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2017-18
Genética Molecular

Genética de Bactérias e
seus Vírus- Parte 3

Gene
expression in
prokaryotes:
The operon
model

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Operon Model of Regulation

• In bacterial systems, when several enzymes act in

sequence in a single metabolic pathway, usually
either all or none of the enzymes are produced

• This coordinate regulation results from control of

the synthesis of one or more mRNA molecules that
are polycistronic

• Polycistronic mRNAs are transcribed from an

operon: a collection of adjacent structural genes
regulated by an operator

The lac operon

and its
regulation

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lac Operon
• Structure

lac Operon
• The genetic regulatory mechanism in bacteria was first
explained by the operon model of François Jacob and
Jacques Monod

• They studied lactose-utilization system in E.coli

• The lactose-utilization system consists of two kinds of

components: structural genes (lacZ , lacY, lacA), which
encode proteins needed for the transport and
metabolism of lactose, and regulatory elements (the
repressor gene lacI, the promoter lacP, and the operator
lacO)
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lac Operon
• The products of the lacZ (enzyme β -galactosidase),
lacY (transporter lactose permease) and lacA
(galactoside transacetylase) genes are encoded by a
single polycistronic mRNA

• The linked structural genes, together with lacP and

lacO, constitute the lac operon

• The promoter mutations (lacP–) eliminate the ability to

synthesize lac mRNA

lac Operon
• The product of the lacI gene is a repressor, which
binds to a unique sequence of DNA bases
constituting the operator

• When the repressor is bound to the operator,

initiation of transcription of lac mRNA by RNA
polymerase is prevented

• Because the repressor is necessary to shut off

mRNA synthesis, regulation by the repressor is
negative regulation

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lac Operon
• Structure

lac Operon

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cyclic AMP is a second messenger

important for intracellular signaling

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In bacteria, the level of cAMP varies depending on the medium used for growth.
In particular, cAMP is low when glucose is the carbon source. This occurs through
inhibition of the cAMP-producing enzyme, adenylate cyclase, as a side-effect of
glucose transport into the cell. The transcription factor cAMP receptor protein (CRP)
also called CAP (catabolite gene activator protein) forms a complex with cAMP and
thereby is activated to bind to DNA. CRP-cAMP increases expression of a large
number of genes, including some encoding enzymes that can supply energy
independent of glucose.

cAMP is involved in the positive regulation of the lac operon.

In an environment with a low glucose concentration, cAMP accumulates and binds to
the allosteric site on CRP (cAMP receptor protein), a transcription activator protein.
The protein assumes its active shape and binds to a specific site upstream of the lac
promoter, making it easier for RNA polymerase to bind to the adjacent promoter to
start transcription of the lac operon, increasing the rate of lac operon transcription.
With a high glucose concentration, the cAMP concentration decreases, and the CRP
disengages from the lac operon.

For details; see Science. 1970 Jul 24;169(3943):339-44.

Cyclic adenosine monophosphate in bacteria.
Pastan I, Perlman R. 16

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c-di-AMP metabolism
and effector functions

Membrane bound and/or

soluble DACs synthesize
c-di-AMP from ATP.

CChem. Commun., 2016, 52, 9327-9342

c-di-AMP binds to
receptor/target proteins
and RNA to modulate
physiological functions.

Phosphodiesterases
(PDE) degrade c-di-AMP
into the linear pApA or
AMP and the second
messenger could also be
secreted by the efflux
pumps of some
intracellular bacteria.

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Sequence of the lac operon control region

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Sequência da região controlo do operão lac

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Concentration of cyclic AMP in cells growing in media with the

indicated carbon sources

Table 9.2.

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Transcription Regulation: Prokaryotes

• In prokaryotes, on–off gene activity is often

controlled through transcription

• In negative regulation, the default state of

transcription is “on” until it is turned “off” by
a repressor protein that binds to the DNA
upstream from the transcriptional start site

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Negative regulation: mechanisms of induction & repression

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Transcription Regulation: Prokaryotes

• In inducible transcription, a repressor

keeps transcription in the “off” state
(repressor + inducer → on)

• In repressible transcription, the default

state is “on” until an active repressor is
formed to turn it “off” (aporepressor + co-
repressor → off)
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Transcription Regulation: Prokaryotes

• In positive regulation, the default state of transcription is

“off” and binding with a transcriptional activator protein
is necessary to turn it “on”

• Many systems are both positively and negatively

regulated

• Negative regulation is more common in prokaryotes

•
• Positive regulation is more common in eukaryotes

• Some genes exhibit autoregulation - the protein product

of a gene regulates its own transcription 31

Positive Regulation: transcription is “off”

and needs to be activated

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lac Operon
• lac operon is also subject to positive
regulation

• Positive regulation of the lac operon involves

cAMP-CRP (cyclic AMP receptor protein),
which binds to the promoter to activate
transcription by RNA polymerase

• In the absence of the cAMP–CRP complex,

RNA polymerase binds only weakly to the
promoter
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IN SUMMARY: Four regulatory states of the lac operon

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Characteristics of partial diploids containing several

combinations of lacI, lacO, and lacP alleles

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Examples of how genotype affects phenotype:

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Facts to remember about

Mechanisms of
transcription and
translation in bacteria

In prokaryotes, the transcription is terminated by two major

mechanisms: Rho-independent (intrinsic) and Rho-dependent.
Rho-dependent mechanism
• Rho is a ~ 50 kD protein, involved in about half of E. coli transcriptional
terminations. It has been well established that six Rho proteins form a
hexamer to terminate transcription, but the precise mechanism is not clear.
• Experiments suggest that two components are essential: (i) the upstream
Rho loading site and (ii) the downstream termination site.
• The Rho hexamer first binds to the RNA transcript at the upstream site
which is 70-80 nucleotides long and rich in C residues. Upon binding, the
Rho hexamer moves along the RNA in the 3' direction. If movement of the
polymerase is slow, the Rho hexamer will catch up and terminate the
transcription at the downstream termination site.
• Rho has ATPase activity which can induce release of the polymerase from
DNA.

http://www.web-books.com/MoBio/Free/Ch4D2.htm

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The Rho-independent termination signal is a stretch of 30-

40 bp sequence, consisting of many GC residues followed by
a series of T ( "U" in the transcribed RNA). The resulting RNA
transcript will form a stem-loop structure to terminate
transcription. Ex: The trp operon

http://www.web-books.com/MoBio/Free/Ch4D2.htm

Facts to remember ABOUT

TRANSLATION INITIATION in
procaryotes:
- Translation starts at each
ribosome binding site (or
Shine-Dalgarno sequence)
- The initiation complex
assembles at this region,
prior to each ATG codon
- -The initiation complex is
composed by the small
ribosome unit (30s) +
initiation factors+ tRNA of
fMet.
- Once positioned the initiation
complex, the large subunit
(50s) will assemble and the
ribosome complex is ready to
translate

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• With the formation of the initiation complex, the fMet-tRNA

occupies the P site of the ribosome and the A site is left
empty.
• This entire initiation process is facilitated by extra proteins,
called initiation factors that help with the binding of ribosomal
subunits and tRNA to the mRNA chain.

http://www.sparknotes.com/biology/molecular/translation/section3.rhtml

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www.sparknotes.com/biology/molecular/translation/section3.rhtml • With tRNA bearing a chain

of amino acids in the P site
and tRNA containing a
single amino acid in the A
site, the addition of a link to
the chain can be made.

• This addition occurs

through the formation of a
peptide bond, the nitrogen-
carbon bond that forms
between amino acid
subunits to form a
polypeptide chain.

• This bond is catalyzed by

the enzyme peptidyl
transferase.

With the A site open again, the

next appropriate aminoacyl
tRNA can bind there and the
same reaction takes place,
yielding a three-amino acid
peptide chain.

This process repeats, creating

a polypeptide chain in the P
site of the ribosome.

A single ribosome can translate

60 nucleotides per second.
This speed can be vastly
augmented when ribosomes
link up to form polyribosomes.

http://www.sparknotes.com/biology/molecular/translation/section3.rhtml

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Ribosomes link up to form

polyribosomes

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Termination of
Translation involves
the recognition of the
stop codon by
releasing factors
(RFs)

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Termination of translation

Translation ends when one of three stop

codons, UAA, UAG, or UGA, enters the A site
of the ribosome.

There are no aminoacyl tRNA molecules that

recognize these sequences. Instead, release
factors bind to the P site, catalyzing the
release of the completed polypeptide chain
and separating the ribosome into its original
small and large subunits.
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Differences between Prokaryote and Eukaryote Translation

Remember that only eukaryotes, and not prokaryotes, undergo post-
transcriptional RNA modifications. These modifications are responsible for
one difference between prokaryotic and eukaryotic translation. Whereas
prokaryotic initiation, which we have just covered, begins with the ribosomal
recognition of the ribosome binding site on the mRNA, eukaryotic initiation
begins with the ribosomal recognition of the 5' cap.

Eukaryotic mRNA need not contain a ribosome binding cap because the
post-transcriptionally added 5' cap suffices for recognition.

Once the eukaryote ribosome binds to the mRNA, further differences

appear

• Whereas in prokaryotes the initiator codon can be either GUG or AUG, in

eukaryotes the codon must be AUG.

• In eukaryotes, the tRNA responsible for recognizing the initiator codon is

not the special tRNA, fMet, but rather the normal met-tRNA.
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