1
Q
What is an operon?
A
An operon is a group of bacterial structural genes under the control of a single promoter
2
Q
How are operons transcribed?
A
Operons are transcribed all together at once
3
Q
What does an operon produce?
A
Operon transcription produces a single mRNA molecule that encodes different proteins
4
Q
What do operons regulate?
A
Operons regulate the expression of genes by controlling transcription
5
Q
What is meant by PROG? What is this supposed to help remember?
A
PROG stands for promotor, regulator, operator and gene, which are the several components of an operon
6
Q
What are the regulator proteins?
A
Regulator proteins bind to the operators of the operon
7
Q
What control can regulator proteins have?
A
They can have negative or positive control on the operon
8
Q
What happens if the regulator protein has negative control?
A
This means that it will decrease transcription when it binds.
9
Q
What happens if the regulator protein has positive control?
A
This means that it will increase transcription when it binds
10
Q
What is the role of a regulator molecule?
A
Regulator molecules are able to alter the expression of the operon
- they’re normally metabolites
- they regulate the regulators
11
Q
Inducible?
A
If the regulatory molecule is on an inducible operon, then the precursor product is used to induce the transcription of the operon
12
Q
Repressible?
A
If the regulatory molecule is for a repressible operon, then the final product is used to repress the transcription of the operon
13
Q
What is the role of products of the operon?
A
The products of mRNA catalyze reactions in a biochemical pathway and alter the expression of the pathway
14
Q
What is the role of a regulator gene?
A
A regulator gene helps to control the expression of the structural genes of the
operon by increasing or decreasing their transcription.
15
Q
Is the regulator part of the operon?
A
Although it affects operon function, the regulator gene is not considered part of the operon
16
Q
Operons have what two types of control?
A
Operons have two types of transcriptional control:
1. Negative control
2. Positive control
17
Q
What does negative control mean?
A
Negative control, in which a regulator protein is a repressor, binding to DNA and inhibiting transcription.
18
Q
What does positive control mean?
A
Positive control, in which a regulator protein is an activator, binding to DNA and stimulating transcription.
19
Q
What is a regulator molecule?
A
Regulator Molecule = metabolite (precursor or product of metabolic pathway)
20
Q
What is an inducible operon?
A
Inducible Operon
transcription is
normally OFF
(not taking place)
21
Q
What happens when a regulator molecule binds inducible operon?
A
When Regulator Molecule binds to Regulator Protein, transcription is turned ON
22
Q
What is a repressible operon?
A
Repressible Operon
transcription is
normally ON
(taking place)
23
Q
What happens when regulator molecule binds to repressible?
A
When Regulator Molecule
binds to Regulator Protein, transcription is turned OFF
24
Q
How do we typically name the operon?
A
Effect of regulator protein + effect of molecule + operon
negative/positive + repressible/inducible
25
What happens with a negative inducible operon?
Negative Inducible Operons regulate the synthesis of the enzymes economically:
they are synthesized only when their substrate (V) is available.
26
Negative inducible: no inducer present?
The regulator protein is a repressor that binds to the operator and prevents the transcription of the structural genes
27
Negative inducible: inducer present?
When inducer is present it binds to the regulator, thereby making it unable to bind the operator, transcription takes place
28
What is allosteric inhibition?
Something binds to an allosteric (away) site, causing a change in the active site of the molecule.
29
Allosteric inhibition effect on negative inducible?
Molecule interacts with regulatory protein, no longer sable to bind to operator and transcription occurs
30
What does a negative repressible operon do?
Negative Repressible Operons can use the Product (U) to provide
negative feedback … turning off the genes involved in synthesis
31
Negative repressible: no product present?
Regulator protien is an inactive repressor, unable to bind to the operator, therefore transcription and translation takes place
32
Negative repressible: product u present?
Product u binds the regulator protein making it active and able to bind the operator, preventing transcription
33
What is the lac operon an example of?
The lac operon of E. coli is an example of a negative inducible operon
34
Who described the lac operon of E. coli?
François Jacob and Jacques Monod first described the “operon model” for the genetic control of lactose metabolism in E. coli.
35
How does lactose metabolism occur?
1. permeate transports lactose into the cell
2. B-galactosidase breaks it into galactose and glucose
3. lactose is also broken down into allolactose
4. allolactose is also converted to galactose and glucose
36
What is the role of permease?
Permease allows the lactose into the cell
37
What is the role of B-galactosidase?
This enzyme cleaves the bond between galactose and glucose
38
What are the different enzymes encoded by?
The enzymes β-galactosidase, permease, and transacetylase are
encoded by adjacent structural genes in the lac operon and have a common promoter (lacP)
39
lacZ, lacY, and lacA role?
β-Galactosidase - lacZ gene,
permease - lacY gene, and
transacetylase - lacA gene
40
What occurs with the lac operon in the absence of lactose?
In the absence of lactose, the regulator protein (a repressor) binds to the operator and inhibits transcription
41
What happens when lactose is present?
When lactose is present, some is converted to allolactose, which then binds to the regulator protein making the protein inactive
- the regulator protein cannot bind to the operator
- structural genes are transcribed and translated
42
How did Jacob and Manod determine the function of the lac operon?
Jacob and Monod worked out the structure and function of the lac operon by analyzing mutations that affected lactose metabolism.
43
What did they use?
Partial-diploid strains of E. coli were used.
44
What are the two ways that the lac operon can act?
Mutations on bacterial DNA and the plasmid showed some parts of lac operon are cis acting (able to control the expression of genes on
the same piece of DNA), whereas other parts are trans acting (able to control the expression of genes on other DNA molecules)
45
What is meant by cis acting?
able to control the expression of genes on
the same piece of DNA
46
What is meant by trans acting?
able to control the expression of genes on other DNA molecules
47
What is a plasmid?
A plasmid is a small DNA molecule within a cell that is physically separated from a
chromosomal DNA and can replicate independently.
48
What are the 4 kinds of mutations of the lac operon?
* Structural-gene mutations
* Regulator-gene mutations
* Operator mutations
* Promoter mutations
49
What do mutations on lacZ and lacY cause?
Mutations on lacZ or lacY structural genes altered the amino acids and affected
the structure of the proteins.
50
Independence of structural mutations?
Mutations of the lacZ and lacY genes were independent and usually affected only the product of the gene in which the mutation occurred.
51
What happens in the presence of lactose?
β-galactosidase and permease were produced normally in the presence of lactose
52
How is the genotype of a partial diploid written?
The genotype of a partial diploid is written by separating the genes on each DNA molecule
with a slash
functional/nonfunctional / nonfunctional/functional
53
Regulator gene mutations?
A mutation in the regulator gene (lacI) causes constitutive expression because the regulator is unable to bind the operator region.
54
Regulator gene mutation - super repressor?
If there's a super repressor, it will NOT bind lactose, meaning that it will be permanently repressed, as lactose presence will never cause the repressor to fall off.
55
What happens if the lacI gene is trans dominant?
The partial diploid lacI+ lacZ−/ lacI− lacZ+ produces β-galactosidase
only in the presence of lactose because the lacI gene is trans dominant.
56
What happens with the LacS gene?
The partial diploid lacIs IacZ+/ lacI+ lacZ+ fails to produce β-galactosidase in the presence and absence of lactose because the lacIs gene encodes a superrepressor.
57
What is an operator mutation?
Operator Mutation (lacOc) is constitutive & cis acting.
Even in the absence of lactose transcription is turned on.
58
Operator mutation causes?
The repressor is prevented from binding to the operator, therefore as long as lactose is present, the operon is transcribed.
59
What does designated lacP- cause (promotor mutation)?
Designated lacP−, interferes with the binding of RNA
polymerase to the promoter (cis acting).
60
What happens with E. coli that has lacP-?
E. coli strains with lacP− mutations don’t produce lac
proteins either in the presence or in the absence of lactose.
61
What partial diploid exhibits normal lacZ transcription?
The partial diploid
lacI+lacP+lacZ+ / lacI+lacP−lacZ+ exhibits normal synthesis of β-galactosidase
62
What diploid fails to produce lacZ transcription?
lacI+lacP−lacZ+ / lacI+lacP+lacZ− fails to produce β-galactosidase whether or not lactose is present.
63
Location of structural gene mutations?
lacZ, lacY, affecting only the two of them
64
Effect of structural gene mutations?
Alter amino acid sequence of protein encoded by gene in which mutation occurs
65
Location of regulator gene mutation?
lacI - trans mutation
66
Effect of regulator gene mutation?
Affects transcription of structural genes
67
Operator mutations location?
lacO - cis
68
Effect of operator mutations?
Affect transcription of structural genes
69
Promotor mutation location?
lacP - cis
70
Effect of promotor mutation?
Affects transcription of structural genes
71
What does E. coli preferentially metabolize?
E. coli metabolize glucose preferentially, even in the
presence of lactose and other sugars.
72
Why does E. coli prefer glucose?
They do so because glucose enters glycolysis without further modification and
therefore requires less energy to metabolize than do other sugars.
73
What is catabolite repression?
When glucose is available, genes that participate in the
metabolism of other sugars are turned off through a process known as catabolite repression.
74
When does efficient transcription of the lac operon occur?
Efficient transcription of
the lac operon, for example, takes place only if lactose is
present and glucose is absent (or very low).
75
How does the lac operon exhibit catabolite repression?
lac Operon exhibits Catabolite Repression by High Glucose Positive (by CAP) Inducible (by cAMP) Control
76
At what levels can gene expression be controlled?
1. Alteration of DNA/chromatin structure
2. Transcriptional control
3. RNA processing & degradation
4. Translational control
5. Post-translational modification
77
What are the two methods of altering DNA/chromatin structure?
1. Chromatin remodelling
2. Histone modification and DNA methylation
78
What is chromatin remodelling?
Chromatin-remodeling complexes bind directly to particular sites on DNA and reposition the nucleosomes,
allowing other transcription factors and RNA polymerase to bind to promoters and initiate transcription
79
What is histone methylation?
Addition of methyl groups (CH3) to the tails of histone proteins and/or DNA brings
about either the activation or the repression of transcription.
80
What is acetylation of histones?
Addition of acetyl groups (CH3CO) to histones usually stimulates transcription
81
What is the typical result of histone methylation?
Methylation of DNA and histones causes nucleosomes to pack tightly together
- transcriptional factors cannot bind the DNA, and genes are not expressed.
82
What is the typical result of histone acetylation?
Histone acetylation results in loose packing of nucleosomes.
- transcriptional acts can bind the DNA and genes are expressed
83
What are the two types of transcriptional control?
1. Transcriptional Factors and regulator proteins
2. Enhancers and Insulators
84
What is the role of transcriptional factors and regulator proteins?
Transcriptional activator proteins stimulate and stabilize the basal transcription apparatus.
- They interact directly or
indirectly through coactivator proteins
85
What is the role of enhancers?
Enhancers are regulatory elements that affect the transcription of distant genes.
- They can stimulate any promoter in their vicinity .
86
What is the role of insulators?
Insulators block the effect of enhancers in a position dependent manner.
87
What does an insulator block?
An insulator blocks the action of an enhancer on a promoter when the
insulator lies between the enhancer and the promoter.
88
What are the 2 types of RNA processing and degradation?
1. RNA splicing and multiple 3’ cleavage sites
2. RNA Interference (siRNA, miRNA and methylation)
89
How does gene regulation occur through RNA splicing?
Alternative splicing allows pre-mRNA to be spliced in multiple ways, generating
different proteins in different tissues or at different times in development.
90
How does gene regulation occur through mutliple 3' cleavage sites?
Multiple 3’ cleavage sites use different cleavage sites to produce mRNA’s of
different length.
91
Example of alternative splicing in Drosophila sex development?
Sexual development in
Drosophila involves a cascade of gene regulation that controls alternative splicing
- Retention of exon B in the male fly results in a non-functional Tra protein
- Exon B carries a stop codon.
92
What are the three different types of RNA interference we looked at?
RNA Interference (siRNA, miRNA and methylation)
93
How does each mechanism of RNAi work?
a) Small interfering RNAs (siRNAs) degrade mRNA by cleavage.
(b) MicroRNAs (miRNAs) lead
to the inhibition of translation.
(c) Some siRNAs bring about methylation of histone proteins or DNA, inhibiting transcription.
94
How does siRNA work?
1. dsRNA cleaved by dicer enzyme
2. produces siRNAs
3. siRNAs combine with protein to form RISC
4. RISC pairs with complementary mRNA
5. complex cleaves mRNA
6. RNA is degraded
95
How does miRNA work?
1. Other dsRNA regions cleaved by dicer
2. produces miRNA
3. miRNA combines with RISC and imperfectly binds to mRNA
4. translation inhibited
96
How does siRNA cause methylation?
1. other siRNAs attach to complementary sequences in DNA and attract methylating enzymes
2. This methylates the DNA or histones and inhibits transcription
97
What are the 2 main mechanisms of translational control?
1. miRNA regulation of translation
2. Availability of components for translation
98
How does miRNA regulation of translation work?
miRNAs inhibit the translation of complementary mRNAs.
- Researchers suggest that miRNA can inhibit the initiation step of translation as well as steps after initiation, such as ribosome stalling or premature termination
99
What is the role of translational components in controlling translation?
Ribosomes, charged tRNAs, initiation factors, and
elongation factors are all required for the translation of mRNA molecules. The availability of these components affects the rate of translation and therefore influences gene expression.
100
Example of translational control by available components?
Virus exposure causes an increase In availability of initiation factors for translation
- Rate of translation and protein synthesis is increased
101
What are the types of post-translational modification?
1. Protein modifications and degradation
102
How does protein modification and degradation work?
Many eukaryotic proteins are extensively modified after translation by the selective cleavage and trimming of amino acids from the ends, by acetylation, or by the addition of phosphate groups, carboxyl groups,
methyl groups, carbohydrates, or ubiquitin (a small protein).
- Control of these modifications, which affect the transport, function, stability, and activity of the proteins.
103
At what levels are genes regulated in bacteria vs. eukaryotes?
Bacterial: mainly transcription
Eukaryotes: many levels
BIOL2030
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