NPTEL – Biotechnology- Systems Biology
Genetic Switches-2
Dr. M. Vijayalakshmi
School of Chemical and Biotechnology
SASTRA University
Joint Initiative of IITs and IISc – Funded by MHRD
Page 1 of 11
NPTEL – Biotechnology- Systems Biology
Table of Contents
1 THE CONCEPT OF THE LAC OPERON- TRANSCRIPTION ACTIVATION
AND REPRESSION CONTROL ..................................................................................... 3
1.1 LACTOSE OPERON .................................................................................................... 3
1.1.1 When both glucose and lactose are absent ..................................................... 5
1.1.2 When both glucose and lactose are present ................................................... 5
1.1.3 When lactose is present and glucose is absent ............................................... 5
2 RIBOSWITCHES- REGULATORY FUNCTIONS OF RNA ......................................... 6
2.1 WHAT DO WE KNOW ABOUT RIBOSWITCHES? ............................................................... 7
2.2 MECHANISM OF FUNCTION OF RIBOSWITCHES ............................................................. 9
2.3 HOW RELEVANT IS THE STUDY OF GENETIC SWITCHES TO SYSTEMS BIOLOGY? ............ 10
3 REFERENCES ........................................................................................................... 11
3.1 TEXT BOOKS .......................................................................................................... 11
3.2 LITERATURE REFERENCES ...................................................................................... 11
Joint Initiative of IITs and IISc – Funded by MHRD
Page 2 of 11
NPTEL – Biotechnology- Systems Biology
1 The Concept of the lac operon- Transcription Activation
and Repression Control
Our earlier discussion on genetic switches gave us an idea of how genetic switches
function in the cell and how they elegantly control cellular functions. We discussed the
specific case of the simple tryptophan repressor which functions as a switch in bacterial
systems. Let us now shift our discussions to complicated types of switching circuits in
nature. The complexities in switching in such circuits are brought about by positive and
negative controls in the circuit. The classic example of such a switching is the lac
operon. The lac operon in E.coli is regulated by the lac repressor and CAP (Catabolite
Activator Protein) and is controlled both negatively and positively at the transcriptional
state.
1.1 Lactose operon
The lac operon consists of a cluster of functionally related genes controlled by a single
promoter. This operon includes the promoter and operator apart from three structural
genes lacZ, lacy and lacA. LacZ codes for beta galactosidase, lacY codes for permease
and lacA codes for transacetylase. Fig 1(a) explains the gene regulation in Lac operon.
Here Beta galactosidase is a cytoplasmic protein which hydrolyses lactose, Permease
acts as lactose importer, and transacetylase detoxifies toxic beta galactosides.
Lactose is a disaccharide which upon hydrolysis forms glucose and galactose in the cell
and eventually to break it down while the CAP facilitates bacteria to utilise lactose in the
absence of glucose. In the absence of lactose, it is not required for CAP to induce the
expression of the lac operon. Here the lac repressor ensures that the lac operon is shut
off. This facilitates the controlled region of lac operon to integrate two different signals,
ensuring that the operon is expressed only when both conditions are met- lactose
present and glucose absent. The other three possible combinations of signals keep the
cluster of genes in the OFF state. The lac operon codes for proteins that transport
lactose into small amount of permease is found even under repressive condition. The
Joint Initiative of IITs and IISc – Funded by MHRD
Page 3 of 11
NPTEL – Biotechnology- Systems Biology
regulator produces a repressor which binds to operator and prevents RNA polymerase
from binding to promotor and thus inhibits transcription of three structural genes. The
gene regulation process in lac operon is illustrated in Fig 1 (b).
(a)
(b)
Fig 1. Gene regulation through the Lac operon (a) In the presence of Lactose; (b) In the absence of
Lactose
Joint Initiative of IITs and IISc – Funded by MHRD
Page 4 of 11
NPTEL – Biotechnology- Systems Biology
1.1.1 When both glucose and lactose are absent
Since the lactose (inducer) is not available to bind to the repressor protein, hence the
repressor binds to the promoter region and terminates the process of transcription. As a
result there is no gene expression.
1.1.2 When both glucose and lactose are present
Under a such condition, the bacteria prefer glucose and utilise lactose only when
glucose is exhausted, thus recording two growth curves. This is called diauxic growth.
1.1.3 When lactose is present and glucose is absent
Lactose is taken in with the help of permease and is converted into allo-lactose. Allolactose binds to the repressor and makes it non functional and thus the three structural
genes are transcribed.
Thus the lac– operon is an example of a negative inducible operon -negative with
reference to effect of repressor on transcription of structural genes and inducible with
reference to effect of lactose on structural gene transcription. Under this situation the
levels of cAMP are high.
Fig 2: a. Glucose present (cAMP low); no lactose; no lac mRNA
b. Glucose present (cAMP low); lactose present
c. No glucose present (cAMP high); lactose present
Joint Initiative of IITs and IISc – Funded by MHRD
Page 5 of 11
NPTEL – Biotechnology- Systems Biology
Video1 – Function of the Lac Operon
Joint Initiative of IITs and IISc – Funded by MHRD
Page 6 of 11
NPTEL – Biotechnology- Systems Biology
2 Riboswitches- Regulatory functions of RNA
The central dogma of life has always portrayed the nucleic acids as the blue print for a
cell assigning the regulatory and enzymatic functions to the proteins sysnthesised in the
cell. Recent work on RNA over the past decade and explorations of its newer functions
in the cell strongly challenge the text book view of the central dogma. Though RNA
molecules have been shown to be involved in cleavage, splicing and translation and
novel gene regulatory mechanisms operating at both the DNA and mRNA level have
been explored in detail. Newer functions of specific RNAs that can function as sensors
of vitamin B1, B2 and B12 cofactors, have taken center stage. Riboswitches are a
fascinating type of RNA structures that regulate gene expression both at the
transcription and translation levels by binding to small molecules (ligands). These are
structures that form in a messenger RNA and are involved predominantly in gene
regulation events in bacteria.
These riboswitches regulate gene expression through the formation of alternative
structures which either prematurely terminate transcription or inhibit the initiation of
translation when they are in the repressing conformation. Riboswitches regulate the
synthesis of Riboflavin, Thiamin and Cobalamin and the metabolism of Methionine,
Lysine and Purines. Riboswitches are present in bacterial species, in fungi and in
plants. More than 2% of the Bacillus subtilis genome has been shown to be regulated
by riboswitches.
2.1 What do we know about riboswitches?
Riboswitches fold into compact RNA secondary structures which comprise a base stem,
a central multi loop and several branching hair pins as shown in Fig 3.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 7 of 11
NPTEL – Biotechnology- Systems Biology
(a)
(b)
(c)
Fig 3. Structure of riboswitches; (a) RFN-element; (b) G-box; (c) B12-element
The riboswitches distinguish themselves strikingly form other regulatory systems
through two features. The first deals with the fact that riboswitches are present across
diverse organisms. For example the THI elements are observed in eubacteria, archea
and eukaryotes. The S-boxes, G-boxes and L-boxes are observed in gram-positive
bacteria from the Bacillus, Thermotogale and Bacteroidetes species. The next
outstanding feature of the riboswitches is that they regulate diverse processes as
Joint Initiative of IITs and IISc – Funded by MHRD
Page 8 of 11
NPTEL – Biotechnology- Systems Biology
riboflavin and transport, thiamin synthesis and transport, purine metabolism and
synthesis etc as in Table 1.
Table 1
Riboswitches and their properties
Riboswitches Functional system
RFN-element
Riboflavin biosynthesis and
transport
Thiamin biosynthesis ;
THI-element
transport of thiamin and related
compounds
Ligand
FMN (flavin mononucleotide)
TPP (thiamin
pyrophosphate)
Cobalamin biosynthesis; transport
of cobalamin and related
B12-element
compounds; cobalt transport;
Coenzyme B12
cobalamin-independent
(adenosylcobalamin)
isoenzymes of cobalamindependent enzymes
S-box
Methionine biosynthesis and
SAM (S-
transport SAM metabolism
adenosylmethionine)
2.2 Mechanism of function of Riboswitches
As stated earlier, regulation through riboswitches involves the formation of alternative
structures.
Condition 1: Repressed state
During repression two RNA structures are formed, the small molecule ligand binds to
the structure and stabilizes the switch forming the regulatory hairpin. This hairpin
Joint Initiative of IITs and IISc – Funded by MHRD
Page 9 of 11
NPTEL – Biotechnology- Systems Biology
sequesters to the ribosome binding site and can function either to terminate the
transcription process or to inhibit the initiation of translation.
Condition 2: Derepressed state
During the derepressed state, the riboswitches is not involved in ligand binding and
therefore forms an alternative structure comprising the complementary regions in the
riboswitches base stems and a portion of the regulatory hairpin. When the riboswitches
directly sequesters the site of translation initiation variations occur in this switch. On
change of parity the riboswitches functions as an alternative to the regulatory hairpin,
activating gene expression in the presence of the ligand and repressing gene
expression when it is not bound.
2.3 How relevant is the study of genetic switches to Systems Biology?
Molecular Biology techniques and precise genetics over the past decades have
established the genetic switch as a leading theme of gene regulation. The availability of
vast amounts of data from genomics, proteomics and high throughput experimentation,
have enabled biology to move from the component centric paradigm to a systems level
quest to understand how specific parts of system function together to carry out complex
functions. Systems Biology approaches would help illustrate the factors that regulate the
efficiency of the switch. Systems Biology can help in modelling the long range
interactions between the regulatory proteins in the network and the cooperatively
involved in such tight negative auto regulatory networks. The evolution of the switch is
an interesting paradigm to be explored using Systems Biology. A question like how
robust is the switch against molecular level alterations, how stochastic is the gene
regulatory process under such switching would throw interesting discussions on these
themes. The construction of genetic toggle switches in vivo would also be an off shoot
of such explorations.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 10 of 11
NPTEL – Biotechnology- Systems Biology
3 References
3.1 Text Books
1. Alberts B, Bray D, Lewis J. et al., Molecular Biology of the Cell, Garland Science,
(1994),
2. Mark Ptashne , A Genetic Switch-Phage Lambda Revisited, CSHL Press, U.S.A,
(2004).
3.2 Literature References
1. Gardener Timothy S et al., Construction of a genetic toggle switch in Escherichia
coli, Nature, (2000), 403, 339-342.
2. Alexander Serganov, Evgeny Nudler, A decade of riboswitches, Cell, (2013),
152, 17-24.
3. Breaker RR, Complex Riboswitches, Science, (2008), 319, 1795-1797.
Joint Initiative of IITs and IISc – Funded by MHRD
Page 11 of 11