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 Molecular Biology

 Douglas W. Smith
York 2722

 BIMM 100

 5254 Muir Biology Building
Fall, 2000  

 x42620; dsmith@ucsd.edu

 

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18. Prokaryotic Gene Regulation: Operons

 

Readings: Brown, 8:185-188; 9:206, 208

Outline:

 

 

Operons and the resulting transcriptional regulation of gene expression permit prokaryotes to rapidly adapt to changes in the environment: new carbon sources, lack of an amino acid, etc. The genes involved in the catabolism of the carbon source or in the biosynthesis (anabolism) of the amino acid can be rapidly turned on or turned off.
Negative control of expression of the Operon is the dominant case.

A. Principles of Operon Theory:
... [Brown, Section 8.3.1]

An Operon is a set of genes that are expressed in unison, ie they are Coordinately expressed, together with the DNA control elements used for their expression.

Usually, an Operon is a set of genes which are adjacent to each other on the chromosome and are Coordinately Expressed at the transcription level via a single mRNA molecule.

A negatively-acting protein called the Repressor regulates expression of the Operon by binding to an Operator DNA site near the promoter for transcription.

Expression of the gene encoding the Repressor is usually NOT itself controlled by a Repressor. It then is expressed at all times, ie is constitutively expressed.

This Operator binding site for the Repressor is the major type of DNA control element.

The genes that are Coordinately expressed are called Structural Genes and encode proteins that function enzymatically in a common process such as a biosynthetic or catabolic pathway.

The genes that encode regulatory proteins such as Repressors are called Regulator Genes and are NOT usually part of the set of coordinately expressed operon genes.

A small molecule (metabolite) usually interacts with the Repressor. This small molecule is either an Inducer or a Co-Repressor:

An Inducer inactivates the Repressor in the Inducer-Repressor complex.

A Co-Repressor activates the Repressor in the Inducer-Repressor complex.

Thus binding of this small molecule to one site of Repressor alters its ability to bind Operator DNA at a second site on Repressor ... example of Allosterism

NOTE: Products of Regulatory Genes can then diffuse or move to their targets and hence are Trans-acting Factors.

Operators and DNA control elements are the targets and affect only the DNA to which they are attached; they are Cis-acting elements. Any protein binding site on DNA, e.g. transcription promoters and terminators, are cis-acting elements.

 

B. Two general Operon Classes:
Catabolic and Anabolic Pathways

1. Catabolic Operons:

The small molecule interacting with the Repressor is an Inducer, and is usually the sugar or metabolic which is broken down.

Note the rationale: the operon should be expressed only when Inducer is present, to express the genes needed to break down (catabolize) the Inducer, eg sugar

General Example:

Structural genes A, B, C with Operator O near Promoter P.
Regulator gene R constitutively expressing Repressor R inactivated by Inducer I, as shown in the following Figure:

2. Anabolic (biosynthetic) Operons:

The small molecule interacting with the Repressor is a Co-Repressor, and is usually the end-product of the biosynthetic pathway, eg an amino acid.

Note the rationale: the operon should be expressed only when Co-Repressor is absent, to express the genes needed to synthesize the Co-Repressor, eg amino acid

General Example:

Structural genes A, B, C with Operator O near Promoter P.

Regulator gene R constitutively expressing Repressor R activated by Co-Repressor Co, as shown in the following Figure:

 

 

C. Lac Operon - Paradigm Catabolic Operon
... [Brown, Fig 8.15]

1. Basic Features:

Three structural genes: lacZ - lacY - lacA

encoding b-galactosidase, lac permease, and lac transacetylase respectively.

b-galactosidase hydrolyzes lactose to galactose and glucose;
permease transports lactose into the cell;
and transacetylase is important only in some environments.

Gene encoding Lac Repressor: lacI ... active as a Tetramer

fortuitously adjacent to Lac Operon ... constitutively expressed: 10 copies/cell

Inducer: b-galactosides, including non-metabolizable molecules such as IPTG
Xgal: a colorometric inducer ... when cleaved yields a blue color

Induction occurs when Inducer is added to growth medium: Repressor is inactivated

1000-fold increase in gene expression in minutes
with rapid return to Basal Level (few molecules expressed under repression) when Inducer is removed

NOTE: unstable mRNA is important here! but still have enzyme molecules present ...
Prokaryotic mRNA is usually unstable, with a half-life of 2-4 minutes

 

2. Genetics: ... Mutations in Regulation Processes

a. Noninducible Mutants: Mutations that prevent induction, i.e. lac operon always turned off

1) lacIs mutants: point mutations that yield a "Super Repressor", one that binds the Operator even in the presence of Inducer

lacIs mutants are dominant to both lacI- mutations and lacI+ wildtype in merodiploids: lacIs/lacI+ or lacIs/lacI- are phenotypically LacIs

 

2) lacP mutants: nearly all Promoter mutations are "down" mutants, i.e. that promoter does not support transcription by HoloRNApol. In this case, the lac operon is turned off independent of presence or absence of Inducer...

 

b. Constitutive Mutants: Mutations in which the Lac Operon is always turned on

1) lacOc operator constitutive mutants: point mutations in the Operator that prevent Repressor binding ... Lac operon is always turned on ...

These mutants are Cis-dominant, ie they affect ONLY the DNA (lac genes) to which the Operator is attached.

Thus, lacOc/lacO+ merodiploids are phenotypically LacOc, ie are constitutive, but only via expression of the lac genes on the same DNA as the lacOc mutation ...

For cis vs trans, see Brown, Box 8.4


2) lacI- mutants, eg deletions: Repressor is inactive and Lac operon is on ...

These can be complemented in trans by product of a lacI+ gene on a plasmid ...
Thus, lacI encodes a trans-acting factor and is a Gene ...

That is: Phenotype of lacI-/ lacI+ is LacI+ or lacI+ is Dominant to lacI-

(such duplications of parts of the chromosome on a plasmid yield mero-diploid or partial diploid cells ... traditionally done with F plasmid as F-primes ... now can be done with any recombinant DNA chimeric plasmids ...)

 

3) lacI-d mutants: Defective repressor that cannot bind operator ...

These mutants are similar to lacI- mutants,
BUT lacI-d are dominant to lacI+ ...
due to Tetramer with bad subunits ... such a Tetramer is unable to bind Operator

DNA Mapping of the lacI mutations identifies Repressor Domains, the domain that binds inducer versus the domain that binds Operator DNA.

 

c. "Knockout" mutants are the key! ... Best: delete entire gene ...next best: large deletion or insertion, eg Transposon ... next best: frameshift mutations (short insertions or deletions)

If the knockout mutant cells are constitutive, the control is Negative Control.

If the knockout mutant cells are noninducible, the control is Positive Control.

Without a Knockout mutant, the mutation may either have inactivated the gene or increased the activity of the gene product, ie made a "super" product.

Example: LacI product could be positive acting, ie an Activator, in which case LacI- mutants could be a "super" Activator and LacIs mutants could be an inactive Activator.

 

3. Binding of Lac Repressor to Operator: ... [similar to Brown, Fig 7.20]

Operator defined by lacOc mutants ... 26 bp region, nuc palindrome (in part)

Binding defined by DNA footprinting and protection assays

Binding of Inducer to Repressor causes conformational change in Repressor, probably via a Hinge motion between Head piece and rest of Repressor protein and Inducer-Repressor complexes binds much less strongly to Operator

Repressor falls off Operator, bind DNAs elsewhere

 

4. Mechanism of Inhibition of Transcription:

Repressor actually causes RNA polymerase to bind more readily to Lac Promoter !!

Recent evidence: Repressor decreases affinity of rNTPs for RNA polymerase; in their absence, RNA polymerase falls off at a nearby pause site during transcription elongation ...

 

5. Catabolite Repression: "glucose effect" ... [Brown, Fig 11.6]

Complex of cAMP (3',5'-cyclic-AMP; Brown, Fig 11.6) and Catabolite Activator Protein (CAP) binds as Dimer to CAP binding site usually within or adjacent to Promoter of Catabolic Operons, e.g. in the Lac operon promoter at left end.

Binding of CAP-cAMP is required for expression of these Catabolite Operons, i.e. Transcription of these Operons is activated by CAP-cAMP binding

CAP Binding Site: ~22 bp, pentamer palindrome

 

CAP-cAMP binding affects both RNA polymerase and DNA structure:

CAP-cAMP interacts directly with Alpha subunit of RNA polymerase ... geometry of the precise location of the CAP binding site is important here (operons differ...)

CAP-cAMP binding bends the DNA nearly 90°

 

Function of Catabolite Repression:

Facilitate use of Glucose over Lactose (or other sugars) when both Glucose and Lactose are available to the bacteria

Mechanism:

When Glucose is present, cAMP levels are low, little CAP-cAMP complex is formed, and low expression of Lac operon even when Lactose is present.

When Glucose is absent, cAMP levels are high, much CAP-cAMP complex is formed, and Lactose induces Lac operon to high level of expression

 

 

 

D. Tryptophan (Trp) Operon - Paradigm Anabolic Operon

1. Basic Features: ... [Brown, Fig 8.6]

5 structural genes, enzymes used for Trp biosynthesis; 2 normal terminators

Control Region: Promoter, Operator, Leader sequence, Attenuator

Regulator Gene: trpR ... gene unlinked to Trp Operon ... Trp Repressor

Tryptophan is a Co-Repressor: Repressor-Trp complex is active Repressor

Repressor by itself is inactive ... Derepression: 70-fold increase in expression

Typical anabolic Operon ...

2. Attenuation: ... [Brown, Fig 9.10]

additional Control in Trp operon and other aa operons ... 10-fold effect

Intrinsic transcription Terminator at the beginning of the Operon

Principle: alternative hairpin structures, one of which is active Terminator

 

Mechanism:

1. RNA polymerase initiates and transcribes a short coding region for a Leader peptide of 14 amino acids ... this leader is probably translated

2. Two of the 14 amino acids are Tryptophanes ...

3. The Attenuator region can form two alternative Hairpin structures:

Regions 1 and 2 paired, and 3 and 4 paired; or Regions 2 and 3 paired

4. Pairing 3:4 forms the transcription Terminator for Leader mRNA

Model: Presence or absence of Trp amino acid the key

In absence of Trp: ribosome stalls at the two Trp codons, permitting 2:3 pairing in Attenuator before 4 is transcribed ... no 3:4 pairing, no transcription termination, and Trp operon is transcribed completely

In presence of Trp: ribosome moves through the two Trp codons, disrupting 2:3 pairing in Attenuator ... 3:4 pairing is permitted, transcription termination occurs, and Trp operon is not transcribed.

Thus: coupled transcription-translation important; termination depends on stem-loop structure in the mRNA rather than in the DNA ... RNA polymerase pauses AFTER the stem, releases the mRNA due to presence of string of U's pairing with DNA A's

 

3. Feedback Inhibition:

Tryptophane, as the End Product of the trp operon, can interact with the first enzyme of the pathway, causing an Allosteric Conformational change in the structure of the first enzyme, inactivating its catalytic activity ...

If first enzyme is inactive, no intermediates (and no product) are synthesized. ...

 






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