to the ACE-2 entry receptor on the surface of the host cell
- it's an S protein trimer
- targets most vaccines and neutralizing antibodies made by your body
- promotes fusion between host cell plasma membrane and viral envelope
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What are the series of events that drive viral entry into the host cell?
1) virus randomly comes in contact with the surface of a potential host cell
2) spike protein binds to the ACE-2 receptor on host cell
3) lid of spike protein is removed by a protease on the surface of the host cell
4) fusion machinery unfolds and inserts into the cell membrane
5) hydrophobic portion of spike inserts into the cellular membrane
6) as the spike protein refolds, it pulls membranes together promoting fusion
7) a pore is created that allows viral genome entry into the host cell cytoplasm
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What is the alternative mode of entry for the coronavirus?
the viral particle can trigger endocytosis too
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Why isn't hydroxychloroquine effective against the coronavirus?
While it can inhibit endocytosis of the viral particle, the virus can still enter via its spike proteins.
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What are the characteristics of the CoV-2 Genome?
- ss(+)RNA
- 27 Kb long
- 5' cap and leader sequence that is needed to initiate translation
- encodes 13 genes but makes 27 proteins
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How do you get >27 proteins from 13 genes in the coronavirus?
1) poly proteins and proteases: virus encodes its own protease (Mpro) and is used in a giant fusion protein to make lots of individual protein from one gene
2) translate from sub-genomic mRNAs
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What is paxlovid?
AKA nirmatrelvir, a protease inhibitor that stops viral replication by inhibiting Mpro.
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What is RdRp?
RNA-depemdemt RNA polymerase
- all RNA viruses encoded their own
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What is special about the coronavirus' RdRp?
they have a proofreading activity using ExoN, which is not common amongst most RNA viruses
- it can fix its errors
- needs ExoN because of the size of its genome
- proofreading allows for longer, more sophisticated genomes
-this is something humans don't havez
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Why is it important for the coronavirus to have double membrane vesicles where the viral RNA is copied?
1) hides viral RNA from cellular sensors
2) increase local concentration of RNA replication and processing machinery to make the amplification efficient
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What is Nsp3?
a protein that forms pores in the coronavirus vesicles for the viral RNA to pass through undetected
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What happens when the viral gene expression increases?
the cellular gene expression is shut off
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What do accessory genes do in the coronavirus?
influence how the virus behaves in its host cell and our body
- currently very unknown
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From a chemist perspective, what makes a cell "living"?
molecular composition
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From a physicist perspective, what makes a cell "living"?
Thermodynamics
278
From a biologist perspective, what makes a cell "living"?
evolution
279
From a philosopher's perspective, what makes a cell "living"?
transcendental
280
What are the basic building blocks of life from the understanding of bacteria?
-Genesis: how the first cell was made (LUCA)
- Blueprints: a minimal genome
- Confinement: making the cellular container
- division: splitting the container into two
- Organization: partitioning cellular components
- Central Dogma: replication, transcription, and translation
- Re-Genesis: How far are we and how far should we be?
281
What are the reasons/benefits for making a cell from scratch?
- fundamental principles underlying cellular function
- understand the link between molecular machineries and a living system
- allows for cellular engineering
- we can then expand the capabilities of natural systems
282
What are the collection of molecules that can make a synthetic cell?
- need to be simple enough to self-assemble (look towards simpler systems)
- complex enough to take on the essential properties of a living organism
- synthesize de novo
- 100s of genes
- 1000s of proteins
283
What are the two requirements to making a synthetic cell?
1) the informational genome - RNA and DNA
2) 3D structure - the container for the cell
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What is the working definition of a living cell?
Entities that can autonomously replicate both their information-carrying molecules and the container in which these molecules reside and that can undergo Darwinian Evolution.
285
What is the triad of life?
- Information: allows a cell to replicate, transcribe, and translate genetic material that can be faithfully passes down to future generations
- self-organization: the ability of cells to sequester themselves and their genomes from the environment
- metabolism: metabolic networks for nutrient synthesis, phospholipid synthesis for volume,e expansion, and energy regeneration based on carbon source
286
What are the two approaches towards synthesizing life from scratch?
1) top-down: existing biological cells are minimized to identify the smallest set of genes that sustain life
- remove genes that are not essential
2) Bottom-up: starting from scratch, assemble basic components one-by-one to build a cell de novo
- nothing but essential genes
- lack of detailed information on gene networks
- high number of possible constructs that can be assembled from a gene pool
- hard to assess which essential gene is missing
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What is a minimal genome?
one that only includes the genes absolutely necessary to satisfy the requirements for a cell to be considered a living thing
all components are:
1) essential
2) well-understood
3) accounted for
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What is special about host-associated bacteria?
have near-minimal genomes due to genome erosion
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Why is genome erosion possible?
due to nutrient-rich environment of the host
290
What are mycoplasmas?
- have no cell wall
- have the smallest genomes capable of host-independent growth
- only has 828 genes (how many are essential?)
291
What is the progress being made toward a minimal genomes?
1) fast, efficient and cheap assembly of whole genomes
2) "booting-up" in the host cell
3) high-throughput genome modification
292
How did the first synthetic speciation event occur?
Mycoplasma mycoses genomic DNA was transplanted into mycoplasma capricolum, yielding mycoplasma mycoides cells
293
What were the steps in the synthetic speciation of mycoplasma mycoses cells?
1) move bacterial genome into yeast
2) engineer using yeast genetics
3) install back into bacterium
4) repeat
294
What does the term booting up mean?
we can replace an entire genome of a cell and change the organism
295
How was synthia, the first fully synthetic Mycoplasma Mycoides genome created?
The first bacterium whose parent is a computer
1) 1Kb overlapping DNA fragments synthesized from oglionucleotides
2) fragments joined by homologous recombination to generate 10 kb and 100kb intermediated
3) Fragments joined in yeast
4) whole assembled M. mycoides genome transplanted into M. capricolm
296
Is synthia considered a living cell?
no, because it cannot divide, which is required in order to be considered living
297
Why is it difficult to synthesize a dividing cell?
the variability in division machinery makes it difficult to pinpoint an ideal division system
298
What is FtsZ in bacterial division?
It is a tubulin-like GTPase that polymerizes upon GTP binding
- forms a Z-ring at the division site
- the Z-ring provides a scaffold for recruiting other division proteins
299
What is the importance of liposome assembly in creating a live cell?
liposomes can be used as containers for processes that make a cell "alive"
300
How does FtsZ interact with liposomes?
It can pinch them
301
What is death by dilution?
progenies lack one or more essential components and die due to asymmetric inheritance
- a serious threat to the vitality
- spatial and temporal regulation is needed to control for the site for cell division in synthetic cells
302
What is the min system?
division-site-regulation mechanism
- name is derived from a mini-cell phenotype, division leads to small cells without a copy of the chromosome (absence of Min proteins)
303
What is MinD?
ATPase
ATP form binds to the membrane
304
What is MinC?
-Cell division inhibitor
- Prevents Z-ring formation
- Follows MinD
305
What is MinE?
- ATPase Stimulator
- Releases MinD from membrane
306
What is special about the MinD and MinC complex?
When they bind together, they oscillate
- self organize into oscillations
307
How do Min and FtsZ polymers interact in an artificial cell?
FtsZ polymers are positioned by Min
308
What will be the likely confinement material in artificial cells?
liposomes
309
What are the 3 main difficulties with Synthia 3.0?
1) don't know the function of 1/3 of genes in the minimal genome
20 many self-organizing systems have not been reconstituted
3) many self-organizing systems reconstituted in cell-free setups are not present in the 3.0 version
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What are the 6 ways microbial biotechnology is used?
1) Bacterial genes used to save crops
2) Probiotics
3) Phage Therapy
4) Bioremediation
5) Targeted gene editing using CRISPR
6) BioMaterials Science
311
What are insecticidal proteins used for in/on plants?
-engineered into plant genomes to protect crops from insect devastations
- bacterium can be sprayed on plants or the plants can be engineered to express these genes
312
How are Cas used against foreign phage DNA?
gRNAs bind to Cas proteins and allow for highly targeted cleavage of phage DNA
- CRISPR Cas9 from streptococcus pyogenes has been adapted as a tool to quickly and efficiently edit eukaryotic genes
-gRNA directs endonuclease Cas9 to knock out eukaryotic genes or replace them with modified forms
313
What is synthetic biology?
the design and construction of new biological parts, devices, and systems for a desired purpose
314
What is a genetic logic gate?
a promoter and gene complex
315
What are the 3 common logic gates used in synthetic biology?
1) Buffer gate - amplifies signals
2) NOT gate - represses protein production
3) OR gate - involves several genes
316
What are toggle switches?
A circuit involves two repressor genes (NOT gates) whose products can repress each other's transcription
- switch depends on whether one or two different inducer signals is present
317
What are Kill Switches?
made to commit suicide once the organism's job is done to ensure that GMOs are safe
- still are also subject to evolution
318
What are examples of kill switches?
1) a synthetic organism is programmed to destroy toxins in the environment
2) toxin breakdown is coupled to flipping the kill switch on
3) as soon as the toxin is removed from the environment, the microbe dies.
319
What are encapsulin?
protein nano compartments derived from bacteria that have potential to be used as a drug delivery mechanism with many advantages
320
What can genetically-modified probiotics be used for?
to treat illnesses
321
What is ActoBio Therapies?
GM microbes used to treat Type 1 diabetes
322
What is Oragenics of Tamp?
GM Bacteria to treat mouth sores caused by cancer chemotherapy
323
What is Osel?
GM microbes to prevent HIV infections
324
What is Synlogic?
GM E.coli to rid life-threatening levels of ammonia from the bodies of people with cirrhosis of the liver or PKU
325
How does our gut microbiome play a role in how our immune system responds to food allergies?
- LPS from our gut bacteria trains our immune system to not overreact
- Mice mutants non-responsive to LPS became anaphylactic to peanuts
- mice treated with broad-spectrum antibiotics also had severe reactions
326
How can we use microbes to manipulate food allergies?
- adding S. variable was sufficient in protecting from egg allergy in mice, and hopefully in people too
327
What two types of bacteria are depleted in people with depression?
1) coprococcus
2) Dialister
328
What is cyano-concrete?
a concrete that is alive and reproduce with cyanobacteria that deposits binding materials
- it yields self-repairing concrete and takes in a lot of CO2
329
What has been used to rescue neuronal activity?
Injecting O2-producing cyanobacteria upon illumination.
330
What is being used for bomb detection?
1 strain of bacteria is the detector and the second is the signifier
- the two strains work together ro indicate the location of buried explosives that would be invisible above ground
- the second strain receives the signal from the first bacteria and emits a glowing light, locating potential threats
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