Final Exam 2 Flashcards
Wong Portion
1
Q
What is Bacterial Genome
A
The entire set of genetic information of a bacterium. Chromosomal and plasmid DNA
2
Q
What are the types of genetic material?
A
- Chromosomal
2. Plasmid
3
Q
What are the major differences between chromosomes and plasmids?
A
- chromosomes carry essential genes / plasmids do not
- plasmids are smaller
- chromosome replication initiation tightly linked with cell cycle / plasmids is not
4
Q
What are essential genes?
A
- chromosomes only carry these
- required for: DNA replication, RNA polymerases, rRNA, tRNA, ribosomal proteins (need these for translation process), DNA repair
- what defines if something is a chromosome
5
Q
What is the replication time for E.Coli
A
40 minutes
6
Q
What is the Bacterial Cell Cycle
A
"BCD" BIRTH - new - makes enough enzymes for replication - length of period depends on medium that its grown on
CHROMOSOME
- chromosomal replication
- length determined by genome length and ability of how fast to replicate
- this period is unique to each organism
- chromosomal DNA linked to this period
DIVISION
- separation of cells
- 2 copies
*plasmids can replicate in any of the cycles
7
Q
How many copy numbers do bacterial chromosomes have compared to plasmids?
A
Chromosomes = 1-2 copies Plasmids = 1-Many
8
Q
What is an exonuclease
A
- enzyme that removes successive nucleotides at the end of a sequence
- binds to the end of a DNA strand and starts to digest from there
9
Q
What are the advantages to having Circular DNA rather than linear?
A
- Shorter
- compacted - No exonucleases
- extra removal of genetic material - only need one primer
- you don’t have telomeres
10
Q
What are some disadvantages to Linear DNA
A
- exonucleases
- more mechanisms needed to replicate (bc telomeres)
- longer
11
Q
How long is E.Coli
A
1 Circular Chromosome
4.6 X 10^6 bp
12
Q
What is a Chromid?
A
originally a plasmid that has converted into a chromosome; Large plasmids can be converted into chromosomes
13
Q
How many bacteria have a divided genome?
A
10%
14
Q
Plasmid –> Chromid
A
- When a plasmid is large
- cell wall will transfer essential genes
- replication cycle linked to cell cycle
- DNA repairing genes localized on the chromosome 2
15
Q
Coordination of Vibro Chromosomal DNA Replication
divided genome
A
- larger chromosome gets replicated 1st
- cells finish replication at the same time
- 2nd replicated 2/3 in
- Dimer will bind to the CrtS location when replication has occurred because if it is too early it will loosely bind, to compared to if it has bound after. It will tightly bind allowing for a conformational change to a monomer.
16
Q
What are the advantages of having a divided genome?
A
- Faster replication time
- More genetic information without sacrificing cell growth
- Larger genome = more genes = more fitness
17
Q
Burkholderia Cepacia
A
- Opportunistic Bacteria
- 3-4 CC
- Causes Pneumonia
18
Q
Agrobacterium
A
1 CC
1 LC
19
Q
Borellia
A
1 LC + Many Plasmids
20
Q
General Features of Chromosomal Bacterial DNA
A
- Haploid
- any mutations is not good
- promoter goes along gene direction
- recombination process
- 97% of genome = coding purposes
- usually has same copies of genes many times over (redundancy)
- genes that code for RNA operons = 1 copy
- rarely have introns
- compacted into nucleoid
21
Q
In bacteria, how many copies of genes code for RNA operons
A
- ONE single copy
- mRNA = amplification effect
- rRNA = one cannot translate another; however bc of this there is a high copy number
22
Q
Dynamic Loops
A
- compacts bacterial chromosomal DNA
- supercoiled by gyrase
- 50-500 dynamic loops
- compact ~1600 fold
- facilitate by Heat Unstable Nucleoid Proteins
- number of loops can vary
23
Q
How many proteins does E.Coli have?
A
4,288
24
Q
Average size of coding sequence for E.Coli
A
951 bp (317 amino acids)
25
Details of E.Coli Chromosomal DNA
```
4.6 X 10^6 BP
4,288 Proteins
Average gene size = 1kb [includes intergenic region]
Circular
Double stranded
Average size of coding sequence = 951 bp (317aminoacids)
21% Metabolism
10% Transport
38% Unknown
```
26
Operon
A cluster of genes that is under the control of a single promoter
(they share the same promoter)
27
4 Basic Gene Organization
1. Genes that have related functions are grouped together to form operons
2. Roughly equal number of genes on each strand
3. Highly expressed genes are transcribed in the same direction as DNA replicates
4. Highly expressed genes are located close to OriC
28
Why do we need multiple promoters?
- if we want a higher gene to product, we would place a promoter in front of the gene
- this gives us a higher abundance of transcription
29
__% of 2+ genes / operon
~30%
30
__% of 1 gene as the transcription unit
~70%
31
__% of 1 promoter / operon
68%
32
__% of 2+ promoters
32%
33
Why does each strand have roughly an equal number of genes?
If equal # = coding is similar for each strand
34
What is OriC
Origin Replication
C = Chromosome
- There is only one replication origin
35
What is TerC
Termination for DNA replication
36
What is the highest expressed operon
rMD
37
Explain OPPOSITE DIRECTION transcription
- faster than unidirectional
- RNA & DNA polymerase move along the same DNA track
- "head-on" collision
CONSEQUENCES:
- bc of head on collision, it slows transcription + replication
- the polymerase falls off;
> takes time to put it back on
> can leave a transcription bubble where water can get into and can cause mutations (because at that point it is in a single strand form where it is less stable)
38
Which direction does bacteria transcribe genes in?
Organizes genes in a co-directional manner to avoid collisional impacts
(minimizes problems to go in the same direction)
39
Explain SAME DIRECTION transcription
- co-directional collision
- DNA poly. and RNA poly. go in the same direction
> DNA poly. moves faster (1000 nts/sec) and RNA poly is slower (50 nts/sec)
> if DNA poly. is in front, it moves quicker than RNA poly. and it does not catch up
> if RNA poly. is in front, DNA can hit it and cause a co-directional collision --> this results in RNA falling off, not DNA
> RNA will return to the promoter
> no effect on DNA replication - cause it stays on and continues replication
40
What has a genome size smaller than 1 Mgb?
Mycoplasma
41
What is Free Living
- requires glucose in order to support growth
| - needs minimal medium + carbon to grow
42
What is Parasitic
- requires host to provide nutrients
43
What is Endosymbiotic
- cell within a cell
- bacterium supplies amino acids
- cannot produce certain amino acid; the other cell will provide it but will exchange nutrients + other amino acids
44
What is the smallest genome?
Nasula
137 genes
112 kb`
45
What is a bacteriophage
- virus that infect bacteria
46
Basic Definition of Transposable Element
"jumping genes"
- genetic elements that jump from one location to another within the genome
- can move from one DNA molecule to another, or on the same DNA molecule
47
Basic definition of Mobliome
- MOBILE genetic elements that are present in the bacterial genome
- i.e. plasmid
> it can be transferred from one cell to another
48
What is the Jumping Rate
10^-5
- this is the rate in which mutation occurs
49
Transposable Elements: Basics
- every cell has it
- amounts vary
- 45% of human genome is TE
- 75% of corn genome is TE
- They jump from one location to another within a genome
- can jump from chromosome to plasmid (vice versa)
- "molecular parasite"
- cannot replicate on its own
- represents a piece of DNA
- possible to jump into an essential gene causing death
- jumps into intergenic regions
50
What is Transposase
The enzyme that catalyzes the jumping of transposable elements
Many Transposase: Higher frequency of jumps
Low Transposase: Lower frequency of jumps
51
5 Mechanisms that ensure TE jumping is not random, but rare...
1. Location
- between gene spacing
2. Poor Translation Process
- even if mRNA is made, it cannot be translated frequently
3. Poor Promoter
- transcription to not occur frequently
4. Tons of Transposases
- catalyze is slow, long to time to catalyze
5. Controlling Transcription Process
- allows the genes to be expressed in a small time period
- when DNA first replicates, it is not methylated, allows RNA poly to bind to DNA allowing for short expression before methylation occurs
52
What is the transposition process?
1. DNA and TE does not have to be homologous
2. Tnp mediates the process
3. Tnp recognizes the short target sequence
>2-24 bp
4. AFTER transposition, target is duplicated and arranged as DIRECT REPEAT
5. Tnp recognizes the IR sequence at the END of the TE
6. Target sites may or may not be random
53
What are the two forms of repeats?
DIRECT REPEAT
- duplication on same strand
- goes in same direction
```
INVERTED REPEAT
- inverted duplication
ABCD -- D'C'B'A'
- can form stem and loop structure
- role is to define boundary of TE
- Tells Tnp where to cut (recognize)
```
54
What are the subunits of Tnp
Subunit A - binds to B; by itself can't recognize; indirect
Subunit B - identify, recognize, binds to IR
[ both A+B are used in overall recognition of IR]
Subunit D - recognizes target, binds to sequence byself
Subunit E - Recognize target
Subunit C - Connects
55
What is Mu
Bacterial phage that is a transposon that is Random
56
Tn7
Non-random transposon
57
What are the two classes of TE?
1. Insertion Sequence
- Must have: Tnp + IR
- Simplest
- 750-2000 bp
2a. Composite Transposon
- Genes inbetween two IDENTICAL IS
- One might be non-autonomous (not able to transcribe Tnp --> not able to jump anymore)
- Autonomous - transcribes Tnp = jumps
- only one gene makes a functional transposase
2b. Complex Transposon
- Not based on IS
- can have additional genes (ie. antibiotic)
58
How long are Inverted Repeats?
9 - 41 bp sequence
59
What are the two things that an autonomous transposon requires?
- IR
| - gene for functioning transposase
60
What are the two mechanisms for 'JUMPING'?
1. Conservative Transposition
| 2. Replicative Transposition
61
Conservative Transposition
- mechanism for jumping
- copy number does not increase
- excision of transposon
1. Tnp recognizes IR boundary
- B recognizes; A positions to cut
2. 2 5' ends are created
- caused by A
3. B creates 3' end
4. gap formed - segment removed
5. D + E recognize target site
6. B will cut target site
7. B joins TE to target
8. recomb. process
9. single strand regions become a primer (DR)
62
Replicative Transposition
- copy numbers increase
- replicative
- requires resolvase enzyme
63
How many nucleotides is the target sequence?
4 Nts
64
Consequences of jumping TE
1. Insertional Mutagenesis
2. Polar Effect
3. Turn On repressed genes
4. Deletion via Recombination: Direct repeat
5. Insertion via Recombination: Inverted Repeat
6. Gene Translocation
65
What are the forms of plasmids
- Circular
- Linear
- Double Stranded
- Single Stranded
66
What are the four types of Plasmids
F Plasmid
- Fertility
- helps in conjugation (gene transfer)
R Plasmid
- resistance
- Hg resistance (cystedine)
M Plasmid
- helps in nitrogen fixation
Col Plasmid
- Colicin
- many forms
- can be rare
1. acts as nuclease (destroys chromosome DNA)
2. Makes nuclease that destroys RNA
3. Act on bacterial cell wall
4. More permeabiliity (but lacks proton gradient)
- need the producer to make antidote
67
What is ORI on a plasmid
- The replication origin
68
What is REP
Replication Protien
69
What are the two mechanisms for replication in plasmids
1. THETA MODE
- bidirectional replication
- REP used to locate ORI and binds to sequence
- unwinds
2. ROLLING CIRCLE MODE
- REP locates ORI and nicks the DNA
- protein attaches to 5' end
- 3' end acts as primer
- unidirectional
70
What is host range?
1. BROAD HOST RANGE
- must have: REP, be able to make primase, RK2 RSF101 must be expressed
2. NARROW HOST RANGE
- used for genetic engineering
71
Plasmid copy numbers
- varies
- each plasmid has their own copy number
- F plasmid = 1-2
- genes on plasmid tell how many copy numbers
- the higher the copy the number, the higher the chance the daughter cell gets it
- if daughter cell doesn't = death
72
Plasma Addiction
Need plasma to survive; if there is no plasma, cell dies
73
2 Genes to maintain/retain plasmids
CcdA
- antidote to controlled cell death
- unstable
- susceptible to protein degredation
CcdB
- controlled cell death
- stable
- stops gyrase (which is an essential gene)
74
What are the types of gene transfer
1. Vertical
(parent --> child)
2. Horizontal
(conjugation, transduction, transformation)
75
Basics of Conjugation
- gene transfer by cell-to-cell contact by conjugation pili
- genes required are plasma encoded
- g- to g+ or vice versa
- cross-domain gene transfer
76
Conjugation Pili
- continuous lengthening and shortening process until they make contact
- apart of the type II pili class
- number of pilis vary (1-10)
- randomly located
77
What are the two models underneath Conjugation Pili?
1st Model
- 6% use it
- DNA Channel
- 3nm
2nd Model
- 94% use it
- attachment and retraction
- Cell to cell contact
- outer membranes fuse together
- more dominant
- pili retract (depolymerization)
78
What is depolymerization of pilus
Shortening of pili
79
What are the two type of plasmids?
1. Self-Transmissible
- 2 sets of genes that carry out gene transference (Dtr + Mpf) - both are needed for pili to form
- narrow host range for short distances
- broad host range for interspecies transfer
2. Mobilizable
- one set of genes (not mpf)
- borrows from self-transmissable (mpf)
- smaller
- no conjugation pili formed
