Download Lecture 14 Gene Regulation

Chapter 19
The Regulation of Gene
Expression
• In bacteria, growth and cell division are
controlled by genes. These genes’
expression is controlled by the needs of
the cell as it responds to its environment
with the goal of increasing in mass and
dividing.
• Genes that are continuously expressed
are constitutive genes or housekeeping
genes. Examples include protein
synthesis and glucose metabolism.
• All genes are regulated at some level, so
that as resources decrease the cell can
respond with a different molecular
strategy.
• Prokaryotic genes are often organized into
operons that are cotranscribed: In a
nutshell, a regulatory protein binds to an
operator sequence in the DNA adjacent to
the gene array, and controls production of
the polycistronic (polygenic) mRNA.
• Gene regulation in bacteria and phage seems to
be similar in many ways to the 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 percent of the genomic ORFs have no
attributed function.
• (ORFs open reading frame are A stretch of DNA
that when translated into an amino acid
sequence doesn’t contain an internal stop
codon. An ORF can be evidence that a DNA
sequence is part of a gene or that codes for a
specific portion of the complete protein).
• There are two classic examples to
understand gene regulation in
prokaryotes. The lac operon, an example
of inducible operons and the tryptophan
(trp)that is an example of repressible
operons.
Inducible operons, The Lac operon
• 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.
Lactose as a Carbon Source for E. coli
• E. coli expresses genes for glucose metabolism constitutively, but
the genes for metabolizing other sugars are regulated in a “sugar
specific” sort of way. When that specific sugar is present, the
presence of the sugar stimulates synthesis of the proteins needed
to metabolize it.
• When the dissacharide lactose is E. coli’s sole carbon source, three
genes are expressed:
• a. β-galactosidase (bacterial lactase) has two functions:
1. Breaking lactose into glucose and galactose. (Galactose is
converted to glucose, and glucose is metabolized by constitutively
produced enzymes.)
2. Converting lactose to allolactose (an isomerization).
Allolactose is involved in regulation of the lac operon (Figure
19.2).
• a. β-galactosidase (bacterial lactase) has two functions:
1. Breaking lactose into glucose and galactose.
(Galactose is converted to glucose, and glucose is
metabolized by constitutively produced enzymes.)
2. Converting lactose to allolactose (an isomerization).
Allolactose is involved in regulation of the lac operon.
• b. Lactose permease (M protein) is required for
transport of lactose across the cytoplasmic membrane.
• c. Transacetylase is poorly understood.
The lac operon shows coordinate induction
• In glucose medium, E. coli normally has very low
levels of the lac gene products.
• 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.
• ii. The mRNA for the enzymes has a short
half-life. When lactose is gone, lac transcription
stops, and enzyme levels drop rapidly.
Experimental Evidence for the
Regulation of lac Genes
• In 1961, Francois Jacob and Jacques Monod
proposed the operon model for the control of
gene expression in bacteria.
• The experiments of Jacob and Monod produced
an understanding of arrangement and control of
the lac genes.
• They worked with mutants strains that allowed
them to understand and hypothesized how that
regulatory mutations had affected the normal
control of gene expression for the operon.
Jacob and Monod’s Operon Model
for the Regulation of lac Genes
• Jacob and Monod’s model of regulation, with
more recent information, follows:
• 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.
• The lacI gene has its own constitutive promoter and
terminator, and repressor protein is always present in low
concentration. Repressor protein binds the operator
(lacO+), and prevents RNA polymerase initiation to
transcribe the operon genes (negative control). (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).
• β-galactosidase in wild-type E. coli
growing with lactose as the sole carbon
source converts lactose into allolactose.
The allolactose binds
to the repressor and
this association
produces allosteric shift
(changes shapes) and
dissociates from the lac
operator. Free
repressor-allolactose
complexes are unable
to bind the operator.
„
• By removing the repressor protein from
the operator, Allolactose induces
expression of the lac operon by allowing
transcription to occur.
Positive control of this operon
• Repressor exerts negative control by preventing
transcription. Positive control of this operon also
occurs when lactose is E. coli’s sole carbon source, with
no glucose present (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 recruits RNA
polymerase to the promoter, leading to transcription.
Positive control of this operon
• When both glucose and lactose are in the medium, E. coli preferentially
uses glucose, due to catabolite repression (glucose effect)
• 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 CAPcAMP 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.
• 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.
The trp operon in 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.
Gene Organization of the
Tryptophan Biosynthesis Genes
• 1. Yanofsky and colleagues characterized the controlling
sites and genes of the trp operon.
• a. There are five structural genes, trpA through trpE.
• 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 7kb. The operon
produces a polygenic transcript with five structural
genes for tryptophan biosynthesis.
Regulation of the trp Operon
• Two mechanisms regulate expression of the trp operon:
a. Repressor–operator interaction.
• When tryptophan is present, it will bind to an
aporepressor protein (the trpR gene product. The trpR
gene is located close to this operon, but it is not part of
it).
• The active repressor (aporepressor-tryptophan) binds the
trp operator, and prevents transcription initiation.
Repression reduces transcription of the trp operon about
70-fold.
b. Regulation by Transcription termination
When there is tryptophan maximal starvation, the expression
of the tryptophan operator is maximal, however when there is
not starvation, transcription is also controlled by attenuation.
Attenuation produces only short (140bp) transcripts that do
not encode structural proteins and it can reduce trp operon
transcription 8- to 10-fold..
Termination occurs at the attenuator site within the trpL
region. The proportion of attenuated transcripts to full-length
ones is related to tryptophan levels, with more attenuated
transcripts as the tryptophan concentration increases.
Together, repression and attenuation regulate trp gene
expression over a 560- to 700-fold range.