Dr. Thomas lectures terms and concepts Flashcards
(185 cards)
1
Q
What is the Messelon and Stahl experiment?
A
the N14/15 experiment that showed that DNA replication was semi conservative
2
Q
What is DNA G
A
aka primase
RNA polymerase that makes RNA primer
3
Q
What does DNA A do?
A
binds to the ori site and starts the open complex
4
Q
What is DNA B?
A
helicase
unwinds DNA ahead of the replication fork
5
Q
What is HU? what does it do?
A
Histone like protein that helps the DNA bind to the ori
6
Q
What two repair mechanisms increase the fidelity of DNA replication?
A
missmatch repair and 3’-5’ exonuclease
7
Q
What happens during the formation of the open complex? (3 steps, what is involved)
A
- DnaA binds specific sequences in oriC (with help from histone-like protein HU)
- DNA unwinds (stands separate) in an ATP-dependendent manner
- SSBs bind to keep open complex open
8
Q
What 2 things happen to form the prepriming complex?
A
- DnaB (helicase) binds to both ends of open complex
2. This gives two replication forks going in opposite directions
9
Q
What happens during the priming stage?
A
- DNA G (primase) lays down a 12 nt piece of RNA
2. DNA pol III initiates synthesis and adds dNTP’s to the 3’ end
10
Q
What is different about extension on the lagging strand than the leading strand?
A
Done in 1000 bp okazaki fragments
Multiple primers laid down
Primers are removed and the space filled in by DNA pol I
Break is sealed by ligase
11
Q
What happens during DNA synthesis termination? What proteins are involved? What is used if there are decantations?
A
- Tus binds Ter and the termination sequence
- Ter stops the replisome when it reaches the complex
- other replisome shows up and dislodges Tus
- Replication is complete
If the chromosomes are decantenated, then topoisomerase IV is used
12
Q
What factors are involved in resolving dimers via recombination? How are they resolved?
A
Dimers resolved by site specific recombination at dif sites
The dif sites are lined up by FtsK
FtsK is localized to the septum and activates XerC/D
XerC/D are the recombinases that act on dif sites
13
Q
what does DNA gyrase introduce?
A
Negative supercoils
14
Q
what is supercoiling?
A
Energy stored in the DNA
15
Q
What does topoisomerase I do?
A
Topoisomerase I removes supercoils
16
Q
What are 5 features of histone like proteins?
A
- Compact DNA
- Small and abundant
- Some are site specific and some bind in a more general way
- Can serve as accessory factors to help initiate processes (replication, recombination)
- Can be general repressors
17
Q
What are the two complexes that are formed during initiation of transcription?
A
the open and the closed complex
18
Q
What leaves and what is formed at the start of elongation?
A
- sigma factor leaves
2. the transcription bubble is formed
19
Q
What are the two forms of transcription termination
A
- intrinsic or factor independent
2. rho dependent termination
20
Q
How is translation initiated? (3 things happen)
A
- shine dalgarno sequence is recognized by the 16s rRNA in the small subunit
- Scans until it finds the first AUG
- fMet binds at the first stop codon
21
Q
What are the two elongation factors involved in translation?
A
EF-G is the translocase
EF-tu is elongation factor tu
Both use GTP
22
Q
What is simple feedback inhibition?
A
A regulatory enzyme is inhibited by the end
product of a pathway
23
Q
What is positive regulation?
A
The product of an enzyme can stimulate the activity of enzymes downstream in the pathway
24
Q
What is cumulative feedback inhibition
A
In a branched pathway with more than one product, multiple products can inhibit the enzyme
25
What is concerted feedback inhibition?
Occurs in pathways with isoenzymes
multiple products of the branched pathway will inhibit each of the isozymes
26
What is forward genetics?
selecting for the phenotype and then identifying the mutation
27
What is reverse genetics?
making a mutation and then observing the phenotypes
28
What is trans complementation?
Providing the extrachromosomal DNA in the form of a plasmid
29
What is cis complementation ?
actually putting the gene back into the chromosome
30
What is a microbiome?
populations of cohabiting and interacting microbes.
31
a healthy microbiome is one that is?
stable and diverse
32
What are 3 ways to study microbiomes?
1. 16s rRNA
2. OTUs
3. Metagenomic analysis
33
Do microbial communities have core functions?
yes
34
increased diversity in the gut leads to ...?
increased colonization resistance
35
How do salmonella disrupt the microbiome to pathogenize the host?
Normally host secretes lipocalin-2 which sequesters enterobactin which is an iron siderophore produced by normal bacteria
- this establishes the iron homeostasis
Salmonella secretes a different iron siderophore called salmochelin which cannot be inhibited by lipocalin-2
- they take up all the iron and kill off the commensal bacteria
36
What is attenuation?
Way to couple transcription to nutrient supply
37
How is attenuation used in the Trp operon?
Low levels of trp, the ribosome stalls during translation which forms the 2'3 hairpin loop
- signals more trp needs to be made
- does not inhibit transcription
In high conditions, the ribosome translates quickly leading to the formation of the 3,4 hairpin loop and transcription is inhibited
38
What are repressors and where do they bind to?
bind to operators and black transcription by RNA polymerase
39
What factors are involed in catabolite repression?
Glucose regulates the activity of adenylate cyclase which makes cAMP
cAMP binds catabolite activator protein CAP
CAP is an activator that recruits RNA polymerase to promoters that would not be turned on efficiently otherwise
40
What is one of the most abundant proteins synthesized by the cell?
Flagellin
41
What sigma factor is involved in flagellar biosynthesis? What genes does it regulate?
sigma 28 regulates transcription of late genes
42
What do the early flagella genes code for?
make foundation and transcriptional activation complex to allow the middle genes to be transcribed
43
what is the middle gene that codes for sigma 28?
FliA
44
What sequesters sigma28?
FlgM
45
When is sigma28 uninhibited?
when the hook and basal body are complete and FlgM is secreted
46
What does sigma 54 need the help of?
NtrC bound at “enhancer” sequences far upstream from promoter
47
What is the homology of cis sRNAs like?
extensive homology with their target and are very specific
48
What is the homology of trans sRNAs like? what do they use?
have limited homology with their target and require the assistance of Hfq
49
what is Hfq?
A protein that stabilizes RNA RNA hybrids
50
What are riboswitches?
are transcripts where the RNA is regulatory and controls gene expression
51
What are 4 ways that bacteria can destroy incoming foreign DNA
1. Restriction modification systems
2. Block phage attachment
3. Block phage infection
4. CRISPR
52
What are restriction modification systems?
cleavage of foreign DNA because it is unmethylated by restriction endonucleases
53
How do cells block phage attachment?
can mask structural features like pili that many phages use to infect
54
How do cells block phage infection?
by committing cell suicide
55
What do CAS genes code for? where are they located?
they are found adjacent to the CRISPR locus
| They code for enzymes that process the CRISPR locus
56
What is the structure of the CRISPR locus?
Repeated sequences of host DNA interspaced with an array of DNA fragments isolated from phage DNA
This locus has the cas genes on the 5' end of it that are transcribed first
57
What are the three stages of CRISPR?
1. immunization/adaptation
2. crBiogenesis
3. Interference
58
What happens during the immunization phase?
During an infection, a new spacer is derived from the virus/phage genome and incorporated into the CRISPR array along with a new repeated unit
59
What happens during the biogenesis stage? What is generated?
1. CRISPR locus is transcribed
2. CAS proteins process the crRNAs by endoribonuclease action
- generates small cRNAs
60
What happens during the interference phase?
the crRNAs guide a complex of Cas proteins to the matching target DNA to initiate nucleolytic cleavage
61
What is the role of the PAM sequence? what does it stand for?
Stands for protospacer adjacent motif
Sequence of DNA that is near to the protospacer in foreign DNA but not present in the CRISPR locus
How the crRNAs recognize foreign DNA from self and cleave
Absence of a PAM sequence protects against cleavage
62
What is the protospacer?
the target DNA that will be acquired from the phage or plasmid and incorporated as a spacer
63
How many types of CRISPR systems are there ? How are they classified?
3
| By the way that the cRNAs are processed
64
What is the most common type of CRISPR? what does it use?
Type II that uses Cas9 endonuclease
65
What is the interaction between the crRNA and the Cas9 complex?
After the Cas genes have processed the array products, there is a piece of crRNA bound by endonuclease Cas9
the complex can then go recognize and destroy specific DNA sequences
66
What gene editing method has CRISPR largely replaced?
RNAi
67
What 3 scientists were part of the phage group?
Max Delbrück, Salvador Luria, Alfred Hershey
68
Who figured out how lambda phage works? what else did they develop?
Mark Ptashne
Developed idea of “modular” nature of proteins
69
What is a plaque?
A zone of lysed bacteria
70
What are turbid plaques?
Areas where lysogenic phages have inserted themselves into the host genome and so the bacteria have replicated
71
What is the prophage?
The dormant phage within the host
72
What is the lysogen?
The host bacteria with a phage
73
What does cI encode? what does the product repress?
the CI repressor
represses activation of other lambda genes and can activate its own transcription
74
What is cro
Another repressor that represses the transcription for cI and allows for transcription of genes leading to the lytic cycle
75
How many operators are there between the cI and cro genes?
3
76
What is the affinity of binding of CI for the operators?
Or1>Or2>Or3
77
What is the point of cooperative binding?
as more CI (or cro) bind to the operators, more is recruited and it results in the shutting of of the respective genes
- how to control the amount of CI and cro
78
What does binding of cI repressor at Or2 activate/recruit?
Repressor bound at OR2 acts as an activator. It recruits polymerase to PRM
- the maintenance promoter
- transcription of cI to remain in the lysogenic state
79
What is the promotor for repressor maintenance?
Or3
80
what is the promoter for repressor establishment? What is it activated by?
activated by the presence of one of the late lysogenic genes cII which binds to a different promoter for cI
81
how does the decision occur to either go to the lysogenic or lytic states following infection?
1. after infection, N protein is made
2. N protein stimulates production of CII
3. If the cell is healthy and there are lots of nutrients, bacterial proteases will cleave CII. The repressor is then not made and the lytic cycle is engaged
If there are not as many nutrients, CII is stable. It binds to the promotor of repressor establishment and stimulated the transcription of cI and the lysogenic cycle is initiated
82
What is the enzyme involved integrating DNA into the host genome?
Integrase
83
What two enzymes are invovled in excising the DNA from the host chromosome?
integrase and exisionase
84
What is M13?
A filamentous phage with a single + strand of DNA that is surrounded by a single layer of protein
85
How is M13 passed into the host cell?
The entire phage enters the cell
86
Why are the M13 phages called "male specific"
because the adsorb to the sex pilus
87
What are the steps of filamentous phage replication?
1. + sense ssDNA is present in cell
2. Copied into the dsDNA replicative form
3. the - sense strand is used as a template to make phage proteins
4. the RF is used to make more + strand by rolling circle replication
- can be semi conservative
88
What is the filamentous phage useful for in a laboratory setting?
making site directed mutations
89
What two gene products are involved in the rolling circle replication process of ssDNA viruses? What do they do?
gene II product makes nick in the + strand and stays bound while a new + strand is synthesized
packaging of the + sense ssDNA strand is done with the help of both gene II and gene V
90
What features in some bacteria originally arose from phages?
Pathogenicity islands
91
What are colicins? what are the two types
toxins secreted by bacteria that either form pores in other bacteria or act as nucleases
92
What are colicin systems encoded on?
Plasmids
93
What prevents the bacteria secreting the colicins from being killed?
plasmid with the colicin also encodes the immunity product that neutralizes it
94
What are 4 features of high copy number plasmids and what is one example of this?
1. “Relaxed” control of copy number
2. Generally small size <10 kb
3. Replication is unlinked to cell division
4. Random partitioning
ColE1 plasmid is best studied of this type. ColE1 make colicins that kill species related to E. coli
95
What are 3 features of low copy number plasmids and what is one example?
1. "stringent" control of copy number
2. Usually larger (like up to 300 kb)
3. Directed partitioning systems with the replication linked to cell division
R100 plasmid is example
96
What feature do low copy number plasmids often have to ensure that the progeny have it?
They are often conjugative which is a backup mechanisms in case there is an error in partitioning
97
What is plasmid amplification? what is used/how does it work?
Adding drugs like chloramphenicol to to the media prevents the host cell from making new proteins
All the machinery needed to copy the plasmid is there though and so many copies can still be made
98
Explain how copy number is controlled by antisense RNA. What playerzzz are involved? What type of plasmid does it control?
Controls high copy number plasmids like ColE1
Normally RNA II stimulates replication of the plasmid
replication of the plasmid results in the production of RNA I and rop proteins
As concentration of plasmid rise, so do rop and RNAI concentrations
rop helps RNA I (which is antisense to part of RNAII) bind to it
this stops plasmid replication
99
What is the role of RepA in the replication of plasmids?
Binds to the OriC
100
How is R100 plasmid replication controlled? What factors are involved?
RepA is required for initiation of replication at oriV
CopB is a repressor of repA
when the plasmid first enters the cell there are low levels of CopB and so replication proceeds
CopB initiates transcription of copA which is antisense RNA to the repA message
101
What is plasmid incompatibility?
R100 cannot coexist with related plasmids because the copA genes are similar. Therefore they will repress the replication of the related plasmid
102
Explain how replication is controlled by the presence of iterons? what are they?
Iterons are DNA repeats
RepA binds iterons and “handcuffs” 2 plasmids together
- prevents them from replicating again
103
Can RepA cuff related plasmids?
Yes, form of plasmid incompatibility
104
What is the stability of naturally occuring plasmids vs artificial plasmids?
the naturally occurring ones are more stable
105
What 3 factors are associated with plasmid stability ?
1. Plasmid integrity
2. partitioning
3. differential growth rates
106
What sequences do plasmids often have that allow for deletions or inversions?
insertion sequences or other recombination “hotspots”
107
What is the point of toxin antitoxin systems?
way to ensure that plasmids are inherited
108
Which is usually more stable, the toxin or the antitoxin?
the toxin
109
What are the two toxin-antitoxin systems involved in partitioning?
1. CcdB-CcdA toxin-antitoxin system
| 2. sok and hok system
110
what kind of plasmid is the CcdB/CcdA system encoded on?
low copy plasmid
111
How does the CcdB/CcdA work? what does each factor do?
CcdB inhibits topoisomerase which is essential for cell functioning
CcdA (antitoxin) inhibits the action of CcdB
112
What are the functions of the two factors in the sok and hok system?
hok encodes protein that is toxic to host (host killing)
sok encodes antisense RNA that prevents hok translation (supressor of killing)
113
What happens if during partitioning the daughter cell does not receive the plasmid with the toxin antitoxin genes on it?
The cell will die because there will still be CcdB or hok present in the cell but no genes to transcribe CcdA or sok
114
How does differential growth rate impact the stability of the plasmid?
if the plasmid is large and codes for lots of non-essential genes, there will be selective pressure to lose the plasmid
115
What is transformation?
uptake of naked DNA
116
What are 2 kind of extreme ways of transforming DNA?
1. Biolistic transformation: shoot DNA in on gold particles
- often done in plant cells
2. Electroporation: shock in the DNA using a capacitor
- used for some complex strains
117
What do naturally competent bacteria become competent in response to?
growth phase
118
what do Neisseria and Haemophilus have that allows them to selectively take up DNA?
proteins that bind preferred DNA sequences (usually from relates or same species)
119
How does the DNA enter the bacteria during transformation?
As ssDNA
120
Who coined the term plasmid?
Joshua Lederberg
121
what is a self-transmissable plasmid?
encode machinery to initiate conjugation and transfer
122
what is a mobilizable plasmid?
can be transferred but need Tra functions supplied in trans
123
What is transconjugant?
progeny from a successful conjugation
124
What genes encode transfer functions? which of the two cells in a conjugation event possess them?
tra genes
the donor has them on the plasmid
125
What does the donor cell do to transfer the plasmid?
forms pilus and brings cells in close contact
| Pilus forms to form bridge to transfer DNA
126
What 2 things are needed if you have a mobilizable plasmid?
Non conjugating plasmids can be transferred too if they have mob genes and bom site (often called oriT)
127
What are two components of Tra genes?
1. Dtr component (DNA transfer and replication): genes needed to process the DNA
2. Mpf (mating pair formation): genes that make the sex pilus
128
What 2 things do you need to have a conjugation event?
1. Tra genes
| 2. OriT
129
What factor is encoded by the Dtr component of the Tra genes? What does it do?
Relaxase is an endonuclease that cuts at a nick site in OriT and makes a covalent bond with the 5' end of the DNA
130
What is site specific recombination?
occurs at particular DNA sequences by enzymes that recognize said DNA sequences and catalyze recombination with a specific recipient DNA
131
What is homologous recombination? what does it require?
occurs between DNA sequences that are homologous and requires RecA
132
what is intramolecular recombination and what is it commonly used for?
Looping out of a circular piece of DNA
Commonly used to excise plasmid or phage DNA
133
Increasing what 2 factors will increase the chance of recombination?
1. increasing the amount of homology between the strands
| 2. increasing the distance between the two sites
134
the frequency of co-inheritance is inversely proportional to?
to the distance between two DNA markers
135
What are the 2 jobs of RecA overall
1. cleave LexA repressor to initiate the SOS response
| 2. Pair homologous gene segments together so that they can exchange strands
136
Describe the process of RecA mediated recombination?
1. ssbp open up the DNA to be single stranded
2. RecA binds cooperatively to the strand of DNA
3. formation of a nucleo-protein filament
4. RecA filament then goes looking for homology to pair with and recombine
137
What are 2 features of the F plasmid?
1. it can integrate into the chromosome
| 2. its a conjugative plasmid
138
What are F' plasmids?
Plasmids that incorporated some host DNA when they were excising
139
What are F' plasmids useful for?
constructing merodiploids
140
bacteria that have integrated the F plasmid are called?
Hfr strains “high frequency of recombination"
141
Explain how Hfr mapping works
lol I ain't explaining,
get Bryn to tell you
142
What is specialized transduction? when does it occur?
Occurs when a lysogen is induced to go into the lytic state
In excising the prophage, some host DNA is incorporated
The phage then replicates
- also replicated the host DNA
Cell lysis and phage release
The phages then go on to infect other cells
143
What is generalized transduction ? when does it occur?
Occurs when a phage infects a cell and immediately enters the lytic cycle
Host DNA is degraded by phage enzymes
Replication of phage DNA and synthesis of new phages
- some of which accidentally take up host DNA
Transducing phages are released and will go infect other cells
- some of which contain donor DNA
144
What are the 3 common transducing phages?
P1, P22, Mu
145
what do insertion sequences encode?
a transposase flanked by inverted repeats
146
What do transposases do?
duplicate a target sequence and inserts the element
147
Where do insertion sequences insert themselves?
randomly
148
What makes transposons different from insertion sequences?
they have a selectable marker
149
What are the 2 kinds of transposons?
1. composite
| 2. non composite
150
what are composite transposons?
Contain 2 copies of insertion sequence flanking gene(s) for antibiotic resistance
151
What two factors limit the activity of transposase?
1. acts preferentially on hemimethylated DNA so works better following replication
2. preferentially acts in cis
152
Who developed the notion of gene regulation ?
Barbara McClintock
153
How could you deliver a transposon into a cell?
Encode transposon on a plasmid or phage that cannot replicate in the recipient
154
What is the suicide plasmid? What special site does it have and what does it need for replication?
Conjugative plasmid with a R6K origin of replication
Requires Pi protein supplied in trans by a temperate phage
155
What is encoded on the suicide plasmid?
contains a transposon with antibiotic selection
156
Why will the transposon be incorporated into the host DNA? how can you select for this?
there won't be any Pi protein in the cell it is introduced into
Since it can't replicate, the transposon will "hop" to the host DNA
you can select because the transposon will encode antibiotic resistance genes
157
What is the Luria and Delbruck Fluctuation Test? What were the outcomes
T1 is a phage that can kill E-coli
mutants that are resistant are easily isolated
Showed that mutations are spontaneous because each time they did the experiment, there were a different number of mutants
158
What would have been the result of the fluctuation test if mutation was directed?
you would have had the same number of mutants each time
159
What do deletions and rearrangements arise from?
recombination
160
What are conditional mutants?
Have a mutation where they can live in one condition but die in another
161
What 2 kinds of mutations are often polar?
nonsense and insertion mutations
162
Why would mutations affect translational coupling?
the recognition of an initiation codon may be dependent on translation of the gene upstream
163
What are 3 types of "pseudoreversion" or intragenic supression?
1. same codon or same nucleotide
2. different codon but in the same gene
3. frame shift
164
What are the stop codon "alternate names" and what do they correspond to
```
amber = UAG
opal = UAA
ochre = UGA
```
165
What are tRNA suppressors?
tRNA that have mutations in the anticodon that allow them to bind to the mutated codon and still incorporate the correct amino acid
166
What is a bypass suppressor?
If you have a mutation in one pathway you can have other products from other pathways "bypassing" the mutation to still give you your product
167
What are 3 common types of mutation types in bacteria?
1. require additional supplements
2. loss of ability to use a nutrient source
3. resistance
168
What do E-coli mutator strains have?
A defect in proofreading
169
What does ethidium bromide do ?
Inserts between paired bases, acts as an intercalating agent
170
What is nitrosoguanadine (NTG)?
An alkylating agent
171
What is EMS and what does it do?
Ethyl methanesulfonate
Reacts with guanine to make O-6-methylguanine
Causes polymerase to pair it with a thymine instead of a cytosine
172
What does nitrous acid do?
Deaminates bases
173
What does UV radiation cause?
crosslinks between adjacent pyrimidines
174
What is the result of a thymine dimer occurring?
causes polymerase to see them as one base instead of two
Results in a frameshift mutation
175
How does the Ames test work? what is it testing for?
tests to see if something is a mutagen
If you plate mutant bacteria on a deficient medium with the carcinogen, you can count the number of "revertants" that are able to now grow
176
How does the blockage of DNA replication stimulate the SOS response?
If the polymerase is blocked, the helicase is going to keep unwinding the DNA, leaving more ssDNA to be bound by RecA
This binding of RecA activates its protease activity and it cleaves LexA to relieve repression
177
What is the first stage of the SOS response (after cleavage of the repressor)
Nucleotide excision repair
178
What are the 3 proteins involved in the nucleotide excision repair?
UvrA, B, C, and D
179
What occurs during nucleotide excision repair?
1. two UvrA and one UvrB protein binds to DNA nonspecifically
2. translocate via an ATP dependent process until they find the mutation
3. when they find the mutation, UvrA leaves and a stable UvrB-DNA complex is formed
4. UvrC comes and binds to the UvrB-DNA complex and makes a nick on either side of the damage, resulting in a 12nt fragment
5. UvrD helicase action then removes the fragment
6. the space is filled in by Pol I and ligase
180
What are the 2 alternate polymerases?
Pol IV and PolV
181
What are the 2 subunits of Pol IV?
UmuD and UmuC
182
What gene codes for PolV? What kind of gene is it?
DinB, an SOS induced gene
183
Which of the 2 alternate polymerases causes a frameshift mutation?
Pol IV
184
What factors are involved in mismatch repair
Mut H, Mut S, Mut L, DNA helicase II and exonucleases
185
What are the steps in mismatch repair?
1. MutH binds to hemimethylated DNA at GATC sequences following replication
2. complex of MutS and MutL bind to mismatched DNA between the two
3. DNA is threaded through the MutS MutL complex until it reaches the MutHs
4. MutH cleaves unmethylated DNA
5. DNA helicase II and exonucleases complex
degrades the unmethylated DNA towards the mismatch
6. gap is filled in by DNA polymerase III
and the nick is sealed by DNA ligase
