Regulation of gene expression in bacteria

lac Operon of E. coli

trp operon of E. coli

Gene expression in bacteria - some useful distinctions

Regulated genes

Control cell growth and cell division.

Expression is regulated by the needs of the cell and the

environment as needed (not continuously).

Constitutive genes

Continuously expressed.

Housekeeping genes (such as those required for protein synthesis

and glucose metabolism).

*All genes are regulated at some level!

Operon - what is it?

Cluster of genes in which expression is regulated by operator-repressor

protein interactions, operator region, and the promoter.

• Contents of an operon:

Promoter
Repressor
Operator (controlling site)
Coding sequences
Terminator

• Adjacent polycystronic coding sequences (e.g., bacteria, mtDNA) are

co-transcribed to make a polygenic mRNA.

Inducers and induction:

• Inducer = chemical or environmental agent that initiates

transcription of an operon.

• Induction = synthesis of gene product(s) in response to an inducer.

Organization of an inducible gene containing an operon
E. coli’s lac operon:

• E. coli expresses genes for glucose metabolism continuously.

• Metabolism of other alternative types of sugars (e.g., lactose) are

regulated specifically.

• Lactose = disaccharide (glucose + galactose), provides energy.

• Lactose acts as an inducer (effector molecule) and stimulates

expression of three proteins at 1000-fold increase:

-galactosidase (lacZ)

•Breaks lactose into glucose + galactose.

•Converts lactose to the allolactose, regulates lac operon.

•Lactose permease (lacY)

•Transports lactose across cytoplasmic membrane.

•Transacetylase (lacA)

•transfers an acetyl group from acetyl-CoA to -galactosides.

General organization of the lac operon of wild-type E. coli

Order of controlling elements and genes:

lacI: promoter-lacI-terminator

operon: promoter-operator-lacZ-lacY-lacA-terminator
Functional state of the E. coli lac operon in the absence of lactose
Functional state of the E. coli lac operon growing on lactose
Model of lac repressor tetramer (4 polypetides) protein
Summary of Jacob-Monod E. coli lac operon model
1. Operon is a cluster of genes; expression is regulated by operator-
repressor protein interactions, operator, and a promoter.

2. lac I gene possesses its own weak promoter and terminator; lacI
repressor proteins always exist in low concentration.

• Repressor protein is a tetramer (4 polypeptides).

• Repressor binds the operator (lacO) and prevents RNA

polymerase initiation of transcription.

• Binding is not complete, so low levels of lacZ, lacY, and lacA

proteins are always synthesized.

• As soon as lactose occurs in high concentration, lac operon

switches to the “on” position.

3. -galactosidase in wild-type E. coli growing on lactose converts

lactose to allolactose.

• Allolactose binds to repressor proteins, which in turn are

“disabled” and unable to bind the operator.

• Allolactose induces expression of the lac operon.

Summary of Jacob-Monod E. coli lac operon model (cont.):

4. RNA polymerase initiates synthesis of a single polygenic mRNA

containing mRNA for lacZ, lacY, and lacA.

5. mRNA is translated as a single molecule by a string of ribosomes.

6. lac operon is said to be under negative control (lacI blocks RNA

polymerase if inducer is absent).

7. Different types of mutations occur in lacO, lacI, and promotor:

lacO -change repressor binding site (repressor does

not bind)
-continuously expressed

lacI -change repressor conformation (cannot bind

operator)
-continuously expressed

-super-repressors bind operator but not allolactose

-lactose does not induce the operon

promoter -alter affinity for RNA polymerase

-increase or decrease transcription rate
Positive control also occurs in the lac operon:

• Positive control occurs when lactose is E. coli’s sole carbon source

(but not if glucose also is present).

• Catabolite activator protein (CAP) binds cAMP, activates, and binds to

a CAP recognition site upstream of the promoter (cAMP is greatly
reduced in presence of glucose).

• CAP changes the conformation of DNA and facilitates binding of RNA

polymerase and transcription.

• When glucose and lactose are present, E. coli preferentially uses

glucose due to low levels of active CAP (low cAMP).

• Adding cAMP to cells restore transcription of the lac operon even

when glucose is still present.
Positive control of the
lac operon with CAP
Sequence of the lac operon was the first well-characterized molecular
model of gene regulation:

• lac operon promoter begins at -84 bp immediately after the lacI stop
codon and ends at -8 bp from the transcription start site.

• CAP-cAMP binding site occurs at -54 to -58 and -65 to -69.

• RNA polymerase binding site spans -47 to -8.

• Operator is next to the promoter at -3 to +21.

• mRNA transcript begins at +1 bp within the operator.

• -galactosidase gene has a leader sequence before the start codon.

• -galactosidase start codon (AUG) is at position +39 to +41

Base pair sequence of controlling sites, promoter, and operator for
lac operon of E. coli
The Trp operon of E. coli:

• If amino acids are present in the growth medium E. coli will “import”
amino acids before it makes them.

• Genes for amino acid synthesis are repressed, repressible operons.

• When amino acids are absent in the growth medium, genes are
“turned on” (or expressed) and amino acid synthesis occurs.

• The tryptophan (Trp) operon of E. coli is one of the most extensively

studied repressible operons.
The Trp operon of E. coli; first characterized by Charles Yanofsky et al.:

• Trp operon spans ~7 kb and produces 5 gene products required for

synthesis of the amino acid tryptophan.

• Trp operon contains 5 biosynthetic coding genes, trpA-E.

• Promoter and operator are upstream of trpE.

• Leader region (trpL) occurs between trpA-E coding genes and the
operator.

• Within trpL is an attenuator region (att).

• TrpR (repressor protein gene) occurs upstream of the promoter.

General organization of the Trp operon of E. coli
Regulation of the trp operon

Two mechanisms regulate the trp operon:

1. Repressor/operator interaction

2. Termination of initiated transcripts

Regulation of the trp operon:

1. Repressor/operator interaction

• When tryptophan is present, tryptophan binds to trpR gene product.

• trpR protein binds to the trp operator and prevents transcription.

• Repression reduces transcription of the trp operon ~70-fold.

Regulation of the trp operon:

2. Termination of initiated transcripts

• Transcription also is controlled by attenuation, process of translating

a short, incomplete polypeptide.

• When cells are starved for tryptophan, trp genes are expressed
maximally.

• Under less severe tryptophan starvation, trp genes are expressed at

lower than maximum levels.

• Attenuation can regulate transcription level by a factor of 8 to 10,

and combined with the repression mechanism, 560-700 fold.
Molecular model for attenuation:

• Recall that a leader region (trpL) occurs between the operator and
the trpE sequence.

• Within this leader is the attenuator sequence (att).

• att sequence contains a start codon, 2 Trp codons, a stop codon,

and four regions of sequence that can form three alternative
secondary structures.

Secondary structure Signal

• Paired region 1-2 pause

• Paired region 2-3 anti-termination

• Paired region 3-4 termination

Organization of the leader/attenuator trp operon sequence
Molecular model for attenuation (cont.):

• Recall that transcription and translation are tightly coupled in

prokaryotes and occur simultaneously.

• Pairing of mRNA regions 1 and 2 causes RNA polymerase to pause

just after these regions are synthesized.

• Pause is long enough for a ribosome to load onto the mRNA and
begin translating just behind RNA polymerase.
Molecular model for attenuation (cont.):

Position of the ribosome plays an important role in attenuation:

When Trp is scarce or in short supply (and required):

1. Trp-tRNAs are unavailable, ribosome stalls at Trp codons and

covers attenuator region 1.

2. Region 1 cannot pair with region 2, instead region 2 pairs with

region 3 when it is synthesized.

3. Region 3 (now paired with region 2) is unable to pair with

region 4 when it is synthesized.

4. RNA polymerase continues transcribing region 4 and beyond

synthesizing a complete trp mRNA.
Molecular model for attenuation (cont.):

Position of the ribosome plays an important role in attenuation:

When Trp is abundant (and not required):

1. Ribosome does not stall at the Trp codons and continues

translating the leader polypeptide, ending in region2.

2. Region 2 cannot pair with region 3, instead region 3 pairs with

region 4.

3. Pairing of region 3 and 4 is the “attenuator” sequence and

acts as a termination signal.

4. Transcription terminates before the trp synthesizing genes are

reached.
Attenuation model in Trp starved cells
Attenuation model in Trp non-starved cells
Predicted amino acid sequences of attenuators for
Phe, His, Leu, Thr, and Ile operons in E. coli