3. Types of Control
Negative ControlNegative Control Product of regulatory geneProduct of regulatory gene
inhibits transcriptioninhibits transcription
Positive ControlPositive Control Product of regulatory geneProduct of regulatory gene
enhances transcriptionenhances transcription
4. Operon
• Unit of coordinate gene expression
• Includes structural genes and their adjacent
regulatory elements
• We will consider
– Lac operon (inducible)
– Ara operon (inducible)
– Trp operon (repressible)
5. Types of Operons
InducibleInducible Initial condition: OFFInitial condition: OFF
Inducer switches operon ONInducer switches operon ON
RepressibleRepressible Initial condition: ONInitial condition: ON
Repressor switches operon OFFRepressor switches operon OFF
10. Introduction
• regulator gene – A gene that codes for a
product (typically protein) that controls the
expression of other genes (usually at the level
of transcription).
• structural gene – A gene that codes for any
RNA or protein product other than a
regulator. Figure 26.01: A regulator gene
codes for a protein that acts at
a target site on DNA.
11. • In negative regulation, a repressor protein binds to an
operator to prevent a gene from being expressed.
• In positive regulation, a transcription factor is required
to bind at the promoter in order to enable RNA
polymerase to initiate transcription.
Figure 26.02: In negative control, a trans-
acting repressor binds to the cis-acting
operator to turn off transcription.
Figure 26.03: In positive control, a trans-
acting factor must bind to cis-acting site in
order for RNA polymerase to initiate
transcription at the promoter.
12. • The lac operon contains three genes: lacZ, lacY,
and lacA. These genes are transcribed as a single
mRNA, under control of one promoter.
• Genes in the lac operon specify proteins that
help the cell utilize lactose.
• lacZ encodes an enzyme that splits lactose into
monosaccharides (single-unit sugars) that can be
fed into glycolysis.
• Similarly, lacY encodes a membrane-embedded
transporter that helps bring lactose into the cell.
14. • In addition to the three genes, the lac operon
also contains a number of regulatory DNA
sequences. These are regions of DNA to which
particular regulatory proteins can bind,
controlling transcription of the operon.
15. • The promoter is the binding site for RNA polymerase, the enzyme
that performs transcription.
• The operator is a negative regulatory site bound by
the lac repressor protein. The operator overlaps with the promoter,
and when the lacrepressor is bound, RNA polymerase cannot bind
to the promoter and start transcription.
• The CAP binding site is a positive regulatory site that is bound by
catabolite activator protein (CAP). When CAP is bound to this site, it
promotes transcription by helping RNA polymerase bind to the
promoter.
16. The lac repressor
• The lac repressor is a protein that represses (inhibits) transcription of the lac
operon.
• It does this by binding to the operator, which partially overlaps with the
promoter. When bound,the lac repressor gets in RNA polymerase's way and
keeps it from transcribing the operon.
• When lactose is not available, the lac repressor binds tightly to the operator,
preventing transcription by RNA polymerase.
• However, when lactose is present, the lac repressor loses its ability to bind
DNA. It floats off the operator, clearing the way for RNA polymerase to
transcribe the operon.
•
17.
18. allolactose
• This change in the lac repressor is caused by the small
molecule allolactose, an isomer (rearranged version) of
lactose.
• When lactose is available, some molecules will be
converted to allolactose inside the cell. Allolactose binds to
the lac repressor and makes it change shape so it can no
longer bind DNA.
• Allolactose is an example of an inducer, a small molecule
that triggers expression of a gene or operon.
• The lac operon is considered an inducible operon because
it is usually turned off (repressed), but can be turned on in
the presence of the inducer allolactose.
19. Catabolite activator protein (CAP)
• When lactose is present, the lac repressor loses its
DNA-binding ability.
• This clears the way for RNA polymerase to bind to the
promoter and transcribe the lac operon.
• CAP isn't always active (able to bind DNA). Instead, it's
regulated by a small molecule called cyclic
AMP (cAMP). cAMP is a "hunger signal" made by E.
coli when glucose levels are low.
• cAMP binds to CAP, changing its shape and making it
able to bind DNA and promote transcription. Without
cAMP, CAP cannot bind DNA and is inactive
20. • CAP is only active when glucose levels are low
(cAMP levels are high). Thus, the lac operon
can only be transcribed at high levels when
glucose is absent.
• This strategy ensures that bacteria only turn
on the lac operon and start using lactose after
they have used up all of the preferred energy
source (glucose).
23. • Glucose present, lactose absent: No
transcription of the lac operon occurs. That's
because the lac repressor remains bound to
the operator and prevents transcription by
RNA polymerase. Also, cAMP levels are low
because glucose levels are high, so CAP is
inactive and cannot bind DNA.
•
24.
25. • Glucose present, lactose present: Low-level
transcription of the lac operon occurs.
The lac repressor is released from the
operator because the inducer (allolactose) is
present. cAMP levels, however, are low
because glucose is present. Thus, CAP remains
inactive and cannot bind to DNA, so
transcription only occurs at a low, leaky level.
26.
27. • Glucose absent, lactose absent: No transcription
of the lac operon occurs. cAMP levels are high
because glucose levels are low, so CAP is active
and will be bound to the DNA. However,
the lac repressor will also be bound to the
operator (due to the absence of allolactose),
acting as a roadblock to RNA polymerase and
preventing transcription.
•
28.
29. • Glucose absent, lactose present: Strong
transcription of the lac operon occurs.
The lac repressor is released from the operator
because the inducer (allolactose) is present.
cAMP levels are high because glucose is
absent, so CAP is active and bound to the DNA.
CAP helps RNA polymerase bind to the
promoter, permitting high levels of
transcription.
34. 2. When lactose is present
• A small amount of a sugar allolactose is formed within
the bacterial cell. This fits onto the repressor protein at
another active site (allosteric site)
• This causes the repressor protein to change its shape
(a conformational change). It can no longer sit on the
operator site. RNA polymerase can now reach its
promoter site
Promotor site
z y a
DNA
I O
40. Alternative RNA Structures from 5’ UTR
Termination signal due toTermination signal due to
hairpin formed by 3+4 pairinghairpin formed by 3+4 pairing
followed by string of uracilsfollowed by string of uracils
No terminationNo termination
signal formedsignal formed
Formation of termination signal depends onFormation of termination signal depends on
level of tryptophan carried by tRNA in the cell.level of tryptophan carried by tRNA in the cell.
43. Summary of Trp Operon Regulation
Level ofLevel of
TryptophanTryptophan
Trp OperonTrp Operon
LowLow
HighHigh
OnOn
Trp repressor inactiveTrp repressor inactive
Lack of attenuation leads to high rate ofLack of attenuation leads to high rate of
mRNA productionmRNA production
OffOff
Tryptophan + repressor = Active repressorTryptophan + repressor = Active repressor
Reduction of mRNA production by attenuationReduction of mRNA production by attenuation
44. Gene expression in eukaryotes
• In eukaryotic cells, the ability to express biologically active proteins comes
under regulation at several points:
• 1. Chromatin structure The physical structure of the DNA, as it exists
compacted into chromatin, can affect the ability of transcriptional
regulatory proteins (termed transcription factors) and RNA polymerases
to find access to specific genes and to activate transcription from them.
The presence modifications of the histones and of CpG methylation most
affect accessibility of the chromatin to RNA polymerases and transcription
factors.
• 2. Epigenetic control : Epigenesis refers to changes in the pattern of gene
expression that are not due to changes in the nucleotide composition of
the genome. Literally "epi" means "on" thus, epigenetics means "on" the
gene as opposed to "by" the gene.
Sanju kaladharan
45. • 3. Transcriptional initiation This is the most important mode for control of
eukaryotic gene expression (see below for more details). Specific factors
that exert control include the strength of promoter elements within the
DNA sequences of a given gene, the presence or absence of enhancer
sequences (which enhance the activity of RNA polymerase at a given
promoter by binding specific transcription factors), and the interaction
between multiple activator proteins and inhibitor proteins.
• 4. Transcript Processing and Modification: Eukaryotic mRNAs must be
capped and polyadenylated, and the introns must be accurately removed
(see RNA Synthesis Page). Several genes have been identified that
undergo tissue-specific patterns of alternative splicing, which generate
biologically different proteins from the same gene.
Sanju kaladharan
46. • 5. RNA Transport: A fully processed mRNA must leave the nucleus in
order to be translated into protein.
• 6. Transcript Stability: Unlike prokaryotic mRNAs, whose half-lives
are all in the range of 1 to 5 minutes, eukaryotic mRNAs can vary
greatly in their stability. Certain unstable transcripts have sequences
(predominately, but not exclusively, in the 3'-non-translated regions)
that are signals for rapid degradation.
• 7.Transalational initiation Since many mRNAs have multiple
methionine codons, the ability of ribosomes to recognize and initiate
synthesis from the correct AUG codon can affect the expression of a
gene product. Several examples have emerged demonstrating that
some eukaryotic proteins initiate at non-AUG codons. This
phenomenon has been known to occur in E. coli for quite some time,
but only recently has it been observed in eukaryotic mRNAs.
Sanju kaladharan
47. • 8. Small Rna mediated Within the past several years a new model of
gene regulation has emerged that involves control exerted by small
non-coding RNAs. This small RNA-mediated control can be exerted
either at the level of the translatability of the mRNA, the stability of
the mRNA or via changes in chromatin structure.
• 9. Post transalational activation Common modifications include
glycosylation, acetylation, fatty acylation, disulfide bond formations,
etc.
• 10. Protein Transport: In order for proteins to be biologically active
following translation and processing, they must be transported to
their site of action.
• 11. Control of Protein Stability: Many proteins are rapidly degraded,
whereas others are highly stable. Specific amino acid sequences in
some proteins have been shown to bring about rapid degradation.
Sanju kaladharan