1. The document discusses gene regulation in bacteria, focusing on the lac operon in E. coli. which controls lactose metabolism. 2. The lac operon contains 3 genes - lacZ, lacY, lacA - that are regulated together. When lactose is present, it is broken down into allolactose, which binds the lac repressor and prevents it from blocking transcription of the operon. 3. Experiments by Jacob and Monod identified mutations affecting regulation, including mutations in the operator or repressor gene that result in constitutive expression of the lac genes.
SOS response was discovered by Miroslav Radman. It's a part of DNA repair system- synthesizes enzymes required for DNA repair. Cellular response to UV damage.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
Topics included - Introduction; explanation; examples like blue white screening method, antibiotic resistance; Extra information regarding - detection of oncogene in vertebrates and role of sleeping beauty; Merits and demerits of insertional inactivation.
Introduction
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
RNA Polymerase
Introduction
Purification
History
PRODUCTS OF RNAP
Messenger RNA
Non-coding RNA or "RNA genes
Transfer RNA
Ribosomal RNA
Micro RNA
Catalytic RNA (Ribozyme)
prokaryotic and eukaryotic
Transcription by RNA Polymerase
TYPES OF RNA POLYMERASE
Type I
Type II
Type III
Prokaryotic Transcription Unit
EXPRESSION OF A PROKARYOTIC GENE
Prokaryotic Polycistronic Message Codes for Several Different Proteins
Eukaryotic Transcription Unit
ENHANCERS AND SILENCERS
RESULT OF THE TRANSCRIPTION CYCLE
RNAP III TRANSCRIBES HUMAN MICRORNAS
RNAP I–specific subunits promotepolymerase clustering to enhance the rRNA genetranscription cycle
RNAP II–TFIIB STRUCTURE ANDMECHANISM OF TRANSCRIPTION INITIATION
FIVE CHECKPOINTS MAINTAINING THE FIDELITY OFTRANSCRIPTION BY RNAP IN STRUCTURAL ANDENERGETIC DETAILS
This presentation contains information about restriction enzymes, its nomenclature, restriction digestion, and its application. This also contains information about the chemicals used in restriction and also explains the general procedure of restriction digestion of DNA
This presentation provide knowledge about Gene Expression & its regulation in brief.
i hope it gives some information about gene expression in your academic time.
In this presentation mentioned - Lac Operon and its expressor.
SOS response was discovered by Miroslav Radman. It's a part of DNA repair system- synthesizes enzymes required for DNA repair. Cellular response to UV damage.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
Topics included - Introduction; explanation; examples like blue white screening method, antibiotic resistance; Extra information regarding - detection of oncogene in vertebrates and role of sleeping beauty; Merits and demerits of insertional inactivation.
Introduction
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
RNA Polymerase
Introduction
Purification
History
PRODUCTS OF RNAP
Messenger RNA
Non-coding RNA or "RNA genes
Transfer RNA
Ribosomal RNA
Micro RNA
Catalytic RNA (Ribozyme)
prokaryotic and eukaryotic
Transcription by RNA Polymerase
TYPES OF RNA POLYMERASE
Type I
Type II
Type III
Prokaryotic Transcription Unit
EXPRESSION OF A PROKARYOTIC GENE
Prokaryotic Polycistronic Message Codes for Several Different Proteins
Eukaryotic Transcription Unit
ENHANCERS AND SILENCERS
RESULT OF THE TRANSCRIPTION CYCLE
RNAP III TRANSCRIBES HUMAN MICRORNAS
RNAP I–specific subunits promotepolymerase clustering to enhance the rRNA genetranscription cycle
RNAP II–TFIIB STRUCTURE ANDMECHANISM OF TRANSCRIPTION INITIATION
FIVE CHECKPOINTS MAINTAINING THE FIDELITY OFTRANSCRIPTION BY RNAP IN STRUCTURAL ANDENERGETIC DETAILS
This presentation contains information about restriction enzymes, its nomenclature, restriction digestion, and its application. This also contains information about the chemicals used in restriction and also explains the general procedure of restriction digestion of DNA
This presentation provide knowledge about Gene Expression & its regulation in brief.
i hope it gives some information about gene expression in your academic time.
In this presentation mentioned - Lac Operon and its expressor.
BIO cell and molecular bio ANSWER ALL1 which of the fo.pdflongojasperze84880
because of a hurricane, your regular laboratory session was cancelled and the gram stain
procedure was performed on cultures incubated for a longer period of time. Examination of the
stain staphylococcus aureus slides revealed a great deal of color variability ranging from intese
blue to shades of pink. Account for this result.
Solution
Staphylococcus aureus is a Gram positive, non motile bacteria. On Gram staining it will appear
purple to blue color since it will retain crystal violet dye. Again, Gram negative bacteria say for
example, E.coli will appear pink in color.
So, here due to longer period of incubation the culture has been contaminated with Gram
negative bacteria. Therefore showing color variability in Gram staining results..
Bio cell and molecular Answer ALL1 although all of the.pdfarpitcomputronics
Bio: cell and molecular
Answer ALL
1 although all of the steps involved in expressing a gene can in principle be regulated, for most
genes the initiation of __________ is the most important point of control.
2 the general transcription factors that assemble at a eukaryotic promoter are different, depending
on the specific gene being transcribed by polymerase II.
True or False?
3 Which of the following describes the lac operon in E. coli when lactose, but not glucose, is
present in the culture medium?
A) CAP, but not the lac repressor, is bound to the lac operon\'s regulatory DNA, and the lac
operon is expressed.
B) CAP and the lac repressor are both bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
C) The lac repressor, but not CAP, is bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
D) Neither CAP nor the lac repressor is bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
Bio: cell and molecular
Answer ALL
1 although all of the steps involved in expressing a gene can in principle be regulated, for most
genes the initiation of __________ is the most important point of control.
2 the general transcription factors that assemble at a eukaryotic promoter are different, depending
on the specific gene being transcribed by polymerase II.
True or False?
3 Which of the following describes the lac operon in E. coli when lactose, but not glucose, is
present in the culture medium?
A) CAP, but not the lac repressor, is bound to the lac operon\'s regulatory DNA, and the lac
operon is expressed.
B) CAP and the lac repressor are both bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
C) The lac repressor, but not CAP, is bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
D) Neither CAP nor the lac repressor is bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
Answer ALL
1 although all of the steps involved in expressing a gene can in principle be regulated, for most
genes the initiation of __________ is the most important point of control.
2 the general transcription factors that assemble at a eukaryotic promoter are different, depending
on the specific gene being transcribed by polymerase II.
True or False?
3 Which of the following describes the lac operon in E. coli when lactose, but not glucose, is
present in the culture medium?
A) CAP, but not the lac repressor, is bound to the lac operon\'s regulatory DNA, and the lac
operon is expressed.
B) CAP and the lac repressor are both bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
C) The lac repressor, but not CAP, is bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
D) Neither CAP nor the lac repressor is bound to the lac operon\'s regulatory DNA, and the lac
operon is not expressed.
1 although all of the steps involved in expressing a gene can in principle be regulated, for.
The basics of Lactose Operon of prokaryotic cell and its detailed information for students at graduation level. An interesting topic of Molecular Biology. It is mainly for students of Life Science.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
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2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
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Acetabularia acetabulum is a single-celled green alga that in its vegetative state is morphologically differentiated into a basal rhizoid and an axially elongated stalk, which bears whorls of branching hairs. The single diploid nucleus resides in the rhizoid.
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
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Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
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1. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 1
CHAPTER 19
Regulation of Gene Expression in
Bacteria and Bacteriophages
Peter J. Russell
edited by Yue-Wen Wang Ph. D.
Dept. of Agronomy, NTU
A molecular Approach 2nd Edition
2. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 2
The lac Operon of E. coli
1. Growth and division genes of bacteria are regulated genes. Their expression is
controlled by the needs of the cell as it responds to its environment with the
goal of increasing in mass and dividing.
2. Genes that generally are continuously expressed are constitutive genes
(housekeeping genes). Examples include protein synthesis and glucose
metabolism.
3. All genes are regulated at some level, so that as resources dwindle the cell can
respond with a different molecular strategy.
4. Prokaryotic genes are often organized into operons that are cotranscribed. A
regulatory protein binds an operator sequence in the DNA adjacent to the gene
array, and controls production of the polycis-tronic (polygenic) mRNA.
5. Gene regulation in bacteria and phage is similar in many ways to the emerging
information about gene regulation in eukaryotes, including humans. Much
remains to be discovered; even in E. coli, one of the most closely studied
organisms on earth, 35% of the genomic ORFs have no attributed function.
3. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 3
The lac Operon of E. coli
Animation: Regulation of Expression of the lac Operon
Genes
1. An inducible operon responds to an inducer substance
(e.g., lactose). An inducer is a small molecule that joins
with a regulatory protein to control transcription of the
operon.
2. The regulatory event typically occurs at a specific DNA
sequence (controlling site) near the protein-coding
sequence (Figure 19.1).
3. Control of lactose metabolism in E. coli is an example of
an inducible operon.
5. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 5
Lactose as a Carbon Source for E. coli
1. E. coli expresses genes for glucose metabolism constitutively, but the genes for
metabolizing other sugars are regulated in a “sugar specific” sort of way. Presence of the
sugar stimulates synthesis of the proteins needed.
2. Lactose is a disaccharide (glucose 1 galactose). If lactose is E. coli’s sole carbon source,
three genes are expressed:
a. β-galactosidase has two functions:
i. Breaking lactose into glucose and galactose. Galactose is converted to glucose, and glucose
is metabolized by constitutively produced enzymes.
ii. Converting lactose to allolactose (an isomerization). Allolactose is involved in regulation
of the lac operon (Figure 19.2).
b. Lactose permease (M protein) is required for transport of lactose across the cytoplasmic
membrane.
c. Transacetylase is poorly understood.
3. The lac operon shows coordinate induction:
a. In glucose medium, E. coli normally has very low levels of the lac gene products.
b. When lactose is the sole carbon source, levels of the three enzymes increase coordinately
(simultaneously) about 1,000-fold.
i. Allolactose is the inducer molecule (Figure 16.2).
ii. The mRNA for the enzymes has a short half-life. When lactose is gone, lac transcription
stops, and enzyme levels drop rapidly.
7. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 7
Experimental Evidence for the Regulation of lac
Genes
1.The experiments of Jacob and Monod produced
an understanding of arrangement and control of
the lac genes.
8. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 8
Mutations in the Protein-coding Genes
1. Mutagens produced mutations in the lac structural genes that were used to
map their locations.
a. β-galactosidase is lacZ.
b. Permease is lacY.
c. Transacetylase is lacA.
d. The genes are tightly linked in the order: lacZ-lacY-lacA
2. The type of mutation made a difference in expression of the downstream genes:
a. Missense mutations affect only the product of the gene with the mutation.
b. Nonsense mutations show polarity (polar mutations), and affect translation of the
downstream genes as well.
3. The interpretation of gene polarity is that ribosomes translate the first gene in the
polycistronic (polygenic) RNA, and finish in proper position to initiate and
translate the next gene. Premature translation termination prevents this by
reducing translation of the downstream genes (Figure 19.3).
10. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 10
Mutations Affecting the Regulation of Gene
Expression
1. Jacob and Monod found mutants that produced all three lac operon proteins
constitutively, and hypothesized that regulatory mutations had affected the normal
control of gene expression for the operon. Constitutive mutants are in two classes
(Figure 19.4):
a. Mutations in the lac operator (lacO) just upstream from the lacZ gene.
b. Mutations further upstream in the lac repressor gene (lacI).
2. Operator-constitutive (lacOc) mutations were defined by experiments using partial
diploid E. coli F’ strains.
a. An example is the partial diploid F’ lacO+ lacZ- lacY+/lacOc lacZ+ lacY-. (Promoters are normal
for both operons, and the lacA gene is irrelevant to the study.)
b. This strain was tested for β-galactosidase and lactose permease, both in the presence and
absence of the inducer.
c. Without inducer, β-galactosidase is produced, but only inactive permease is made.
i. β-galactosidase is produced from the chromosomal gene under control of the constitutive
promoter.
ii. Permease is produced from the chromosomal gene also, but is inactive because the gene is
mutated.
iii. No products are produced from the F’ DNA, because its promoter is wild-type, and
requires induction for gene expression.
12. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 12
d. With inducer, functional molecules of both β-galactosidase and permease are
produced. This indicates that:
i. β-galactosidase is under constitutive control (on the chromosome).
ii. Permease is under inducible control (on the F’ DNA).
e. In both cases, the promoter controls genes downstream from it on the same DNA
molecule, showing cis-dominance.
3. lacI constitutive genes were also discovered in experiments with partial diploid
strains.
a. An example is the strain lacI+ lacO+ lacZ- lacY+/lacI- lacO+ lacZ+ lacY-, in which
both gene sets have normal operators and promoters.
b. Without inducer, no β-galactosidase or permease is produced.
c. With inducer, both are produced. A lacI+ gene can overcome the lacI- mutation.
d. Since lacI+ and lacI- genes are on different DNA molecules, lacI+ is trans-dominant.
e. Jacob and Monod proposed that lacI+ produces a repressor that controls expression
of both lac operons, making them both inducible.
4. The lac promoter is also affected by mutations (Plac-). Most affect
all three structural genes, which are not made, even when the inducer is present.
a. Plac- mutations affect RNA polymerase binding to the start of the operon.
b. Only genes in the same DNA strand are affected, so Plac- mutations are cis-ominant.
iActivity: Mutations and Lactose Metabolism
13. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 13
Jacob and Monod’s Operon Model for the
Regulation of lac Genes
1. Jacob and Monod’s model of regulation, with more recent information,
follows:
a. An operon is a cluster of genes that are regulated together. The order of the lac
genes is shown in Figure 19.4, and Figure 19.5 shows the operon when lactose is
absent.
b. The lacI gene has its own constitutive weak promoter and terminator, and repressor
protein is always present in low concentration.
i. The repressor functions as a tetramer (Figure 19.6).
ii. Repressor protein binds the operator (lacO+), and prevents RNA polymerase
initiation to transcribe the operon genes (negative control).
iii. Binding of the repressor to the operator is not absolute, and so an occasional
transcript is made, resulting in low levels of the structural proteins.
c. β-galactosidase in wild-type E. coli growing with lactose as the
sole carbon source converts lactose into allolactose (Figures 19.2 and 19.7).
i. Repressor bound with allolactose bound changes shape (allosteric shift) and
dissociates from the lac operator. Free repressor-allolactose complexes are
unable to bind the operator.
ii. Allolactose induces expression of the lac operon, by removing the repressor
and allowing transcription to occur.
16. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 16
Fig. 19.7 Functional state of the lac operon in wild-type E. coli growing in the presence
of lactose as the sole carbon source
17. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 17
2. The lacOc mutations result in constitutive gene expression. They are
cis-dominant to lacO+, because repressor cannot bind to the lacOc
operator sequence (Figure 19.8).
3. The lacI- mutations change the repressor protein’s conformation and
prevent it from binding the operator, resulting in constitutive expression
of the operon (Figure 19.9).
a. In a partial diploid (lacI+ lacO+ lacZ- lacY+/lacI- lacO+ lacZ+ lacY-),
the wild-type repressor (lacI+) is dominant over lacI- mutants.
b. Defective lacI- repressor can’t bind either operator, but normal
repressor from lacI+ binds both operators and regulates transcription,
resulting in functional β-galactosidase and permease.
18. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 18
Fig. 19.8a Cis-dominant effect of lacOc mutation in a partial-diploid strain of E. coli
19. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 19
Fig. 19.8b Cis-dominant effect of lacOc mutation in a partial-diploid strain of E. coli
20. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 20
Fig. 19.9a Effects of a lacI- mutation
21. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 21
Fig. 19.9b Effects of a lacI- mutation
22. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 22
Fig. 19.9c Effects of a lacI- mutation
23. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 23
4. Additional lacI mutants have been identified:
a. Superrepressor (lacIS) mutants that produce no lac enzymes.
i. The mutant repressor cannot bind allolactose, so lactose does not induce the operon.
ii. The lacIS allele is trans-dominant in partial diploids (lacI+/lacIS) (Figure 19.10). The superrepressor protein
binds both operators and transcription cannot occur.
iii. Normal repressor cannot compete, because superrepressor cannot be induced to fall off.
iv. Low levels of transcription will occur (superrepressor is not covalently bound to the DNA operator sequence),
but lacIS E. coli
cannot use lactose as a carbon source.
b. Dominance (lac-d) mutants have missense mutations at the 5’ end of the lacI gene.
i. In haploid cells, the phenotype is constitutive expression of the lac operon.
ii. In partial diploids, lac-d is trans-dominant. The operon is expressed even in the presence of normal repressor.
iii. Normal repressor functions as a tetramer. Mutant and normal subunits combine randomly.
iv. A tetramer containing one or more mutant subunits cannot bind to operator DNA. Repression does not occur,
and so gene expression is constitutive.
c. Mutations in the lad promoter can increase or decrease the gene¡¦s transcription rate, by altering its affinity for RNA
polymerase. Examples are lacIQ and lacSQ:
i. Both of these mutations raise the transcription rate of the repressor gene. (Q stands for “quantity” and SQ for
“super quantity”)
ii. Large amounts of repressor are produced in these mutants, reducing the efficiency of lac operon induction so
that high levels of lactose are needed.
lii. Overproduction of the lad gene has been useful for isolating and characterizing the repressor molecule.
5. These mutants indicate that repressor has three different recognition interactions:
a. Binding to the operator region.
b. Binding with the inducer (allolactose).
c. Binding of repressor polypeptide subunits to form an active tetramer.
24. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 24
Fig. 19.10 Dominant effect of lacIs mutation over wild-type lacI+
25. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 25
Positive Control of the lac Operon
Animation: Positive Control of the lac Operon
1. Repressor exerts negative control by preventing transcription. Positive control of
this operon also occurs when lactose is E. coli’s sole carbon source (Figure
19.11).
a. Catabolite activator protein (CAP) binds cyclic AMP (cAMP) (Figure 19.12).
b. CAP-cAMP complex is a positive regulator of the lac operon. It binds the CAP-site, a
DNA sequence upstream of the operon’s promoter.
c. Binding of CAP-cAMP complex causes the DNA to bend, facilitating protein-protein
interactions between CAP and RNA polymerase, and leading to transcription.
2. When both glucose and lactose are in the medium, E. coli preferentially uses
glucose, due to catabolite repression.
a. Glucose metabolism greatly reduces cAMP levels in the cell.
b. The CAP-cAMP level drops, and is insufficient to maintain high transcription of the lac
genes.
c. Even when allolactose has removed the repressor protein from the operator, lac gene
transcription is at very low levels without CAP-cAMP complex bound to the CAP-site.
d. Experimental evidence supports this model. Adding cAMP to cells restored
transcription of the lac operon, even when glucose was present.
26. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 26
Fig. 19.11 Role of cyclic AMP (cAMP) in the functioning of glucose-sensitive operons
such as the lac operon of E. coli
28. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 28
3. The model is that catabolite repression targets adenylate
cyclase (the enzyme that makes cAMP) (Figure 19.12).
a. In E. coli, the phosphorylated form of IIIGlc enzyme activates
adenylate cyclase.
b. Glucose transport into the cell triggers events including
dephosphorylation of IIIGlc.
c. With IIIGlc protein dephosphorylated, adenylate cyclase is
inactivated, and no new cAMP is produced.
4. Catabolic genes for other sugars are also regulated by
catabolite repression. In all cases, a CAP site in their
promoters is bound by a CAP-cAMP complex, increasing
RNA polymerase binding.
29. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 29
Molecular Details of lac Operon Regulation
1. The sequences of significant lac regulatory regions are known. DNase
protection by regulatory molecules (e.g., repressor) is useful in these studies.
2. The lacI DNA sequence shows the expected transcription and translation
signals, except that the start codon is GUG (not AUG). The single base-pair
mutation of IacIQ is also characterized (Figure 19.13).
3. Operon controlling sites (Figure 19.14) were derived from several types of data:
a. Amino acid sequences of the repressor protein and (β-galactosidase were known,
allowing coding regions for lacI and lacZ+ to be identified.
b. Protection assays identified binding sites for:
i. CAP-cAMP complex.
ii. RNA polymerase.
iii. Repressor protein.
30. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 30
Fig. 19.13 Base pair sequences of the lac operon lacI+ gene promoter (Plac+) and of the
5' end of the repressor mRNA
31. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 31
Fig. 19.14 Base pair sequence of the controlling sites, promoter and operator, for the
lactose operon of E. coli
32. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 32
4. The lac operon promoter region begins at -84, immediately next to the lacI gene
stop codon, and ends at -8, just upstream from the transcription start site.
Features of the promoter region include:
a. The consensus sequence for CAP-cAMP binding is in two regions: -54 to -58, and -
65 to -69.
b. The RNA polymerase binding site (including a Pribnow box) spans DNA from -47
to -8, with consensus sequence matches at -10 and -35.
5. The operator is immediately next to the promoter, with repressor protein
protecting DNA from -3 to +21. With repressor bound to the operator, RNA
polymerase can bind but cannot transcribe.
6. The operon transcript begins at +1, which is within the operator region bound by
repressor. (Part of the operator is transcribed.)
a. The β-ga1actosidase gene has a leader region before the start codon.
b. Start codon for 3-galactosidase (AUG) is at + 39 to +41.
c. Several lacOC mutations have been characterized. All are single base pair
substitutions.
7. The lac operon was the first molecular model for gene regulation. Operons are
common in bacteria and phages, but unknown in eukaryotes.
33. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 33
The trp Operon of E. coli
1.If amino acids are available in the medium, E.
coli will import them rather than make them, and
the genes for amino acid biosynthesis are
repressed. When amino acids are absent, the
genes are expressed and biosynthesis occurs.
2.Unlike the inducible lac operon, the trp operon is
repressible. Generally, anabolic pathways are
repressed when the end product is available.
34. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 34
Gene Organization of the Tryptophan
Biosynthesis Genes
1. Yanofsky and colleagues characterized the controlling
sites and genes of the trp operon (Figure 19.15).
a. There are 5 structural genes, trpA through E.
b. The promoter and operator are upstream from the trpE gene.
c. Between trpE and the promoter-operator is trpL, the leader
region. Within trpL is the attenuator region (att).
d. The trp operon spans about 7 kb. The operon produces a
polygenic transcript with five structural genes for tryptophan
biosynthesis.
36. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 36
Regulation of the trp Operon
Animation: Attenuation in the trp Operon of E. coli
1. Two mechanisms regulate expression of the trp operon:
a. Repressor/operator interaction.
b. Transcription termination.
2. When tryptophan is present, it will bind to an aporepressor protein (the trpR
gene product).
a. The active repressor (aporepressor plus tryptophan) binds the trp operator, and
prevents transcription initiation.
b. Repression reduces transcription of the trp operon about 70-fold.
3. When tryptophan is limited, transcription is also controlled by
attenuation.
a. Attenuation produces only short (140-bp) transcripts that do not encode structural
proteins.
b. Termination occurs at the attenuator site within the trpL region.
c. The proportion of attenuated transcripts to full-length ones is related to tryptophan
levels, with more attenuated transcripts as the tryptophan concentration increases.
d. Attenuation can reduce trp operon transcription 8- to 10-fold. Together, repression
and attenuation regulate trp gene expression over a 560- to 700-fold range.
37. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 37
4. The molecular model for attenuation:
a. Translation of the trpL gene produces a short polypeptide. Near
the stop codon are two tryptophan codons.
b. Within the leader mRNA are four regions that can form
secondary structures by complementary base-pairing (Figure
19.16).
i. Pairing of sequences 1 and 2 creates a transcription pause
signal.
ii. Pairing of sequences 3 and 4 is a transcription termination
signal.
iii. Pairing of 2 and 3 is an antitermination signal, and so
transcription will continue.
c. Tight coupling of transcription and translation in prokaryotes
makes control by attenuation possible.
i. RNA polymerase pauses when regions 1 and 2 base pair just
after they are synthesized (Figure 19.16).
38. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 38
ii. During the pause, a ribosome loads onto the mRNA and begins
translation of the leader peptide. Ribosome position is key to
attenuation:
(1) When tryptophan (Trp) is scarce(Figure 19.17):
(a) Trp-tRNAs are unavailable, and the ribosome stalls at the Trp codons in the leader
sequence, covering attenuator region 1.
(b) When the ribosome is stalled in attenuator region 1, it cannot base pair with region
2. Instead, region 2 pairs with region 3 when it is synthesized.
(c) If region 3 is paired with region 2, it is unable to pair with region 4 when it is
synthesized. Without the region 3-4 terminator, transcription continues through the
structural genes.
(2) When Trp is abundant:
(a) The ribosome continues translating the leader peptide, ending in region 2. This
prevents region 2 from pairing with region 3, leaving 3 available to pair with
region 4.
(b) Pairing of regions 3 and 4 creates a rho-independent terminator known as the
attenuator. Transcription ends before the structural genes are reached.
5. Genetic evidence for attenuation includes:
a. Mutations in the leader sequence within regions 3 or 4 disrupt base
pairing and decrease the efficiency of termination (Figure 19.18)
b. Changes in the Trp codons of the leader peptide sequence, so that they
encode a different amino acid, cause attenuation controlled by levels
of the amino acid specified by the mutant codon, not by Trp
43. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 43
Regulation of Other Amino Acid Biosynthesis
Operons
1.Attenuation is involved in regulating many
operons(Figure 19.19).
a.When the operon is for amino acid biosynthesis,
the leader sequence always includes codons for
that amino acid.
b.Other operons regulated by attenuation include
rRNA (rrn) and E. coli ampicillin resistance
(ampC).
45. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 45
Regulation of Gene Expression in Phage Lambda
1. Phage use many bacterial components for
replication, and control their use with phage gene
products.
2. Bacteriophage λ has two possible pathways when it
enters its E. coli host:
a. The lytic cycle, in which the phage takes over the cell and
produces progeny phage.
b. The lysogenic cycle, where phage chromosome is inserted
into the E. coli chromosome, and replicates with the
bacterial genome.
47. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 47
Early Transcription Events
1. The λ chromosome is linear, with “sticky” ends used to circularize
it in the host cell. The regulatory system for choosing between
the lytic and lysogenic pathways is contained in the λ
chromosome (Figure 19.20).
a. First, transcription begins at promoters PL (leftward transcription)
and PR (rightward) (Figure 19.21).
i. The first gene transcribed from PR is cro (control of repressor
and other). The Cro protein is involved in the genetic switch
to the lytic pathway.
ii. The first protein transcribed from PL is N, which is a
transcription antiterminator that allows RNA synthesis to go
through termination regions into the early genes.
(1) N protein allows expression of the cII protein, which in
turn activates:
(a)cI (λ repressor)
(b)O and P (DNA replication proteins).
(c)Q (activation of late genes for lysis and phage particle proteins, only when Q
protein accumulates to certain levels).
50. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 50
The Lysogenic Pathway
1. Early transcription events determine whether the lytic or lysogenic pathway
occurs.
2. The lysogenic pathway results when cII and cIII are expressed.
a. The action of cII and cIII proteins activates the PRE promoter, causing transcription
of the cI (λ repressor) gene.
b. λ repressor binds to 2 operator regions, OL and OR, whose sequences overlap PL and
PR, respectively. This prevents transcription from PL and PR.
c. Transcription of N and cro genes is blocked, and concentrations of their proteins
drop rapidly.
d. In addition, repressor bound to OR causes more repressor mRNA to be made from
another promoter, PRM.
e. High levels of repressor cause lysogeny by binding operators OL and OR.
f. Integrase protein is used to integrate λ DNA into the E. coli chromosome. Integrase
transcript is initiated at the PI promoter, which is controlled by the cII protein.
g. As cII concentration drops, the PI promoter is shut down, leaving PRM as the only
active promoter.
3. Thus, the lysogenic pathway occurs when enough λ repressor is
made to turn off early promoters. Lytic genes, including Q, are not expressed.
Without the Q protein, phage coat and lysis proteins are not produced.
51. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 51
The Lytic Pathway
1. An example of induction of the lytic pathway is exposure to UV light.
a. UV causes the bacterial RecA protein (normally used in DNA repair) to
stimulate the λ repressor proteins to autocleave and become
inactivated.
b. Absence of repressor at OR allows transcription of the cro gene.
c. Cro protein decreases RNA synthesis from PL and PR, reducing
synthesis of cII, therefore blocking synthesis of λ repressor.
d. Transcription from PR is also decreased, but Q protein levels are
sufficient to allow transcription of the late genes needed for the lytic
pathway.
2. Thus, λ uses complex regulatory systems to control entry into the lytic
or lysogenic pathway. The decision depends on competition between
the repressor and the Cro protein.
a. If repressor dominates, lysogeny takes place.
b. If the Cro protein dominates, the lytic pathway occurs.